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    Pre- and post-puberty expression of genes and proteins in the uterus of Bos indicus heifers: the luteal phase effect post-puberty. Fortes M R S,Zacchi L F,Nguyen L T,Raidan F,Weller M M D C A,Choo J J Y,Reverter A,Rego J P A,Boe-Hansen G B,Porto-Neto L R,Lehnert S A,Cánovas A,Schulz B L,Islas-Trejo A,Medrano J F,Thomas M G,Moore S S Animal genetics Progesterone signaling and uterine function are crucial in terms of pregnancy establishment. To investigate how the uterine tissue and its secretion changes in relation to puberty, we sampled tissue and uterine fluid from six pre- and six post-pubertal Brahman heifers. Post-pubertal heifers were sampled in the luteal phase. Gene expression of the uterine tissue was investigated with RNA-sequencing, whereas the uterine fluid was used for protein profiling with mass spectrometry. A total of 4034 genes were differentially expressed (DE) at a nominal P-value of 0.05, and 26 genes were significantly DE after Bonferroni correction (P < 3.1 × 10 ). We also identified 79 proteins (out of 230 proteins) that were DE (P < 1 × 10 ) in the uterine fluid. When we compared proteomics and transcriptome results, four DE proteins were identified as being encoded by DE genes: OVGP1, GRP, CAP1 and HBA. Except for CAP1, the other three had lower expression post-puberty. The function of these four genes hypothetically related to preparation of the uterus for a potential pregnancy is discussed in the context of puberty. All DE genes and proteins were also used in pathway and ontology enrichment analyses to investigate overall function. The DE genes were enriched for terms related to ribosomal activity. Transcription factors that were deemed key regulators of DE genes are also reported. Transcription factors ZNF567, ZNF775, RELA, PIAS2, LHX4, SOX2, MEF2C, ZNF354C, HMG20A, TCF7L2, ZNF420, HIC1, GTF3A and two novel genes had the highest regulatory impact factor scores. These data can help to understand how puberty influences uterine function. 10.1111/age.12721
    Using advanced spatial and single-cell transcriptomics to characterize the human endometrium. Nature genetics 10.1038/s41588-021-00982-0
    The development of the human uterus: morphogenesis to menarche. Human reproduction update There is emerging evidence that early uterine development in humans is an important determinant of conditions such as ontogenetic progesterone resistance, menstrual preconditioning, defective deep placentation and pre-eclampsia in young adolescents. A key observation is the relative infrequency of neonatal uterine bleeding and hormone withdrawal at birth. The origin of the uterus from the fusion of the two paramesonephric, or Müllerian, ducts was described almost 200 years ago. The uterus forms around the 10th week of foetal life. The uterine corpus and the cervix react differently to the circulating steroid hormones during pregnancy. Adult uterine proportions are not attained until after puberty. It is unclear if the endometrial microbiome and immune response-which are areas of growing interest in the adult-play a role in the early stages of uterine development. The aim is to review the phases of uterine development up until the onset of puberty in order to trace the origin of abnormal development and to assess current knowledge for features that may be linked to conditions encountered later in life. The narrative review incorporates literature searches of Medline, PubMed and Scopus using the broad terms individually and then in combination: uterus, development, anatomy, microscopy, embryology, foetus, (pre)-puberty, menarche, microbiome and immune cells. Identified articles were assessed manually for relevance, any linked articles and historical textbooks. We included some animal studies of molecular mechanisms. There are competing theories about the contributions of the Müllerian and Wolffian ducts to the developing uterus. Endometrium features are suggestive of an oestrogen effect at 16-20 weeks gestation. The discrepancy in the reported expression of oestrogen receptor is likely to be related to the higher sensitivity of more recent techniques. Primitive endometrial glands appear around 20 weeks. Features of progestogen action are expressed late in the third trimester. Interestingly, progesterone receptor expression is higher at mid-gestation than at birth when features of endometrial maturation are rare. Neonatal uterine bleeding occurs in around 5% of neonates. Myometrial differentiation progresses from the mesenchyme surrounding the endometrium at the level of the cervix. During infancy, the uterus and endometrium remain inactive. The beginning of uterine growth precedes the onset of puberty and continues for several years after menarche. Uterine anomalies may result from fusion defects or atresia of one or both Müllerian ducts. Organogenetic differentiation of Müllerian epithelium to form the endometrial and endocervical epithelium may be independent of circulating steroids. A number of genes have been identified that are involved in endometrial and myometrial differentiation although gene mutations have not been demonstrated to be common in cases of uterine malformation. The role, if any, of the microbiome in relation to uterine development remains speculative. Modern molecular techniques applied to rodent models have enhanced our understanding of uterine molecular mechanisms and their interactions. However, little is known about functional correlates or features with relevance to adult onset of uterine disease in humans. Prepubertal growth and development lends itself to non-invasive diagnostics such as ultrasound and MRI. Increased awareness of the occurrence of neonatal uterine bleeding and of the potential impact on adult onset disease may stimulate renewed research in this area. 10.1093/humupd/dmaa036
    Morphological features of the rat uterus. Sikora Kateryna,Lyndin Mykola,Hyriavenko Nataliia,Lyndina Yulia,Sikora Vladyslav,Romaniuk Anatolii Polski merkuriusz lekarski : organ Polskiego Towarzystwa Lekarskiego The rat uterus is an important female reproductive organ that has essential for the organism's reproduction. That is why it is necessary to understand all the rat uterus' morphological features as a perfect biomodel for studying the molecular peculiarities of the female reproductive system and pathologies development in experimental studies. AIM:The aim of research was to perform the comprehensive morphological analysis of the uterine in intact female rats. MATERIALS AND METHODS:The uterine of reproductive-aged intact female rats were used in this research. The cytological study of vaginal smears, histological (H and E), and immunohistochemical (estrogen, progesterone, and Ki-67 receptors) analysis of uterus tissues were used for light microscopic examination. RESULTS:The rat's vaginal smears' cytological features showed a specific qualitative cellular composition (variation of leukocytes, nucleated and anucleated cornified epithelial cells) in different estrous cycle phases (proestrus, estrus, metestrus, and diestrus). Uterine histology showed the structural regularities of parenchymal and stromal components with clear differentiation on the endometrium, myometrium and perimetrium. It was presented uterus sensitivity to the influence of the sex hormones (positive to estrogen and progesterone receptors) and the variable cellular proliferation activity (Ki-67 expression) in the organ wall. CONCLUSIONS:Our research demonstrated that the rats« uterus has a unique structural organization, sex hormones sensitivity, and variable proliferation in the parenchymal and stromal components. The rat estrous cycle should be considered while studying the morphological features of the uterus. The rat's uterus may serve as an acceptable object for modeling various pathological processes with the following results' extrapolation.
    Uterine deficiency of Dnmt3b impairs decidualization and causes consequent embryo implantation defects. Long Jing,Li Weike,Chen Mengyue,Ding Yubin,Chen Xuemei,Tong Chao,Li Na,Liu Xueqing,He Junlin,Peng Chuan,Geng Yanqing,Liu Taihang,Mu Xinyi,Li Fangfang,Wang Yingxiong,Gao Rufei Cell biology and toxicology Uterine deficiency of Dnmt3b impairs decidualization and consequent embryo implantation defects. Recent advances in molecular technologies have allowed the unprecedented mapping of epigenetic modifications during embryo implantation. DNA methyltransferase 3a (DNMT3A) and DNMT3B are responsible for establishing DNA methylation patterns produced through their de novo-type DNA methylation activity in implantation stage embryos and during germ cell differentiation. It was reported that conditional knockout of Dnmt3a in the uterus does not markedly affect endometrial function during embryo implantation, but the tissue-specific functions of Dnmt3b in the endometrium during embryo implantation remain poorly understood to investigate the role of Dnmt3b during peri-implantation period. Here, we generated Dnmt3b conditional knockout (Dnmt3b) female mice using progesterone receptor-Cre mice and examined the role of Dnmt3b during embryo implantation. Dnmt3b female mice exhibited compromised fertility, which was associated with defective decidualization, but not endometrial receptivity. Furthermore, results showed loss of Dnmt3b did not lead to altered genomic methylation patterns of the decidual endometrium during early pregnancy. Transcriptome sequencing analysis of uteri from day 6 pregnant mice identified phosphoglycerate kinase 1 (Pgk1) as one of the most variable genes in Dnmt3b decidual endometrium. Potential roles of PGK1 in the decidualization process during early pregnancy were confirmed. Lastly, the compromised decidualization upon the downregulation of Dnmt3b could be reversed by overexpression of Pgk1. Collectively, our findings indicate that uterine deficiency of Dnmt3b impairs decidualization and consequent embryo implantation defects. 10.1007/s10565-021-09664-3
    Stanniocalcin-1 in the female reproductive system and pregnancy. Human reproduction update BACKGROUND:Stanniocalcin-1 (STC-1) is a widely expressed glycoprotein hormone involved in a diverse spectrum of physiological and pathophysiological processes including angiogenesis, mineral homeostasis, cell proliferation, inflammation and apoptosis. Over the last 20 years, numerous studies have reported STC-1 expression within female reproductive tissues including the uterus, ovaries and placenta and implicated STC-1 in processes such as ovarian follicular development, blastocyst implantation, vascular remodelling in early pregnancy and placental development. Notably, dysregulation of STC-1 within reproductive tissues has been linked to the onset of severe reproductive disorders including endometriosis, polycystic ovary syndrome, poor trophoblast invasion and placental perfusion in early pregnancy. Furthermore, significant changes in tissue expression and in maternal systemic concentration take place throughout pregnancy and further substantiate the vital role of this protein in reproductive health and disease. OBJECTIVE AND RATIONALE:Our aim is to provide a comprehensive overview of the existing literature, to summarise the expression profile and roles of STC-1 within the female reproductive system and its associated pathologies. We highlight the gaps in the current knowledge and suggest potential avenues for future research. SEARCH METHODS:Relevant studies were identified through searching the PubMed database using the following search terms: 'stanniocalcin-1', 'placenta', 'ovary', 'endometrium', 'pregnancy', 'reproduction', 'early gestation'. Only English language papers published between 1995 and 2020 were included. OUTCOMES:This review provides compelling evidence of the vital function that STC-1 plays within the female reproductive system. The literature presented summarise the wide expression profile of STC-1 within female reproductive organs, as well as highlighting the putative roles of STC-1 in various functions in the reproductive system. Moreover, the observed link between altered STC-1 expression and the onset of various reproductive pathologies is presented, including those in pregnancy whose aetiology occurs in the first trimester. This summary emphasises the requirement for further studies on the mechanisms underlying the regulation of STC-1 expression and function. WIDER IMPLICATIONS:STC-1 is a pleiotropic hormone involved in the regulation of a number of important biological functions needed to maintain female reproductive health. There is also growing evidence that dysregulation of STC-1 is implicated in common reproductive and obstetric disorders. Greater understanding of the physiology and biochemistry of STC-1 within the field may therefore identify possible targets for therapeutic intervention and/or diagnosis. 10.1093/humupd/dmab028
    Bone Morphogenetic Protein 15 Knockdown Inhibits Porcine Ovarian Follicular Development and Ovulation. Qin Yufeng,Tang Tao,Li Wei,Liu Zhiguo,Yang Xiaoliang,Shi Xuan,Sun Guanjie,Liu Xiaofeng,Wang Min,Liang Xinyu,Cong Peiqing,Mo Delin,Liu Xiaohong,Chen Yaosheng,He Zuyong Frontiers in cell and developmental biology Bone morphogenetic protein 15 () is strongly associated with animal reproduction and woman reproductive disease. As a multifunctional oocyte-specific secret factor, BMP15 controls female fertility and follicular development in both species-specific and dosage-sensitive manners. Previous studies found that BMP15 played a critical role in follicular development and ovulation rate in mono-ovulatory mammalian species, especially in sheep and human, but study on knockout mouse model implied that BMP15 possibly has minimal impact on female fertility of poly-ovulatory species. However, this needs to be validated in other poly-ovulatory species. To investigate the regulatory role of BMP15 on porcine female fertility, we generated a BMP15-knockdown pig model through somatic nuclear transfer technology. The BMP15-knockdown gilts showed markedly reduced fertility accompanied by phenotype of dysplastic ovaries containing significantly declined number of follicles, increased number of abnormal follicles, and abnormally enlarged antral follicles resulting in disordered ovulation, which is remarkably different from the unchanged fertility observed in BMP15 knockout mice. Molecular and transcriptome analysis revealed that the knockdown of significantly affected both granulosa cells (GCs) and oocytes development, including suppression of cell proliferation, differentiation, and follicle stimulating hormone receptor () expression, leading to premature luteinization and reduced estradiol (E2) production in GCs, and simultaneously decreased quality and meiotic maturation of oocyte. Our results provide evidence of the essential role of BMP15 in porcine ovarian and follicular development, and new insight into the complicated regulatory function of BMP15 in female fertility of poly-ovulatory species. 10.3389/fcell.2019.00286
    Comparative mRNA and miRNA expression in European mouflon (Ovis musimon) and sheep (Ovis aries) provides novel insights into the genetic mechanisms for female reproductive success. Yang Ji,Li Xin,Cao Yin-Hong,Pokharel Kisun,Hu Xiao-Ju,Chen Ze-Hui,Xu Song-Song,Peippo Jaana,Honkatukia Mervi,Kantanen Juha,Li Meng-Hua Heredity Prolific breeds of domestic sheep (Ovis aries) are important genetic resources due to their reproductive performance, which is characterized by multiple lambs per birth and out-of-season breeding. However, the lack of a comprehensive understanding of the genetic mechanisms underlying the important reproductive traits, particularly from the evolutionary genomics perspective, has impeded the efficient advancement of sheep breeding. Here, for the first time, by performing RNA-sequencing we built a de novo transcriptome assembly of ovarian and endometrial tissues in European mouflon (Ovis musimon) and performed an mRNA-miRNA integrated expression profiling analysis of the wild species and a highly prolific domestic sheep breed, the Finnsheep. We identified several novel genes with differentially expressed mRNAs (e.g., EREG, INHBA, SPP1, AMH, TDRD5, and ZP2) between the wild and domestic sheep, which are functionally involved in oocyte and follicle development and fertilization, and are significantly (adjusted P-value < 0.05) enriched in the Gene Ontology (GO) terms of various reproductive process, including the regulation of fertilization, oogenesis, ovarian follicle development, and sperm-egg recognition. Additionally, we characterized 58 differentially expressed miRNAs and 210 associated target genes that are essential for the regulation of female reproduction cycles through specific regulatory networks [e.g., (miR-136, miR-374a, miR-9-5p)-(EREG, INHBA)]. Furthermore, our integrated mRNA and miRNA expression profiling analysis elucidated novel direct and indirect miRNA/mRNA causal regulatory relationships related to the reproductive traits of the Ovis species. This study provides in-depth insights into the genomic evolution underlying the reproductive traits of the Ovis species and valuable resources for ovine genomics. 10.1038/s41437-018-0090-1
    MiR-378 and BMP-Smad can influence the proliferation of sheep myoblast. Lu Zengkui,Du Lixin,Liu Ruizao,Di Ran,Zhang Liping,Ma Youji,Li Qing,Liu Enmin,Chu Mingxing,Wei Caihong Gene MicroRNA (miRNA) is a sort of endogenous ~20-25 nt non-coding RNAs, and it can regulate a variety of biological events. We found the miR-378 may involve in regulating the muscle development of sheep during our previous research. However, the molecular mechanism of miR-378 regulating myoblast proliferation is still unclear. In this research, we predicted that BMP2 (Bone morphogenetic protein 2) was the target gene of miR-378 and the BMP-Smad signal pathway that BMP2 participated in playing an important role in the muscle development. Therefore, we tried to determine whether miR-378 influence myoblast proliferation of sheep through the BMP-Smad signal pathway. The results indicated that inhibit BMP-Smad signal pathway by interfering Smad4 to promote proliferation of sheep myoblasts; promote BMP-Smad signal pathway by interfering Smad7 to inhibit proliferation of sheep myoblasts; over-expression miR-378 promotes BMP-Smad signal pathway and myoblast proliferation in sheep; interfering miR-378 inhibits BMP-Smad signal pathway and myoblast proliferation in sheep. However, when both of which functioned at the myoblast, miR-378 could not fully depend on BMP-Smad signal pathway to regulate myoblast proliferation. In sum, both miR-378 and BMP-Smad can influence the proliferation of myoblast, but miR-378 does not target the 3' UTR of sheep BMP2. 10.1016/j.gene.2018.06.039
    Integrated analysis of miRNA and mRNA expression profiles in 2-, 6-, and 12-month-old Small Tail Han Sheep ovaries reveals that oar-miR-432 downregulates RPS6KA1 expression. Gu Bo,Liu Hang,Han Yue,Chen Yang,Jiang Huaizhi Gene Small Tail Han Sheep are an excellent local sheep breed in China, and their outstanding reproductive performance is one of their very important biological characteristics. Clarifying the ovary development process of these ewes should provide a theoretical basis for improving their reproductive efficiency. In this study, we identified the differentially expressed (DE) microRNAs (miRNAs) in 2-, 6-, and 12-month-old small-tail Han sheep ovaries by constructing and analyzing the miRNA expression profiles. These findings clarify the molecular mechanisms regulating the excellent reproductive performance of small-tail Han ewes. We used RNA-Seq technology and bioinformatic to analyze these profiles. Eleven, 13, and 19 DE miRNAs were identified in the 2- vs 6-, 6- vs 12-, and 2- vs 12-month-old ovaries, respectively. In total, 54, 37, and 198 predicted target genes of these DE miRNAs were identified in these three groups, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that in the 2- vs 6-month-old ovaries, the target genes of DE known sheep miRNAs were involved in 102 GO terms and seven signaling pathways; in the 6- vs 12-month-old ovaries, the target genes of DE known sheep miRNAs were involved in 52 GO terms and three signaling pathways; and in the 2- vs 12-month-old ovaries, the target genes of DE known sheep miRNAs were involved in 88 GO terms and six signaling pathways. Three miRNA-target regulatory networks were constructed based on these DE miRNA-target interactions. Nine miRNAs were selected to confirm to the accuracy of the miRNA sequencing data with qRT-PCR. The site at which oar-miR-432 binds RPS6KA1 was determined with a dual-luciferase system. This is the first integrated analysis the expression profiles of miRNAs and their targets during ovarian development in small-tail Han sheep. These data clarify the molecular regulatory mechanisms underlying sheep ovarian development and identify biomarkers that influence the reproductive performance of small-tail Han ewes. 10.1016/j.gene.2019.02.095
    Unraveling Stage-Dependent Expression Patterns of Circular RNAs and Their Related ceRNA Modulation in Ovine Postnatal Testis Development. Li Taotao,Luo Ruirui,Wang Xia,Wang Huihui,Zhao Xingxu,Guo Yunxia,Jiang Haitao,Ma Youji Frontiers in cell and developmental biology Circular RNAs (circRNAs) have been shown to function in the reproductive systems including testis. However, their expression, as well as function in testicular development of sheep remain undefined. Herein, we performed RNA sequencing to reveal circRNA temporal expression patterns in testes of Tibetan sheep from different stages of maturation (3M, 3-month-old; 1Y, 1-year-old; 3Y, 3-year-old). A total of 3,982, 414, and 4,060 differentially expressed (DE) circRNAs were uncovered from 3M vs 1Y, 1Y vs 3Y, and 3M vs 3Y, respectively. Functional enrichment assessment indicated that the source genes of DE circRNAs were primarily engaged in spermatogenesis and testicular immune privilege including blood-testis barrier (BTB). We subsequently constructed the core circRNA-miRNA-mRNA interaction network for genes related to testicular function, such as spermatogenesis, germ cell development, BTB, and cell cycle/meiosis. Furthermore, we validated the target associations between either circ_024949, circ_026259 or IGF1, and oar-miR-29b in this network, and revealed their similar expression signatures in developmental testes that they were extensively expressed in germ cells, Leydig cells, and Sertoli cells, thus suggesting their broad functions in the functional maintenance of Leydig cells and Sertoli cells, as well as the development and maturation of male germ cells. Meanwhile, circ_026259 was shown to promote IGF1 expression through inhibition of oar-miR-29b in sheep Sertoli cells. This work gives the first global view for the expression and regulation of circRNAs in sheep testis, which will be helpful for providing new insights into the molecular mechanism of ovine testis function. 10.3389/fcell.2021.627439
    RNA sequencing reveals significant miRNAs in Atypical endometrial hyperplasia. Tang Shiqian,Dai Yinmei European journal of obstetrics, gynecology, and reproductive biology PURPOSE:In this paper, we aimed to investigate the miRNAs that played a regulatory role in the development of atypical endometrial hyperplasia (AEH). METHODS:RNA sequencing was performed for endometrial tissues from 3 AEH patients and 3 endometrial normal hyperplasia patients. RNA sequencing data were processed and differentially expressed (DE) miRNAs were identified between AEH and controls. The target genes for DE miRNAs were identified and mapped to the protein-protein interaction (PPI) network. The miRNA related functions were predicted and miRNA-disease gene network was constructed. RESULTS:Total 18 DE miRNAs were overlapped in three sample groups, among which hsa-miR-577, hsa-miR-182-5p and hsa-miR-183-5p were top three miRNAs that targeting largest number of genes. Function analysis showed that the 18 overlapped miRNAs mainly related with cancer and signaling transduction related pathways. PPI network showed that total 12 genes were among top 20 genes based on three network topological features including BCL2, UMPS, MAPK13, PRKCB, CREB1, IGF1, SP1, SMAD3, IGF1R, NOTCH2, WNT5A, TK2. Top 10 miRNAs in miRNA-disease gene network were identified such as hsa-miR-577 (degree = 17), hsa-miR-182-5p (degree = 16) and hsa-miR-3609 (degree = 13). CONCLUSION:hsa-miR-577 and hsa-miR-182-5p may play regulatory role in AEH through AMPK signal pathway and Wnt signaling pathway. 10.1016/j.ejogrb.2018.03.025
    CREB coactivator CRTC2/TORC2 and its regulator calcineurin crucially mediate follicle-stimulating hormone and transforming growth factor β1 upregulation of steroidogenesis. Fang Wei-Ling,Lee Ming-Ting,Wu Leang-Shin,Chen Yun-Ju,Mason Jian,Ke Ferng-Chun,Hwang Jiuan-Jiuan Journal of cellular physiology In vitro and in vivo studies implicate that follicle-stimulating hormone (FSH) and transforming growth factor β1 (TGFβ1) play crucial physiological roles in regulating ovarian granulosa cell function essential to fertility control in females. FSH induces cAMP and calcium signaling, thereby activating transcription factor CREB to upregulate steroidogenic gene expression, and TGFβ1 greatly enhances FSH-stimulated steroidogenesis. A CREB coactivator CRTC2/TORC2 was identified to function as a cAMP and calcium-sensitive coincidence sensor. This led us to explore the role of CRTC2 and its regulator calcineurin in FSH and TGFβ1-stimulated steroidogenesis. Primary culture of granulosa cells from gonadotropin-primed immature rats was used. Immunoblotting analysis shows that FSH rapidly and transiently induced dephosphorylation/activation of CRTC2, and FSH + TGFβ1 additionally induced late-phase CRTC2 dephosphorylation. Immunofluorescence analysis further confirms FSH ± TGFβ1 promoted CRTC2 nuclear translocation. Using selective inhibitors, we demonstrate that FSH activated CRTC2 in a PKA- and calcineurin-dependent manner, and TGFβ1 acting through its type I receptor (TGFβRI)-modulated FSH action in a calcineurin-mediated and PKA-independent fashion. Next, we investigated the involvement of calcineurin and CRTC2 in FSH and TGFβ1-stimulated steroidogenesis. Calcineurin and TGFβRI inhibitor dramatically reduced the FSH ± TGFβ1-increased progesterone synthesis and protein levels of StAR, P450scc, and 3β-HSD enzyme. Furthermore, chromatin-immunoprecipitation and immunoprecipitation analyses demonstrate that FSH ± TGFβ1 differentially increased CRTC2, CREB, and CBP binding to these steroidogenic genes, and CREB nuclear association with CRTC2 and CBP. In all, this study reveals for the first time that CRTC2 and calcineurin are critical signaling mediators in FSH and TGFβ1-stimulated steroidogenesis in ovarian granulosa cells. 10.1002/jcp.22978
    Effect of CREB1 promoter non-CpG island methylation on its differential expression profile on sheep ovaries associated with prolificacy. Zhao Jie,Li Feng-Zhe,Wu Jie,Yang Hua,Zheng Jian,Pang Jing,Meng Fan-Xing,Wang Feng,Zhang Yan-Li Tissue & cell This study aimed to investigate the effect of different methylated regions of cyclic-AMP response element binding protein 1 (CREB1) by comparing the high prolificacy (HP) group and low prolificacy (LP) group, which was detected in our previous study. The expression level of CREB1 mRNA in the ovaries of the HP group was higher than in the LP group (P <  0.05). The differential methylated region (DMR) had 4 methylated CG dinucleotides(CGs): -1546, -1544, -1494 and -1464. The DNA methylation levels of -1546 CGs and -1464 CGs were significantly higher in the HP group than in the LP group (P <  0.05). The activity from -1296 to +26 (without DMR) was significantly higher than the activity from -1598 to +26 (with DMR) (P <  0.05). The result of 5-aza-2'-deoxycytidine treatment indicated that the inhibition DNA methylation of DMR reduced the transcription of CREB1. The bioinformatics predictive analysis were found that the -1546 CG site was located in the CCAAT/enhancer-binding protein alpha (CEBPA) binding site and the -1464 CG site was located in the Sp1 binding site. Finally, this study revealed the relationship between the methylation of non-CpG sites of the promoter and transcription of CREB1. This study will provide a theoretical basis of the Hu sheep ovaries associated with DNA methylation. 10.1016/j.tice.2019.04.005
    Oxidized fish oils increased lipid deposition via oxidative stress-mediated mitochondrial dysfunction and the CREB1-Bcl2-Beclin1 pathway in the liver tissues and hepatocytes of yellow catfish. Zhang Dian-Guang,Zhao Tao,Hogstrand Christer,Ye Han-Mei,Xu Xiao-Jian,Luo Zhi Food chemistry At present, the harmful effects and relevant mechanism of oxidized fish oils on fish and fish cells remain unknown. Our study found that oxidized fish oils increased lipogenesis, and reduced lipolysis, activated oxidative stress by decreasing glutathione peroxidase (GPX) activity, increasing malondialdhyde (MDA) content and damaging mitochondrial structure, and activated autophagy in the liver of yellow catfish; oxidized eicosapentaenoic acid (oxEPA) induced oxidative stress in yellow catfish hepatocytes. Oxidative stress, mitochondrial dysfunction and lipophagy mediated oxEPA induced-variations in lipid metabolism. Our further investigation indicated that oxEPA-activated lipophagy was via inhibiting the DNA binding capacity of the cAMP-response element binding protein (CREB)-1 to the region of Bcl-2 promoter, which in turn suppressed the binding activity of Bcl-2 to Beclin1 and promoted autophagosome formation. For the first time, our study elucidated the mechanisms of oxidized fish oils-induced lipid deposition by the oxidative stress, mitochondrial dysfunction and CREB1-Bcl-2-Beclin1 pathway in fish. 10.1016/j.foodchem.2021.129814
    Reproductive role of miRNA in the hypothalamic-pituitary axis. Cao Chunyu,Ding Yifei,Kong Xiangjun,Feng Guangde,Xiang Wei,Chen Long,Yang Fang,Zhang Ke,Chu Mingxing,Wang Pingqing,Zhang Baoyun Molecular and cellular neurosciences The hypothalamic-pituitary-gonadal (HPG) axis plays a critical role in regulating reproductive function. Gonadotropin-releasing hormone (GnRH), which is secreted by the hypothalamus, acts on pituitary gonadotrophs to stimulate luteinizing hormone (LH) and follicle-stimulating hormone (FSH) synthesis and secretion, ultimately affecting the animal's fertility. MicroRNAs are small, non-coding RNAs that are widely expressed throughout the brain and can fine-tune gene expression post-transcriptionally. Recently, growing evidence has unveiled the central position of miRNAs within a key regulatory process involving GnRH secretion and subsequent activation in the pituitary. Although transcriptional regulation of reproduction has been well studied, the post-transcriptional processes are less well understood. In this review, we elaborate comprehensively on the critical role of miRNAs in the reproductive process, including both temporal and spatial aspects. A better understanding of how miRNAs impact the neuroendocrine system may improve our knowledge of reproduction and provide novel targets for therapeutic development. 10.1016/j.mcn.2018.01.008
    Identification of Prolificacy-Related Differentially Expressed Proteins from Sheep (Ovis aries) Hypothalamus by Comparative Proteomics. Zhang Zhuangbiao,Tang Jishun,Di Ran,Liu Qiuyue,Wang Xiangyu,Gan Shangquan,Zhang Xiaosheng,Zhang Jinlong,Chen Wei,Hu Wenping,Chu Mingxing Proteomics Reproduction, as a physiologically complex process, can significantly affect the development of the sheep industry. However, a lack of overall understanding to sheep fecundity has long blocked the progress in sheep breeding and husbandry. In the present study, the aim is to identify differentially expressed proteins (DEPs) from hypothalamus in sheep without FecB mutation in two comparison groups: polytocous (PF) versus monotocous (MF) sheep at follicular phase and polytocous (PL) versus monotocous (ML) sheep at luteal phase. Totally 5058 proteins are identified in sheep hypothalamus, where 22 in PF versus MF, and 39 proteins in PL versus ML are differentially expressed, respectively. A functional analysis is then conducted including Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis to reveal the potential roles of these DEPs. The proteins ENSOARP00000020097, ENSOARP00000006714, growth hormone (GH), histone deacetylase 4 (HDAC4), and 5'-3' exoribonuclease 2 (XRN2) in PF versus MF, and bcl-2-associated athanogene 4 (BAG4), insulin-like growth factor-1 receptor (IGF1R), hydroxysteroid 11-beta dehydrogenase 1 (HSD11B1), and transthyretin (TTR) in PL versus ML appear to modulate reproduction, presumably by influencing the activities of gonadotropin-releasing hormone (GnRH). This study provides an alternative method to identify DEPs associated with sheep prolificacy from the hypothalamus. The mass spectrometry data are available via ProteomeXchange with identifier PXD013822. 10.1002/pmic.201900118
    Comprehensive Analysis of Differentially Expressed Profiles of mRNA, lncRNA, and circRNA in the Uterus of Seasonal Reproduction Sheep. La Yongfu,He Xiaoyun,Zhang Liping,Di Ran,Wang Xiangyu,Gan Shangquan,Zhang Xiaosheng,Zhang Jinlong,Hu Wenping,Chu Mingxing Genes Photoperiod is one of the important factors leading to seasonal reproduction of sheep. However, the molecular mechanisms underlying the photoperiod regulation of seasonal reproduction remain poorly understood. In this study, we compared the expression profiles of mRNAs, lncRNAs, and circRNAs in uterine tissues from Sunite sheep during three different photoperiods, namely, the short photoperiod (SP), short transfer to long photoperiod (SLP), and long photoperiod (LP). The results showed that 298, 403, and 378 differentially expressed (DE) mRNAs, 171, 491, and 499 DE lncRNAs, and 124, 270, and 400 DE circRNAs were identified between SP and LP, between SP and SLP, and between LP and SLP, respectively. Furthermore, functional enrichment analysis showed that the differentially expressed RNAs were mainly involved in the GnRH signaling pathway, thyroid hormone synthesis, and thyroid hormone signaling pathway. In addition, co-expression networks of lncRNA-mRNA were constructed based on the correlation analysis between the differentially expressed RNAs. Our study provides new insights into the expression changes of RNAs in different photoperiods, which might contribute to understanding the molecular mechanisms of seasonal reproduction in sheep. 10.3390/genes11030301
    Identification and Characterization of Hypothalamic Alternative Splicing Events and Variants in Ovine Fecundity-Related Genes. Zhang Zhuangbiao,Tang Jishun,He Xiaoyun,Di Ran,Zhang Xiaosheng,Zhang Jinlong,Hu Wenping,Chu Mingxing Animals : an open access journal from MDPI Previous studies revealed that alternative splicing (AS) events and gene variants played key roles in reproduction; however, their location and distribution in hypothalamic fecundity-related genes in sheep without the FecB mutation remain largely unknown. Therefore, in this study, we described the hypothalamic AS events and variants in differentially expressed genes (DEGs) in Small-tailed Han sheep without the FecB mutation at polytocous sheep in the follicular phase vs. monotocous sheep in the follicular phase (PF vs. MF) and polytocous sheep in the luteal phase vs. monotocous sheep in the luteal phase (PL vs. ML) via an RNA-seq study for the first time. We found 39 DEGs with AS events (AS DEGs) in PF vs. MF, while 42 AS DEGs were identified in PL vs. ML. No DEGs with single nucleotide polymorphisms (SNPs) were observed in PF vs. MF, but five were identified in PL vs. ML. We also performed a correlation analysis of transcriptomics and proteomics, and the results suggested several key DEGs/differentially expressed proteins (DEPs), such as LGALS3 in PF vs. MF and aspartoacylase (ASPA) and transthyretin (TTR) in PL vs. ML, could be candidate genes influencing ovine litter size. In addition, further analyses suggested that AS events, SNPs and miRNA-binding sites existed in key DEGs/DEPs, such as ASPA and TTR. All in all, this study provides a new insight into ovine and even other mammalian reproduction. 10.3390/ani10112111
    Transcriptome Analysis Reveals Differentially Expressed Genes and Long Non-coding RNAs Associated With Fecundity in Sheep Hypothalamus With Different FecB Genotypes. Chen Si,Guo Xiaofei,He Xiaoyun,Di Ran,Zhang Xiaosheng,Zhang Jinlong,Wang Xiangyu,Chu Mingxing Frontiers in cell and developmental biology Small-tailed Han sheep, with different genotypes, manifest distinct ovulation rates and fecundities, which are due to differences in reproductive hormones secreted by the hypothalamic-pituitary-ovarian axis. Nevertheless, the function of the hypothalamus against a mutant background on increasing ovulation rate is rarely reported. Therefore, we determined the expression profiles of hypothalamus tissue collected from six wild-type (WW) and six mutant homozygous (BB) ewes at the follicular and luteal phases by whole-transcriptome sequencing. We identified 53 differentially expressed mRNAs (DEGs) and 40 differentially expressed long non-coding RNAs (DELs) between the two estrus states. Functional annotation analysis revealed that one of the DEGs, PRL, was particularly enriched in the hypothalamic function, hormone-related, and reproductive pathways. The lncRNA-target gene interaction networks and KEGG analysis in combination suggest that the lncRNAs LINC-676 and WNT3-AS acting on and in different phases may induce gonadotropin-releasing hormone (GnRH) secretion. Furthermore, there were differences of regulatory elements and WNT gene family members involved in the follicular-luteal transition in the reproductive process between wild-type () and mutant sheep (). We combined the DEG and DEL data sets screened from different estrus states and genotypes. The overlap of these two sets was identified to select the mRNAs and lncRNAs that have major effects on ovulation. Among the overlapping molecules, seven DEGs and four DELs were involved in the follicular-luteal transition regulated by mutation. Functional annotation analysis showed that two DEGs ( and ) were enriched in melanogenesis, oxytocin, and GnRH secretion. LINC-219386 and IGF2-AS were highly expressed in the BB ewes compared with WW ewes, modulating their target genes ( and ) to produce more GnRH during follicular development, which explains why mutated ewes produced more mature follicles. These results from expression profiling of the hypothalamus with the mutation at different estrus states provide new insights into how the hypothalamus regulates ovulation under the effect of the mutation. 10.3389/fcell.2021.633747
    Analysis of pituitary transcriptomics indicates that lncRNAs are involved in the regulation of sheep estrus. Li Xiaoyue,Li Cunyuan,Xu Yueren,Yao Rui,Li Huixiang,Ni Wei,Quan Renzhe,Zhang Mengdan,Liu Li,Yu Shuting,Ullah Yaseen,Hu Ruirui,Li Yaxin,Guo Tao,Wang Xiaokui,Hu Shengwei Functional & integrative genomics Seasonal estrus is a key factor limiting animal fertility, and understanding the molecular mechanisms that regulate animal estrus is important for improving animal fertility. The pituitary gland, which is the most important endocrine gland in mammals, plays an important role in regulating the physiological processes such as growth, development, and reproduction of animals. Here, we used RNA-seq technology to study the expression profile of lncRNAs in the anterior pituitary of sheep during estrus and anestrus. In this study, we identified a total of 995 lncRNAs, of which 335 lncRNAs were differentially expressed in two states (including 38 up-regulated and 297 down-regulated lncRNAs). RT-qPCR verified the expression levels of several lncRNAs. Target predictive analysis revealed that these lncRNAs can act in cis or trans and regulate the expression of genes involved in the regulation of sheep estrus. Target gene enrichment analysis of differentially expressed lncRNAs indicates that these lncRNAs can regulate sheep estrus by regulating hormone metabolism and energy metabolism. Through our research, we provide the expression profile of lncRNAs in the pituitary of sheep, which provides a valuable resource for further understanding of the genetic regulation of seasonal estrus in sheep from the perspective of lncRNAs. 10.1007/s10142-020-00735-y
    Genome-wide transcriptome profiling uncovers differential miRNAs and lncRNAs in ovaries of Hu sheep at different developmental stages. Shabbir Samina,Boruah Prerona,Xie Lingli,Kulyar Muhammad Fakhar-E-Alam,Nawaz Mohsin,Yousuf Salsabeel,Liu Tianyi,Jabeen Farhat,Miao Xiangyang Scientific reports Ovary development is an important determinant of the procreative capacity of female animals. Here, we performed genome-wide sequencing of long non-coding RNAs (lncRNAs) and mRNAs on ovaries of 1, 3 and 8 months old Hu sheep to assess their expression profiles and roles in ovarian development. We identified 37,309 lncRNAs, 45,404 messenger RNAs (mRNAs) and 330 novel micro RNAs (miRNAs) from the transcriptomic analysis. Six thousand, seven hundred and sixteen (6716) mRNAs and 1972 lncRNAs were significantly and differentially expressed in ovaries of 1 month and 3 months old Hu sheep (H1 vs H3). These mRNAs and target genes of lncRNAs were primarily enriched in the TGF-β and PI3K-Akt signalling pathways which are closely associated with ovarian follicular development and steroid hormone biosynthesis regulation. We identified MSTRG.162061.1, MSTRG.222844.7, MSTRG.335777.1, MSTRG.334059.16, MSTRG.188947.6 and MSTRG.24344.3 as vital genes in ovary development by regulating CTNNB1, CCNA2, CDK2, CDC20, CDK1 and EGFR expressions. A total of 2903 mRNAs and 636 lncRNAs were differentially expressed in 3 and 8 months old ovaries of Hu sheep (H3 vs H8); and were predominantly enriched in PI3K-Akt, progesterone-mediated oocyte maturation, estrogen metabolism, ovulation from the ovarian follicle and oogenesis pathways. These lncRNAs were also found to regulate FGF7, PRLR, PTK2, AMH and INHBA expressions during follicular development. Our result indicates the identified genes participate in the development of the final stages of follicles and ovary development in Hu sheep. 10.1038/s41598-021-85245-y
    [Prokaryotic expression and purification of sheep granulocyte colony stimulating factor for granulosa cell culture]. Li Runting,Chen Longxin,Zhang Limeng,He Haiying,Wang Yong,Yang Ruochen,Duan Chunhui,Liu Yueqin,Wang Yuqin,Zhang Yingjie Sheng wu gong cheng xue bao = Chinese journal of biotechnology In order to clarify the regulation of granule cell stimulating factor (GCSF) on granulosa cells, we studied the effect of GCSF on proliferation and apoptosis of in vitro cultured granulosa cells for research on GCSF used in sheep reproduction and breeding. Sheep GCSF protein was prokaryotic expressed and purified. Its bio-activity was measured with M-NSF60 cells. The purified GCSF was added in cell culture medium in experiment groups with non-added as control. Alarmarblue was used to measure cell proliferation, and flow cytometry was used to detect cell cycles and apoptosis. Sheep GCSF could be expressed and purified. Cell activity increased with GCSF concentration from 0.06 to 600 ng/mL at 24 h and 48 h. Cell cycles were significantly different between experiment and control groups at 24 h. Cell ratio of S was significantly reduced (P<0.05) and G2/M phase significantly increased (P<0.05). The apoptosis ratio of experiment group was significantly reduced (P<0.05) at 48 h. In conclusion, GCSF could enhance cell proliferation, inhibit apoptosis, and regulate cell cycles on in vitro cultured sheep granulosa cells. 10.13345/j.cjb.190584
    Differential Expression and Functional Analysis of CircRNA in the Ovaries of Low and High Fecundity Hanper Sheep. Liu Aiju,Chen Xiaoyong,Liu Menghe,Zhang Limeng,Ma Xiaofei,Tian Shujun Animals : an open access journal from MDPI Litter size is a considerable quality that determines the production efficiency of mutton sheep. Therefore, revealing the molecular regulation of high and low fertility may aid the breeding process to develop new varieties of mutton sheep. CircRNAs are the important factors regulating follicular development, but their mechanism role in the regulation of litter size in Hanper sheep is not clear. In the present study, ovarian tissues from the follicular (F) or luteal phase (L) of Hanper sheep that were either consecutive monotocous (M) or polytocous were collected. Then, we performed transcriptome sequencing to screen for differentially expressed circRNAs (DE-circRNAs) and elucidate their function. In total, 4256 circRNA derived from 2184 host genes were identified in which 183 (146 were upregulated, while 37 were downregulated) were differentially expressed in monotocous sheep in the follicular phase versus polytocous sheep in the follicular phase (MF vs. PF). Moreover, 34 circRNAs (14 were upregulated, while 20 were downregulated) were differentially expressed in monotocous sheep in the luteal phase versus polytocous sheep in the luteal sheep (ML vs. PL). This was achieved through DE-circRNAs function enrichment annotation analysis by GESA, GO, and KEGG, which function through the EGF-EGFR-RAS-JNK, TGF-β and thyroid hormone signaling pathway to affect the litter size of Hanper sheep in MF vs. PF and ML vs. PL. STEM results showed that MAPK signaling pathways play a key role in MF vs. PF and ML vs. PL. Through WGCNA analysis, was a core gene in MF vs. PF and ML vs. PL. Moreover, competitive endogenous RNA (ceRNA) network analysis revealed the target binding sites for miRNA such as oar-miR-27a, oar-miR-16b, oar-miR-200a/b/c, oar-miR-181a, oar-miR-10a/b, and oar-miR-432 in the identified DE-cirRNAs. 10.3390/ani11071863
    TGIF1 and SF1 polymorphisms are associated with litter size in Small Tail Han sheep. Zhang Zhuangbiao,He Xiaoyun,Liu Qiuyue,Tang Jishun,Di Ran,Chu Mingxing Reproduction in domestic animals = Zuchthygiene TGF-β induced factor homeobox 1 (TGIF1) and splicing factor 1 (SF1) are important for mammalian reproduction; however, the effects of these genes on litter size in sheep remain unexplored. In this study, we genotyped 768 ewes from seven sheep breeds at two loci: g.37871539C>T, a synonymous mutation of TGIF1; and g.42314637T>C, a 3'UTR variant of SF1. Our analysis of polymorphism revealed only two genotypes at locus g.37871539C>T in TGIF1, with most sheep populations being moderately polymorphic (0.25 < PIC < 0.5) at this site. In contrast, most breeds exhibited low polymorphism (PIC ≤0.25) at the SF1 locus g.42314637T>C. The association analysis revealed that a synonymous mutation at g.37871539C>T in TGIF1 was highly associated with litter size in Small Tail Han sheep, in which it causes a significant decrease in litter size. Conversely, while the SF1 3'UTR variant g.42314637T>C was also highly associated with litter size in sheep, it causes a significant increase in the number of litter size. Combined, these data provide valuable information regarding candidate genetic markers for sheep breeding programs. 10.1111/rda.13753
    Transcriptome analysis reveals corresponding genes and key pathways involved in heat stress in Hu sheep. Li Y X,Feng X P,Wang H L,Meng C H,Zhang J,Qian Y,Zhong J F,Cao S X Cell stress & chaperones Heat stress (HS) seriously affects animal performance. In view of global warming, it is essential to understand the regulatory mechanisms by which animals adapt to heat stress. In this study, our aim was to explore the genes and pathways involved in heat stress in sheep. To this end, we used transcriptome analysis to understand the molecular responses to heat stress and thereby identify means to protect sheep from heat shock. To obtain an overview of the effects of heat stress on sheep, we used the hypothalamus for transcriptome sequencing and identified differentially expressed genes (DEGs; false discovery rate (FDR) < 0.01; fold change > 2) during heat stress. A total of 1423 DEGs (1122 upregulated and 301 downregulated) were identified and classified into Gene Ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Heat stress triggered dramatic and complex alterations in gene expression in the hypothalamus. We hypothesized that heat stress induced apoptosis and dysfunction in cells and vital organs and affected growth, development, reproduction, and circadian entrainment via the calcium signaling pathway, which influences ribosome assembly and function. Real-time PCR was used to evaluate the expression of the genes regulating important biological functions or whose expression profiles were significantly changed after acute heat stress (FDR < 0.01; fold change > 4), and the results showed that the expression patterns of these genes were consistent with the results of transcriptome sequencing, indicating that the credibility of the sequencing results. Our data indicated that heat stress induced calcium dyshomeostasis, blocked biogenesis, caused ROS accumulation, impaired the antioxidant system and innate defense, and induced apoptosis through the P53 signaling pathway activated by PEG3, decreased growth and development, and enhanced organ damage. These data is very important and helpful to elucidate the molecular mechanism of heat stress and finally to find ways to deal with heat stress damage in sheep. 10.1007/s12192-019-01019-6
    Pineal gland transcriptomic profiling reveals the differential regulation of lncRNA and mRNA related to prolificacy in STH sheep with two FecB genotypes. Li Chunyan,He Xiaoyun,Zhang Zijun,Ren Chunhuan,Chu Mingxing BMC genomic data BACKGROUND:Long noncoding RNA (lncRNA) has been identified as important regulator in hypothalamic-pituitary-ovarian axis associated with sheep prolificacy. However, little is known of their expression pattern and potential roles in the pineal gland of sheep. Herein, RNA-Seq was used to detect transcriptome expression pattern in pineal gland between follicular phase (FP) and luteal phase (LP) in FecB (MM) and FecB (ww) STH sheep, respectively, and differentially expressed (DE) lncRNAs and mRNAs associated with reproduction were identified. RESULTS:Overall, 135 DE lncRNAs and 1360 DE mRNAs in pineal gland between MM and ww sheep were screened. Wherein, 39 DE lncRNAs and 764 DE mRNAs were identified (FP vs LP) in MM sheep, 96 DE lncRNAs and 596 DE mRNAs were identified (FP vs LP) in ww sheep. Moreover, GO and KEGG enrichment analysis indicated that the targets of DE lncRNAs and DE mRNAs were annotated to multiple biological processes such as phototransduction, circadian rhythm, melanogenesis, GSH metabolism and steroid biosynthesis, which directly or indirectly participate in hormone activities to affect sheep reproductive performance. Additionally, co-expression of lncRNAs-mRNAs and the network construction were performed based on correlation analysis, DE lncRNAs can modulate target genes involved in related pathways to affect sheep fecundity. Specifically, XLOC_466330, XLOC_532771, XLOC_028449 targeting RRM2B and GSTK1, XLOC_391199 targeting STMN1, XLOC_503926 targeting RAG2, XLOC_187711 targeting DLG4 were included. CONCLUSION:All of these differential lncRNAs and mRNAs expression profiles in pineal gland provide a novel resource for elucidating regulatory mechanism underlying STH sheep prolificacy. 10.1186/s12863-020-00957-w
    Integrated Hypothalamic Transcriptome Profiling Reveals the Reproductive Roles of mRNAs and miRNAs in Sheep. Zhang Zhuangbiao,Tang Jishun,Di Ran,Liu Qiuyue,Wang Xiangyu,Gan Shangquan,Zhang Xiaosheng,Zhang Jinlong,Chu Mingxing,Hu Wenping Frontiers in genetics Early studies have provided a wealth of information on the functions of microRNAs (miRNAs). However, less is known regarding their functions in the hypothalamus involved in sheep reproduction. To explore the potential roles of hypothalamic messenger RNAs (mRNAs) and miRNAs in sheep without FecB mutation, in total, 172 and 235 differentially expressed genes (DEGs) and 42 and 79 differentially expressed miRNAs (DE miRNAs) were identified in polytocous sheep in the follicular phase versus monotocous sheep in the follicular phase (PF vs. MF) and polytocous sheep in the luteal phase versus monotocous sheep in the luteal phase (PL vs. ML), respectively, using RNA sequencing. We also identified several key mRNAs (e.g., , , , , , and ) and mRNA-miRNAs pairs (e.g., co-regulated by oar-miR-379-5p, oar-miR-30b, oar-miR-152, oar-miR-495-3p, oar-miR-143, oar-miR-106b, oar-miR-218a, oar-miR-148a, and regulated by oar-miR-432) through functional enrichment analysis, and the identified mRNAs and miRNAs may function, conceivably, by influencing gonadotropin-releasing hormone (GnRH) activities and nerve cell survival associated with reproductive hormone release direct and indirect ways. This study represents an integral analysis between mRNAs and miRNAs in sheep hypothalamus and provides a valuable resource for elucidating sheep prolificacy. 10.3389/fgene.2019.01296
    Genome-Wide Analysis and Function Prediction of Long Noncoding RNAs in Sheep Pituitary Gland Associated with Sexual Maturation. Yang Hua,Ma Jianyu,Wang Zhibo,Yao Xiaolei,Zhao Jie,Zhao Xinyue,Wang Feng,Zhang Yanli Genes Long noncoding RNA (lncRNA) plays a crucial role in the hypothalamic-pituitary-testis (HPT) axis associated with sheep reproduction. The pituitary plays a connecting role in the HPT axis. However, little is known of their expression pattern and potential roles in the pituitary gland. To explore the potential lncRNAs that regulate the male sheep pituitary development and sexual maturation, we constructed immature and mature sheep pituitary cDNA libraries (three-month-old, TM, and nine-month-old, NM, respectively, n = 3) for lncRNA and mRNA high-throughput sequencing. Firstly, the expression of lncRNA and mRNA were comparatively analyzed. 2417 known lncRNAs and 1256 new lncRNAs were identified. Then, 193 differentially expressed (DE) lncRNAs and 1407 DE mRNAs were found in the pituitary between the two groups. Moreover, mRNA-lncRNA interaction network was constructed according to the target gene prediction of lncRNA and functional enrichment analysis. Five candidate lncRNAs and their targeted genes , , , and that enriched in growth and reproduction related pathways were further filtered. Lastly, the interaction of candidate lncRNA TCONS_00066406 and its targeted gene were validated in in vitro of sheep pituitary cells. Our study provided a systematic presentation of lncRNAs and mRNAs in male sheep pituitary, which revealed the potential role of lncRNA in male reproduction. 10.3390/genes11030320
    Expression characteristics of pineal miRNAs at ovine different reproductive stages and the identification of miRNAs targeting the AANAT gene. Di Ran,Liu Qiu-Yue,Song Shu-Hui,Tian Dong-Mei,He Jian-Ning,Ge Ying,Wang Xiang-Yu,Hu Wen-Ping,Mwacharo Joram-Mwashigadi,Pan Zhang-Yuan,Wang Jian-Dong,Ma Qing,Cao Gui-Ling,Jin Hui-Hui,Liang Xiao-Jun,Chu Ming-Xing BMC genomics BACKGROUND:Many recent studies have shown that miRNAs play important roles in the regulation of animal reproduction, including seasonal reproduction. The pineal gland is a crucial hub in the regulation of seasonal reproduction. However, little is known about the expression characteristics of pineal miRNAs in different reproductive seasons (anestrus and breeding season). Therefore, the expression profiles and regulatory roles of ovine pineal miRNAs were investigated during different reproductive stages using Solexa sequencing technology and dual luciferase reporter assays. RESULTS:A total of 427 miRNAs were identified in the sheep pineal gland. Significant differences in miRNA expression were demonstrated between anestrus and the breeding season in terms of the frequency distributions of miRNA lengths, number of expressed miRNAs, and specifically and highly expressed miRNAs in each reproductive stage. KEGG analysis of the differentially expressed (DE) miRNAs between anestrus and the breeding season indicated that they are significantly enriched in pathways related to protein synthesis, secretion and uptake. Furthermore, transcriptome analysis revealed that many target genes of DE miRNAs in the ribosome pathway showed relatively low expression in the breeding season. On the other hand, analyses combining miRNA-gene expression data with target relationship validation in vitro implied that miR-89 may participate in the negative regulation of aralkylamine N-acetyltransferase (AANAT) mRNA expression by targeting its 3'UTR at a unique binding site. CONCLUSIONS:Our results provide new insights into the expression characteristics of sheep pineal miRNAs at different reproductive stages and into the negative regulatory effects of pineal miRNAs on AANAT mRNA expression. 10.1186/s12864-021-07536-y
    Exploring the roles of fecundity-related long non-coding RNAs and mRNAs in the adrenal glands of small-tailed Han Sheep. Xia Qing,Li Qiuling,Gan Shangquan,Guo Xiaofei,Zhang Xiaosheng,Zhang Jinlong,Chu Mingxing BMC genetics BACKGROUND:Long non-coding RNAs (lncRNAs) can play important roles in uterine and ovarian functions. However, little researches have been done on the role of lncRNAs in the adrenal gland of sheep. Herein, RNA sequencing was used to compare and analyze gene expressions in adrenal tissues between follicular phases and luteal phases in FecB (MM) and FecB (WW) sheep, respectively, and differentially expressed lncRNAs and genes associated with reproduction were identified. RESULTS:In MM sheep, 38 lncRNAs and 545 mRNAs were differentially expressed in the adrenal gland between the luteal and follicular phases; In WW sheep, 513 differentially expressed lncRNAs and 2481 mRNAs were identified. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses indicated that differentially expressed lncRNAs and their target genes are mainly involved in the circadian rhythm, the mitogen activated protein kinase, thyroid, ovarian steroidogenesis and transforming growth factor beta signaling pathways. Differentially expressed lncRNAs can regulate reproduction by modulating genes involved in these signaling pathways and biological processes. Specifically, XLOC_254761, XLOC_357966, 105,614,839 and XLOC_212877 targeting CREB1, PER3, SMAD1 and TGFBR2, respectively, appear to play key regulatory roles. CONCLUSION:These results broaden our understanding of lncRNAs in adrenal gland of sheep and provide new insights into the molecular mechanisms underlying sheep reproduction. 10.1186/s12863-020-00850-6
    Orexin A promotes progesterone secretion in luteinized granulose cells of Mongolian ovary by and genes. Xie Mengyuan,Han Dong,Xu Xiaojing Zygote (Cambridge, England) To study the role of orexin A in the reproductive regulation of Mongolian sheep, ovine ovarian granulosa cells were cultured in vitro. The cells were divided into groups after luteinization, the experimental group was given orexin A and the transcriptome was sequenced together with that of the control group. The different genes related to reproduction were screened out. qRT-PCR, western blot and enzyme-linked immunosorbent assay (ELISA) were used to verify the selected genes and detect the effect on progesterone secretion. In total, 123 differentially expressed genes were obtained by sequencing. Six genes with high expression related to reproduction (PRRT2, ABCG1, SOX4, TBX3, ID1 and ATP8) were screened. The results of qRT-PCR were consistent with those of sequencing; western blot and ELISA were used to verify the protein levels of steroidogenic acute regulatory protein (StAR) and its related PRRT2 and ABCG1, and to detect their effect on progesterone secretion. Validation results were consistent with those of qRT-PCR and sequencing. The experimental group was given orexin A and compared with the control group. Expression of PRRT2 protein was significantly increased (P < 0.05), ABCG1 protein expression was significantly decreased (P < 0.05), StAR expression was significantly increased (P < 0.05), and progesterone secretion was significantly increased (P < 0.05). The results showed that orexin A promoted the expression of StAR by upregulating PRRT2 and downregulating ABCG1, therefore affecting secretion of progesterone. Gene expression characteristics of orexin A affecting progesterone secretion were preliminarily explored; this study provides a theoretical basis for further study on signalling pathways and reproductive regulation in Mongolian sheep. 10.1017/S096719942000088X
    Comparison between cultured and wild Pacific white shrimp () vitellogenesis: next-generation sequencing and relative expression of genes directly and indirectly related to reproduction. Montes-Dominguez Araceli Lorena,Avena-Soto Jesus Arian,Lizarraga-Rodriguez Jorge Luis,Perez-Gala Rodrigo de Jesus,Jimenez-Gutierrez Stephanie,Sotelo-Falomir Jesus Alberto,Pinzon-Miranda Fernando Marino,Martinez-Perez Francisco,Muñoz-Rubi Horacio Alberto,Chavez-Herrera Dario,Jimenez-Gutierrez Laura Rebeca PeerJ Shrimp fisheries are among the most important fisheries worldwide, and shrimp culture has increased considerably in recent years. Most current studies on reproduction-related genes have been conducted on cultured shrimp. However, gene expression is intimately linked to physiological and environmental conditions, and therefore an organism's growth environment has a great influence on reproduction. Thus, gene expression profiling, should be applied in fisheries studies. Here, we identified the expression patterns of 76 reproduction-related genes in via the analysis of pooled transcriptomes from a time-series experiment encompassing a full circadian cycle. The expression patterns of genes associated both directly (, , and ) and indirectly (, , and ) with reproduction were evaluated, as these genes could be used as molecular markers of previtellogenic and vitellogenic maturation stages. The evaluated genes were prominently upregulated during vitellogenic stages, with specific expression patterns depending on the organism's environment, diet, and season. , , , and could serve as molecular markers for both wild and cultured organisms. 10.7717/peerj.10694
    Genome-wide scans identify known and novel regions associated with prolificacy and reproduction traits in a sub-Saharan African indigenous sheep (Ovis aries). Dolebo Asrat Tera,Khayatzadeh Negar,Melesse Aberra,Wragg David,Rekik Mourad,Haile Aynalem,Rischkowsky Barbara,Rothschild Max F,Mwacharo Joram M Mammalian genome : official journal of the International Mammalian Genome Society Maximizing the number of offspring born per female is a key functionality trait in commercial- and/or subsistence-oriented livestock enterprises. Although the number of offspring born is closely associated with female fertility and reproductive success, the genetic control of these traits remains poorly understood in sub-Saharan Africa livestock. Using selection signature analysis performed on Ovine HD BeadChip data from the prolific Bonga sheep in Ethiopia, 41 candidate regions under selection were identified. The analysis revealed one strong selection signature on a candidate region on chromosome X spanning BMP15, suggesting this to be the primary candidate prolificacy gene in the breed. The analysis also identified several candidate regions spanning genes not reported before in prolific sheep but underlying fertility and reproduction in other species. The genes associated with female reproduction traits included SPOCK1 (age at first oestrus), GPR173 (mediator of ovarian cyclicity), HB-EGF (signalling early pregnancy success) and SMARCAL1 and HMGN3a (regulate gene expression during embryogenesis). The genes involved in male reproduction were FOXJ1 (sperm function and successful fertilization) and NME5 (spermatogenesis). We also observed genes such as PKD2L2, MAGED1 and KDM3B, which have been associated with diverse fertility traits in both sexes of other species. The results confirm the complexity of the genetic mechanisms underlying reproduction while suggesting that prolificacy in the Bonga sheep, and possibly African indigenous sheep is partly under the control of BMP15 while other genes that enhance male and female fertility are essential for reproductive fitness. 10.1007/s00335-019-09820-5
    Review on Genomic Regions and Candidate Genes Associated with Economically Important Production and Reproduction Traits in Sheep (). Gebreselassie Gebremedhin,Berihulay Haile,Jiang Lin,Ma Yuehui Animals : an open access journal from MDPI Sheep () is one of the most economically, culturally, and socially important domestic animals. They are reared primarily for meat, milk, wool, and fur production. Sheep were reared using natural selection for a long period of time to offer these traits. In fact, this production system has been slowing the productivity and production potential of the sheep. To improve production efficiency and productivity of this animal through genetic improvement technologies, understanding the genetic background of traits such as body growth, weight, carcass quality, fat percent, fertility, milk yield, wool quality, horn type, and coat color is essential. With the development and utilization of animal genotyping technologies and gene identification methods, many functional genes and genetic variants associated with economically important phenotypic traits have been identified and annotated. This is useful and presented an opportunity to increase the pace of animal genetic gain. Quantitative trait loci and genome wide association study have been playing an important role in identifying candidate genes and animal characterization. This review provides comprehensive information on the identified genomic regions and candidate genes associated with production and reproduction traits, and gene function in sheep. 10.3390/ani10010033
    Comparative Transcriptomics Identify Key Hypothalamic Circular RNAs that Participate in Sheep () Reproduction. Zhang Zhuangbiao,Tang Jishun,He Xiaoyun,Zhu Mingxia,Gan Shangquan,Guo Xiaofei,Zhang Xiaosheng,Zhang Jinlong,Hu Wenping,Chu Mingxing Animals : an open access journal from MDPI Circular RNA (circRNA), as an emerging class of noncoding RNA, has been found to play key roles in many biological processes. However, its expression profile in the hypothalamus, a powerful organ initiating the reproductive process, has not yet been explored. Therefore, we used RNA sequencing to explore the expression of circRNAs in the hypothalamus of sheep with the ++ genotype. We totally identified 41,863 circRNAs from sheep hypothalamus, in which 333 (162 were upregulated, while 171 were downregulated) were differentially expressed in polytocous sheep in the follicular phase versus monotocous sheep in the follicular phase (PF vs. MF), moreover, 340 circRNAs (163 were upregulated, while 177 were downregulated) were differentially expressed in polytocous sheep in the luteal phase versus monotocous sheep in the luteal sheep (PL vs. ML). We also identified several key circRNAs including oar_circ_0018794, oar_circ_0008291, oar_circ_0015119, oar_circ_0012801, oar_circ_0010234, and oar_circ_0013788 through functional enrichment analysis and oar_circ_0012110 through a competing endogenous RNA network, most of which may participate in reproduction by influencing gonadotropin-releasing hormone (GnRH) activities or affecting key gene expression, indirectly or directly. Our study explored the overall expression profile of circRNAs in sheep hypothalamus, which potentially provides an alternative insight into the mechanism of sheep prolificacy without the effects of mutation. 10.3390/ani9080557
    Photoperiod induced the pituitary differential regulation of lncRNAs and mRNAs related to reproduction in sheep. He Xiaoyun,Tao Lin,Zhong Yingjie,Di Ran,Xia Qing,Wang Xiangyu,Guo Xiaofei,Gan Shangquan,Zhang Xiaosheng,Zhang Jinlong,Liu Qiuyue,Chu Mingxing PeerJ The pituitary is a vital endocrine organ that regulates animal seasonal reproduction by controlling the synthesis and secretion of the hormone. The change of photoperiod is the key factor affecting the function of the pituitary in animals, but the mechanism is unclear. Here, we studied the transcriptomic variation in pars distalis (PD) of the pituitary between short photoperiod (SP) and long photoperiod (LP) using RNA sequencing based on the OVX+E sheep. 346 differentially expressed (DE) lncRNAs and 186 DE-mRNA were found in the PD. Moreover, function annotation analysis indicated that the reproductive hormones and photoperiod response-related pathways including aldosterone synthesis and secretion, insulin secretion, thyroid hormone synthesis, and circadian entrainment were enriched. The interaction analysis of mRNA-lncRNA suggested that MSTRG.240648, MSTRG.85500, MSTRG.32448, and MSTRG.304959 targeted and , which may be involved in the photoperiodic regulation of the PD. These findings provide resources for further study on the seasonal reproductive in ewes. 10.7717/peerj.10953
    Identification and characterization of miRNAs during early pregnancy in domestic sheep. Pokharel Kisun,Peippo Jaana,Li Meng-Hua,Kantanen Juha Animal genetics MicroRNA resources in sheep are limited compared with those in other domesticated mammalian species. By sequencing small RNAs of sheep corpus luteum and endometrium, we have generated the largest amount of miRNA-seq data and compiled the most comprehensive list thus far of miRNAs (n = 599) in sheep. Additionally, we observed a highly conserved maternally imprinted cluster of miRNAs on chromosome 18 homologous to that found on chromosome 14 in human and several other eutherian mammals. 10.1111/age.12992
    An insertion/deletion within the CREB1 gene identified using the RNA-sequencing is associated with sheep body morphometric traits. Erdenee Sarantsetseg,Akhatayeva Zhanerke,Pan Chuanying,Cai Yong,Xu Hongwei,Chen Hong,Lan Xianyong Gene In a previous study, the cyclic AMP response element-binding protein 1 (CREB1) gene, which is likely involved in the regulation of fat metabolism in sheep adipose tissue, was identified using RNA sequencing. CREB1 is a transcription factor that participates in the regulation of cell proliferation, differentiation, and survival as well as energy metabolism. Therefore, based on preliminary studies, this study aimed to reveal the correlation between the insertion/deletion (indel) polymorphism of the CREB1 gene and sheep growth traits. One insertion variation of the ovine CREB1 gene, C3-ins-26 bp, was investigated in 1847 Chinese and Mongolian sheep breeds. The minor allele frequencies in the CREB1 gene varied from 0.021 to 0.938. Further, statistical analyses indicated that the C3-ins-26 bp indel in the CREB1 gene was significantly related to various body measurements (body length, height, and index; chest width, depth, and width index; cannon circumference index; and height at the hip cross) in a Tan sheep population (p < 0.05). Collectively, these findings may provide important insights into marker-assisted selection of sheep. 10.1016/j.gene.2021.145444
    Gene Expression Profiling of Reveals Important Insights about Early Pregnancy in Domestic Sheep. Pokharel Kisun,Peippo Jaana,Weldenegodguad Melak,Honkatukia Mervi,Li Meng-Hua,Kantanen Juha Genes The majority of pregnancy loss in ruminants occurs during the preimplantation stage, which is thus the most critical period determining reproductive success. Here, we performed a comparative transcriptome study by sequencing total mRNA from (CL) collected during the preimplantation stage of pregnancy in Finnsheep, Texel and F1 crosses. A total of 21,287 genes were expressed in our data. Highly expressed autosomal genes in the CL were associated with biological processes such as progesterone formation (, , and ) and embryo implantation (e.g., , and ). Among the list of differentially expressed genes, sialic acid-binding immunoglobulin (Ig)-like lectins (, , ), ribosomal proteins (, ) and chemokines (, CCL24, , ) were upregulated in Finnsheep, while four multidrug resistance-associated proteins (MRPs) were upregulated in Texel ewes. A total of 17 known genes and two uncharacterized non-coding RNAs (ncRNAs) were differentially expressed in breed-wise comparisons owing to the flushing diet effect. The significantly upregulated gene indicated potential for embryonic diapause in Finnsheep and F1. Moreover, we report, for the first time in any species, several genes that are active in the CL during early pregnancy (including , , , and ). 10.3390/genes11040415
    Pre-implantation exogenous progesterone and pregnancy in sheep: I. polyamines, nutrient transport, and progestamedins. Hoskins Emily C,Halloran Katherine M,Stenhouse Claire,Moses Robyn M,Dunlap Kathrin A,Satterfield Michael C,Seo Heewon,Johnson Gregory A,Wu Guoyao,Bazer Fuller W Journal of animal science and biotechnology BACKGROUND:Administration of exogenous progesterone (P4) to ewes during the pre-implantation period advances conceptus development and implantation. This study determined effects of exogenous P4 on transport of select nutrients and pathways that enhance conceptus development. Pregnant ewes (n = 38) were treated with either 25 mg P4 in 1 mL corn oil (P4, n = 18) or 1 mL corn oil alone (CO, n = 20) from day 1.5 through day 8 of pregnancy and hysterectomized on either day 9 or day 12 of pregnancy. Endometrial expression of genes encoding enzymes for synthesis of polyamines, transporters of glucose, arginine, and glycine, as well as progestamedins was determined by RT-qPCR. RESULTS:On day 12 of pregnancy, conceptuses from P4-treated ewes had elongated while those from CO-treated ewes were spherical. The mRNA expression of AZIN2, an arginine decarboxylase, was lower in endometria of P4-treated than CO-treated ewes on day 9 of pregnancy. Expression of FGF10, a progestamedin, was greater in endometria of CO and P4-treated ewes on day 12 of gestation in addition to P4-treated ewes necropsied on day 9 of gestation. Treatment with P4 down-regulated endometrial expression of amino acid transporter SLC1A4 on day 12 of pregnancy. CONCLUSIONS:Results indicated that administration of exogenous P4 during the pre-implantation period advanced the expression of FGF10, which may accelerate proliferation of trophectoderm cells, but also was correlated with decreased expression of glycine and serine transporters and polyamine synthesis enzyme AZIN2. Further research with increased sample sizes may determine how differential expression affects endometrial functions and potentially embryonic loss. 10.1186/s40104-021-00554-6
    Optogenetic regulation of embryo implantation in mice using photoactivatable CRISPR-Cas9. Takao Tomoka,Sato Moritoshi,Maruyama Tetsuo Proceedings of the National Academy of Sciences of the United States of America Embryo implantation is achieved upon successful interaction between a fertilized egg and receptive endometrium and is mediated by spatiotemporal expression of implantation-associated molecules including leukemia inhibitory factor (LIF). Here we demonstrate, in mice, that LIF knockdown via a photoactivatable CRISPR-Cas9 gene editing system and illumination with a light-emitting diode can spatiotemporally disrupt fertility. This system enables dissection of spatiotemporal molecular mechanisms associated with embryo implantation and provides a therapeutic strategy for temporal control of reproductive functions in vivo. 10.1073/pnas.2016850117
    Integrin β1 coordinates survival and morphogenesis of the embryonic lineage upon implantation and pluripotency transition. Molè Matteo Amitaba,Weberling Antonia,Fässler Reinhard,Campbell Alison,Fishel Simon,Zernicka-Goetz Magdalena Cell reports At implantation, the embryo establishes contacts with the maternal endometrium. This stage is associated with a high incidence of preclinical pregnancy losses. While the maternal factors underlying uterine receptivity have been investigated, the signals required by the embryo for successful peri-implantation development remain elusive. To explore these, we studied integrin β1 signaling, as embryos deficient for this receptor degenerate at implantation. We demonstrate that the coordinated action of pro-survival signals and localized actomyosin suppression via integrin β1 permits the development of the embryo beyond implantation. Failure of either process leads to developmental arrest and apoptosis. Pharmacological stimulation through fibroblast growth factor 2 (FGF2) and insulin-like growth factor 1 (IGF1), coupled with ROCK-mediated actomyosin inhibition, rescues the deficiency of integrin β1, promoting progression to post-implantation stages. Mutual exclusion between integrin β1 and actomyosin seems to be conserved in the human embryo, suggesting the possibility that these mechanisms could also underlie the transition of the human epiblast from pre- to post-implantation. 10.1016/j.celrep.2021.108834
    Epigenetic control of embryo-uterine crosstalk at peri-implantation. Kong Shuangbo,Zhou Chan,Bao Haili,Ni Zhangli,Liu Mengying,He Bo,Huang Lin,Sun Yang,Wang Haibin,Lu Jinhua Cellular and molecular life sciences : CMLS Embryo implantation is one of the pivotal steps during mammalian pregnancy, since the quality of embryo implantation determines the outcome of ongoing pregnancy and fetal development. A large number of factors, including transcription factors, signalling transduction components, and lipids, have been shown to be indispensable for embryo implantation. Increasing evidence also suggests the important roles of epigenetic factors in this critical event. This review focuses on recent findings about the involvement of epigenetic regulators during embryo implantation. 10.1007/s00018-019-03245-8
    miR-183-5p regulates uterine receptivity and enhances embryo implantation. Akbar Rubab,Ullah Kamran,Rahman Tanzil Ur,Cheng Yi,Pang Hai-Yan,Jin Lu-Yang,Wang Qi-Jing,Huang He-Feng,Sheng Jian-Zhong Journal of molecular endocrinology Receptive endometrium is a prerequisite for successful embryo implantation, and it follows that poor endometrial receptivity is a leading cause of implantation failure. miRNAs play important roles as epigenetic regulators of endometrial receptivity and embryo implantation through post-transcriptional modifications. However, the mechanisms of action of many miRNAs are poorly understood. In this study, we investigated the role of the miR-183 family, comprising three miRNAs (miR-183-5p, miR-182-5p, and miR-96-5p) in endometrial receptivity and embryo implantation. The miR-183 family shows estrogen-dependent upregulation in endometrial Ishikawa (IK) cells. The miR-183 family also has a positive role in migration and proliferation of IK cells. Furthermore, JAr spheroid attachment experiments show that attachment rates were significantly decreased after treatment of IK cells with inhibitors for miR-183-5p and miR-182-5p and increased after treatment with miR-183-5p-mimic and miR-96-5p-mimic, respectively. The downstream analysis shows that catenin alpha 2 (CTNNA2) is a potential target gene for miR-183-5p, and this was confirmed in luciferase reporter assays. An in vivo mouse pregnancy model shows that inhibition of miR-183-5p significantly decreases embryo implantation rates and increases CTNNA2 expression. Downregulation of CTNNA2 in endometrial cells by miR-183-5p may be significant in mediating estrogenic effects on endometrial receptivity. In conclusion, miR-183-5p and the CTNNA2 gene may be potential biomarkers for endometrial receptivity and may be useful diagnostic and therapeutic targets for successful embryo implantation. 10.1530/JME-19-0184
    SOX2 plays a crucial role in cell proliferation and lineage segregation during porcine pre-implantation embryo development. Lee Mingyun,Choi Kwang-Hwan,Oh Jong-Nam,Kim Seung-Hun,Lee Dong-Kyung,Choe Gyung Cheol,Jeong Jinsol,Lee Chang-Kyu Cell proliferation OBJECTIVES:Gene regulation in early embryos has been widely studied for a long time because lineage segregation gives rise to the formation of a pluripotent cell population, known as the inner cell mass (ICM), during pre-implantation embryo development. The extraordinarily longer pre-implantation embryo development in pigs leads to the distinct features of the pluripotency network compared with mice and humans. For these reasons, a comparative study using pre-implantation pig embryos would provide new insights into the mammalian pluripotency network and help to understand differences in the roles and networks of genes in pre-implantation embryos between species. MATERIALS AND METHODS:To analyse the functions of SOX2 in lineage segregation and cell proliferation, loss- and gain-of-function studies were conducted in pig embryos using an overexpression vector and the CRISPR/Cas9 system. Then, we analysed the morphological features and examined the effect on the expression of downstream genes through immunocytochemistry and quantitative real-time PCR. RESULTS:Our results showed that among the core pluripotent factors, only SOX2 was specifically expressed in the ICM. In SOX2-disrupted blastocysts, the expression of the ICM-related genes, but not OCT4, was suppressed, and the total cell number was also decreased. Likewise, according to real-time PCR analysis, pluripotency-related genes, excluding OCT4, and proliferation-related genes were decreased in SOX2-targeted blastocysts. In SOX2-overexpressing embryos, the total blastocyst cell number was greatly increased but the ICM/TE ratio decreased. CONCLUSIONS:Taken together, our results demonstrated that SOX2 is essential for ICM formation and cell proliferation in porcine early-stage embryogenesis. 10.1111/cpr.13097
    miR-23a-3p increases endometrial receptivity via CUL3 during embryo implantation. Huang Kai,Chen Gezi,Fan Wenqian,Hu Linli Journal of molecular endocrinology A receptive endometrium is required in a successful embryo implantation. The ubiquitination-induced β-catenin degradation is related to the implantation failure.This study aimed to elucidate whether miR-23a-3p regulates endometrial receptivity via the modulation of β-catenin ubiquitination.The expressions of miR-23a-3p and CUL3 were detected in endometrial epithelial cells (EECs) isolated from pregnant mice and in hormone-induced EEC-like Ishikawa cells. The ubiquitination experiment was performed to explore the effect of CUL3 and miR-23a-3p on β-catenin ubiquitination level. The trophoblast attachment was detected by co-culturing JAR (choriocarcinoma cell line) spheroids with Ishikawa cell monolayers. miR-23a-3p was upregulated while CUL3 was downregulated in EECs at day 4 after pregnancy compared with day 1, as well as in hormone-induced Ishikawa cells. miR-23a-3p positively regulated the protein level of β-catenin without affecting the mRNA level. The ubiquitination and degradation of β-catenin was suppressed by miR-23a-3p, while it was promoted by CUL3. Immunoprecipitation confirmed the binding between CUL3 and β-catenin. Luciferase reporter assay confirmed the target relationship between miR-23a-3p and CUL3. The ubiquitination of β-catenin was modulated by the miR-23a-3p/CUL3 pathway. The overexpression of miR-23a-3p promoted JAR spheroid attachments in Ishikawa cells. miR-23a-3p is beneficial for the endometrial receptivity and embryo implantation, whose mechanism is partly through the modulation of CUL3/β-catenin. 10.1530/JME-20-0053
    Sequential activation of uterine epithelial IGF1R by stromal IGF1 and embryonic IGF2 directs normal uterine preparation for embryo implantation. Zhou Chan,Lv Meiying,Wang Peike,Guo Chuanhui,Ni Zhangli,Bao Haili,Tang Yedong,Cai Han,Lu Jinhua,Deng Wenbo,Yang Xiaoyu,Xia Guoliang,Wang Haibin,Wang Chao,Kong Shuangbo Journal of molecular cell biology Embryo implantation in both humans and rodents is initiated by the attachment of a blastocyst to the uterine epithelium. For blastocyst attachment, the uterine epithelium needs to transform at both the structural and molecular levels first, and then initiate the interaction with trophectoderm. Any perturbation during this process will result in implantation failure or long-term adverse pregnancy outcomes. Endocrine steroid hormones, which function through nuclear receptors, combine with the local molecules produced by the uteri or embryo to facilitate implantation. The insulin-like growth factor (IGF) signaling has been reported to play a vital role during pregnancy. However, its physiological function during implantation remains elusive. This study revealed that mice with conditional deletion of Igf1r gene in uteri suffered from subfertility, mainly due to the disturbed uterine receptivity and abnormal embryo implantation. Mechanistically, we uncovered that in response to the nidatory estrogen on D4 of pregnancy, the epithelial IGF1R, stimulated by the stromal cell-produced IGF1, facilitated epithelial STAT3 activation to modulate the epithelial depolarity. Furthermore, embryonic derived IGF2 could activate both the epithelial ERK1/2 and STAT3 signaling through IGF1R, which was critical for the transcription of Cox2 and normal attachment reaction. In brief, our data revealed that epithelial IGF1R was sequentially activated by the uterine stromal IGF1 and embryonic IGF2 to guarantee normal epithelium differentiation during the implantation process. 10.1093/jmcb/mjab034
    CBS and MAT2A improve methionine-mediated DNA synthesis through SAMTOR/mTORC1/S6K1/CAD pathway during embryo implantation. Cai Shuang,Ye Qianhong,Zeng Xiangzhou,Yang Guangxin,Ye Changchuan,Chen Meixia,Yu Haitao,Wang Yuming,Wang Gang,Huang Shuo,Quan Shuang,Zeng Xiangfang,Qiao Shiyan Cell proliferation OBJECTIVES:Early pregnancy loss is a major clinical concern in animal and human reproduction, which is largely influenced by embryo implantation. The importance of methionine for embryo implantation is widely neglected. MATERIALS AND METHODS:We performed a series of experiments with primiparous rats fed diets containing different levels of methionine during early pregnancy to investigate the role of methionine in embryonic implantation and pregnancy outcomes, and used them to perform in vivo metabolic assessments and in vitro uterine explant culture. In addition, through transcriptome analysis and silencing the expression of cystathionine β-synthase (CBS, the key enzyme in transsulfuration pathway) and cell adhesion assay, we measured signalling within Ishikawa, pTr and JAR cells. RESULTS:We determined the relevance and underlying mechanism of methionine on embryo implantation. We showed that methionine deprivation sharply decreased embryo implantation sites, expression of CBS and transsulfuration pathway end products, which were reversed by maternal methionine supplementation during early pregnancy. Moreover, we found CBS improved methionine-mediated cell proliferation and DNA synthesis by CBS inhibition or interference. In addition, transcriptome analysis also revealed that CBS influenced the signalling pathway-associated cell proliferation and DNA synthesis, as well as a correlation between CBS and methionine adenosyltransferase 2A (MAT2A), implying that MAT2A was possibly involved in cell proliferation and DNA synthesis. Further analysis revealed that MAT2A influenced S-adenosylmethionine receptor SAMTOR expression, and SAMTOR activated mTORC1 and its downstream S6K1 and CAD, ultimately enhancing DNA synthesis in the embryo and uterus. CONCLUSIONS:Taken together, these studies demonstrate that CBS and MAT2A improve methionine-mediated DNA synthesis through SAMTOR/mTORC1/S6K1/CAD pathway during embryo implantation. 10.1111/cpr.12950
    Endometrial cell-derived small extracellular vesicle miR-100-5p promotes functions of trophoblast during embryo implantation. Tan Qiang,Shi Shuang,Liang Jingjie,Cao Dingren,Wang Shaoyu,Wang Zhengguang Molecular therapy. Nucleic acids Communication between maternal uterus and blastocyst occurs in the early stages of pregnancy, and the interaction influences the success of embryo implantation. Whereas small extracellular vesicles (sEVs) play an essential role in mediating intercellular communication in numerous biological processes, their role in embryo implantation during the window of implantation (WOI) remains poorly defined. Here, we report that endometrial epithelial cells (EECs) secrete sEVs during early pregnancy, which affects the trophoblast behaviors (migration, invasion, and proliferation), thus influencing embryo implantation. We show that microRNA (miR)-100-5p, sEVs containing microRNA (miRNA), activates both focal adhesion kinase (FAK) and c-Jun N-terminal kinase (JNK), as well as contributes to trophoblast migration and invasion. Furthermore, our findings indicate that the sEV miR-100-5p promotes angiogenesis during the implantation process. In conclusion, this study reveals a novel mechanism by which EEC-derived sEV miR-100-5p crosstalks with trophoblasts, leading to an enhanced ability for implantation. 10.1016/j.omtn.2020.10.043
    Targeting the transforming growth factor-β signaling during pre-implantation development in embryos of cattle, sheep and goats. Hajian Mehdi,Hosseini Sayyed Morteza,Ostadhosseini Somayyeh,Nasr-Esfahani Mohammad Hossein Growth factors (Chur, Switzerland) Recently, application of chemical inhibitors against differentiation signaling pathways has improved establishment of mESCs. In this study, we applied inhibitors of TGF-β (SB431542) and BMP4 (Noggin) from cleavage to blastocyst stage in cattle, goat and sheep embryos. SB significantly decreases blastocyst rate and total cell number (TCN) in sheep blastocysts, whereas only TCN was significantly decreased in cattle blastocysts. In contrast to SB, Noggin significantly improved cattle blastocyst development but decreased TCN. However, Noggin treatment led to a significant increase in TCN in sheep blastocysts. Regarding pluripotency triad (OCT4, NANOG, SOX2) and cell lineage commitment (REX1, CDX2, GATA4), SB led to a significant reduction in SOX2 expression in goat and cattle, while Noggin increased at least one or two of pluripotent markers in these species. Taken together, this data suggests that inhibition of TGF-β by Noggin may be more favorable for derivation of stem cells in farm animals. 10.1080/08977194.2016.1206089
    Functional roles of agmatinase during the peri-implantation period of pregnancy in sheep. Lenis Yasser Y,Elmetwally Mohammed A,Tang Wanjin,Satterfield Carey,Dunlap Kathrin,Wu Guoyao,Bazer Fuller W Amino acids This study investigated the effect of agmatine (Agm) in proliferation of ovine trophecdoderm cells (oTr1) as well as the importance of the arginine decarboxylase (ADC) and agmatinase (AGMAT) alternative pathway for synthesis of polyamines in ovine conceptuses during the peri-implantation period of pregnancy. Morpholino antisense oligonucleotides (MAOs) were used to inhibit translation of mRNAs for ODC1 alone, AGMAT alone, and their combination. Rambouillet ewes (N = 50) were assigned randomly to the following treatments on Day 8 of pregnancy: MAO control (n = 10); MAO-ODC1 (n = 8); MAO-ADC (n = 6); MAO-ODC1:MAO-ADC (n = 9); or MAO-ODC1:MAO-AGMAT (n = 9). Ewes were ovario-hysterectomized on Day 16 of pregnancy to obtain uterine flushings, uterine endometrium, and conceptus tissues. Inhibition of translation of both ODC1 and AGMAT resulted in 22% of ewes having morphologically and functionally normal (elongated and healthy) conceptuses designated MAO-ODC1:MAO-AGMAT (A). But, 78% of the MAO-ODC1:MAO-AGMAT ewes had morphologically and functionally abnormal (not elongated and fragmented) conceptuses designated MAO-ODC1:MAO-AGMAT (B). The pregnancy rate was less (22%; P < 0.05) for MAO-ODC1:MAO-AGMAT ewes than for MAO-control (80%), MAO-ODC1 (75%), MAO-ADC (84%), and MAO-ODC1:MAO-ADC (44%) ewes. Moreover, inhibition of translational of both ODC1 and AGMAT mRNAs increased expression of ADC, SLC22A1, SLC22A2, and SLC22A3 mRNAs, as well as abundances of agmatine, putrescine, spermindine, and spermine in conceptus tissue. However, MAO-ODC1:AGMAT(B) ewes had greater abundances of agmatine, putrescine, and spermidine and reduced amounts of spermine in uterine flushes. Thus, in vivo knockdown of translation of ODC1 and AGMAT mRNAs increased expression of genes for the synthesis and transport of polyamines in ovine conceptuses during the peri-implantation period of pregnancy. 10.1007/s00726-017-2515-1
    Immunolocalization of angiogenic growth factors in the ovine uterus during the oestrus cycle and in response to Steroids. Tremaine T D,Fouladi-Nashta A A Reproduction in domestic animals = Zuchthygiene The vascular changes associated with endometrial maturation in preparation for embryo implantation depend on numerous growth factors, known to regulate key angiogenic events. Primarily, the vascular endothelial growth factor (VEGF) family promotes vascular growth, whilst the angiopoietins maintain blood vessel integrity. The aim was to analyse protein levels of VEGFA ligand and receptors, Angiopoietin-1 and 2 (ANG1/2) and endothelial cell receptor tyrosine kinase (TIE-2) in the ovine endometrium in the follicular and luteal phases of the oestrus cycle and in response to ovarian steroids. VEGFA and its receptors were localized in both vascular cells and non-vascular epithelium (glandular and luminal epithelium) and stroma cells. VEGFA and VEGFR2 proteins were elevated in vascular cells in follicular phase endometrium, compared to luteal phase, most significantly in response to oestradiol. VEGFR1 was expressed by epithelial cells and endothelial cells and was stimulated in response to oestradiol. In contrast, Ang-1 and Ang-2 proteins were elevated in luteal phase endometrium compared to follicular phase, and in response to progesterone, evident in vascular smooth muscle cells and glands which surround TIE-2-expressing blood vessels. Our findings indicate that VEGFA is stimulated by oestradiol, most predominantly in follicular phase endometrium, and Ang-1 and 2 are stimulated by progesterone and were increased during the luteal phase of the oestrus cycle, during the time of vascular maturation. 10.1111/rda.13156
    Molecular cloning and expression of FGF2 gene in pre-implantation developmental stages of in vitro-produced sheep embryos. Mor A,Mondal S,Reddy I J,Nandi S,Gupta Psp Reproduction in domestic animals = Zuchthygiene Early embryonic mortality is one of the main sources of reproductive loss in domestic ruminants including sheep. Fibroblast growth factor-2 (FGF-2) is a member of FGFs family that mediates trophoblast activities and regulates embryonic development in various species. In this study, we have cloned, characterized sheep FGF2 cDNA (KU316368) and studied the expression in sheep embryos. Ovaries of non-pregnant sheep were collected from local abattoir and matured in culture medium at 38.5ºC, 5% CO , 95% humidity for 22-24 hr. The matured oocytes were inseminated with capacitated spermatozoa in Brackett and Oliphant medium and resulted embryos were cultured in CO incubator for 6-7 days to complete the developmental stages from two cells to blastocyst stage. Total RNA was extracted from immature oocytes (n = 100), mature oocytes (n = 100) and different stages of embryos such as 2 cell (n = 50), 4 cell (n = 25), 8 cell (n = 12), 16 cell (n = 6), morula (n = 5) and blastocyst (n = 3). The total RNA isolated from the oocytes and embryos was reverse transcribed and subjected to real-time polymerase chain reaction using sequence-specific primers and SYBR green as the DNA dye. On sequence analysis, the nucleotide sequence of sheep FGF2 exhibited highest sequence similarity with cattle (100%) and least with rat and mouse (69.2%). At the deduced amino acid level, a highest degree of similarity was noticed with cattle, buffalo, goat, pig, camel and horse (100%) and lowest degree of identity with rat, human and mouse (98.2%). The FGF2 mRNA expression was higher in immature and mature oocytes and gradually decreases from 2-cell stage of embryo to the blastocyst stage. More over a significant differences in FGF2 mRNA expression (p < .05) were observed between immature oocytes and all pre-implantation stages of embryo. It can be concluded that FGF-2 plays a significant role in pre-implantation and early development of embryos in sheep. 10.1111/rda.13182
    Endometrial luminal epithelial cells sense embryo elongation in the roe deer independent of interferon-tau†. van der Weijden Vera A,Puntar Brina,Rudolf Vegas Alba,Milojevic Vladimir,Schanzenbach Corina I,Kowalewski Mariusz P,Drews Barbara,Ulbrich Susanne E Biology of reproduction Numerous intrauterine changes take place across species during embryo development. Following fertilization in July/August, the European roe deer (Capreolus capreolus) embryo undergoes diapause until embryonic elongation in December/January. Embryonic elongation prior to implantation is a common feature among ungulates. Unlike many other ruminants, the roe deer embryo does not secrete interferon-tau (IFNτ). This provides the unique opportunity to unravel IFNτ-independent signaling pathways associated with maternal recognition of pregnancy (MRP). This study aimed at identifying the cell-type-specific endometrial gene expression changes associated with the MRP at the time of embryo elongation that are independent of IFNτ in roe deer. The messenger RNA (mRNA) expression of genes known to be involved in embryo-maternal communication in cattle, pig, sheep, and mice was analyzed in laser capture microdissected (LMD) endometrial luminal, glandular epithelial, as well as stromal cells. The mRNA transcript abundances of the estrogen (ESR1), progesterone receptor (PGR), and IFNτ-stimulated genes were lower in the luminal epithelium in the presence of an elongated embryo compared to diapause. Retinol Binding Protein-4 (RBP4), a key factor involved in placentation, was more abundant in the luminal epithelium in the presence of an elongated embryo. The progesterone receptor localization was visualized by immunohistochemistry, showing an absence in the luminal epithelium and an overall lower abundance with time and thus prolonged progesterone exposure. Our data show a developmental stage-specific mRNA expression pattern in the luminal epithelium, indicating that these cells sense the presence of an elongated embryo in an IFNτ-independent manner. 10.1093/biolre/ioz129
    NLRP7 is expressed in the ovine ovary and associated with in vitro pre-implantation embryo development. Li Guangdong,Tian Xiuzhi,Lv Dongying,Zhang Lu,Zhang Zhenzhen,Wang Jing,Yang Minghui,Tao Jingli,Ma Teng,Wu Hao,Ji Pengyun,Wu Yingjie,Lian Zhengxing,Cui Wei,Liu Guoshi Reproduction (Cambridge, England) NLRP (NACHT, LRR and PYD domain-containing proteins) family plays pivotal roles in mammalian reproduction. Mutation of NLRP7 is often associated with human recurrent hydatidiform moles. Few studies regarding the functions of NLRP7 have been performed in other mammalian species rather than humans. In the current study, for the first time, the function of NLRP7 has been explored in ovine ovary. NLRP7 protein was mainly located in ovarian follicles and in in vitro pre-implantation embryos. To identify its origin, 763 bp partial CDS of NLRP7 deriving from sheep cumulus oocyte complexes (COCs) was cloned, it showed a great homology with Homo sapiens. The high levels of mRNA and protein of NLRP7 were steadily expressed in oocytes, parthenogenetic embryos or IVF embryos. NLRP7 knockdown by the combination of siRNA and shRNA jeopardized both the parthenogenetic and IVF embryo development. These results strongly suggest that NLRP7 plays an important role in ovine reproduction. The potential mechanisms of NLRP7 will be fully investigated in the future. 10.1530/REP-19-0081
    Transcriptional analyses of endometrial caruncles in sheep with in vivo / in vitro produced embryos during the peri-implantation period. Xing Kai,Qi Xiaolong,Ni Hemin,Wang Xiangguo,Guo Yong,Sheng Xihui Reproductive biology In vitro fertilization (IVF) for ovine embryos is strongly associated with low pregnancy rates and negative effects on embryonic and postnatal development, but the mechanisms remain unclear. We aimed to identify genes essential for successful implantation of IVF embryos in sheep by species-specific microarray analysis. Differential gene expression analysis between caruncles and intercaruncle sites of the ovine uterus with in vivo (IVV) -produced embryos on day 10 of pregnancy found 43 differentially expressed genes (DEGs) with at least a 2-fold change, such as coiled-coil domain-containing 152 (CCDC152) and pregnancy-associated plasma protein A (PAPPA). Gene ontology analysis revealed these DEGs were related to cell proliferation, the immune system process, localization and response to stimulus. Moreover, we identified 93 DEGs in endometrial caruncles with IVV- versus in vitro (IVT)-produced embryos on days 12, 14 and 16 of pregnancy, including prostaglandin-endoperoxide synthase 2 (PTGS2) and cubilin (CUBN). GO analysis revealed that these DEGs were related to cell adhesion, cell proliferation, embryo implantation, embryonic morphogenesis, the immune system process and localization. Two DEGs (PAPPA and RSAD2) in caruncle versus intercaruncle sites at day 10 were also differentially expressed between the IVV and IVT groups at the three implantation stages. Self-organizing feature map cluster analysis of transcript profiles during embryo implantation revealed different gene expression patterns between the IVV and IVT groups. In conclusion, this study identified many genes that may be associated with mechanisms underlying IVF-induced ovine embryo implantation failure during the peri-implantation period, and such genes provide potential candidates for further study. 10.1016/j.repbio.2019.09.008
    Vitamin C treatment of embryos, but not donor cells, improves the cloned embryonic development in sheep. Zhang Yumei,Gao Enen,Guan Hong,Wang Qianqian,Zhang Shuo,Liu Kexiong,Yan Fengxiang,Tian Hao,Shan Dehai,Xu Huijuan,Hou Jian Reproduction in domestic animals = Zuchthygiene Vitamin C is not only an antioxidant but also a regulator of epigenetic modifications that can enhance the activity of the ten-eleven translocation (TET) family dioxygenases and promote the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Here, we investigated the effects of vitamin C in regulating DNA methylation in sheep somatic cells or embryos in an effort to improve the cloned embryo development. Vitamin C treatment of sheep foetal fibroblast cells significantly increased the 5hmC levels but did not affect the 5mC levels in cells. After nuclear transfer, vitamin C-treated donor cells could not support a higher blastocyst development rate than non-treated cells. Although combination of serum starvation and vitamin C treatment could induce significant 5mC decrease in donor cells, it failed to promote the development of resultant cloned embryos. When cloned embryos were directly treated with vitamin C, the pre-implantation development of embryos and the 5hmC levels in blastocysts were significantly improved. This beneficial role of vitamin C on embryo development was also observed in fertilized embryos. Our results suggest that vitamin C treatment of the embryos, but not the donor cells, can improve the development of cloned sheep embryos. 10.1111/rda.13606
    Expression and in situ localization of GATA4, 5 and 6 mRNAs in ovine conceptuses and uterine endometria during the peri-implantation period. Bai Hanako,Sakurai Toshihiro,Godkin James D,Imakawa Kazuhiko Animal science journal = Nihon chikusan Gakkaiho In vertebrates, six GATA transcription factors, GATA1 through GATA6, have been identified and GATA1-3 is known to be involved in hematopoietic developments, while GATA4-6 play roles in cardiac and endoderm developments. Recently, we and others have found that GATA2 and GATA3 found in the trophectoderm plays a role in gene expression specific to this cell type, but GATA4-6 have not been well characterized in early embryonic developments. Using quantitative polymerase chain reaction (qPCR) and in situ hybridization, we examined the expression of GATA4, 5 and 6 messenger RNAs (mRNAs) in ovine conceptuses and uteri during the peri-implantation period. In ovine conceptuses, GATA4, 5 and 6 transcripts were present on days 15, 17 and 21 (day 0 = day of mating), and high GATA5 and 6 mRNAs were found on day 21, most of which were localized in the trophectoderm and endoderm. Moreover, minute and substantial GATA4 and 5 mRNAs were found in days 15 and 21 uterine endometria, respectively. Increase in GATA4-6 transcripts in day 21 uteri indicates that in addition to GATA1-3, GATA4-6 may also play a potentially novel role in the development of ovine trophectoderm, endoderm and/or uterine endometria following conceptus attachment to the uterine epithelium. 10.1111/asj.12156
    Proteomic analysis of the sheep caruncular and intercaruncular endometrium reveals changes in functional proteins crucial for the establishment of pregnancy. Al-Gubory K H,Arianmanesh M,Garrel C,Bhattacharya S,Cash P,Fowler P A Reproduction (Cambridge, England) The expression and regulation of endometrial proteins are crucial for conceptus implantation and development. However, little is known about site-specific proteome profiles of the mammalian endometrium during the peri-implantation period. We utilised a two-dimensional gel electrophoresis/mass spectrometry-based proteomics approach to compare and identify differentially expressed proteins in sheep endometrium. Caruncular and intercaruncular endometrium were collected on days 12 (C12) and 16 (C16) of the oestrous cycle and at three stages of pregnancy corresponding to conceptus pre-attachment (P12), implantation (P16) and post-implantation (P20). Abundance and localisation changes in differentially expressed proteins were determined by western blot and immunohistochemistry. In caruncular endometrium, 45 protein spots (5% of total spots) altered between day 12 of pregnancy (P12) and P16 while 85 protein spots (10% of total spots) were differentially expressed between P16 and C16. In intercaruncular endometrium, 31 protein spots (2% of total spots) were different between P12 and P16 while 44 protein spots (4% of total spots) showed differential expression between C12 and C16. The pattern of protein changes between caruncle and intercaruncle sites was markedly different. Among the protein spots with implantation-related changes in volume, 11 proteins in the caruncular endometrium and six proteins in the intercaruncular endometrium, with different functions such as protein synthesis and degradation, antioxidant defence, cell structural integrity, adhesion and signal transduction, were identified. Our findings highlight the different but important roles of the caruncular and intercaruncular proteins during early pregnancy. 10.1530/REP-13-0600
    Cervical changes in estrogen receptor alpha, oxytocin receptor, LH receptor, and cyclooxygenase-2 depending on the histologic compartment, longitudinal axis, and day of the ovine estrous cycle. Rodríguez-Piñón M,Gonzalez R,Tasende C,Bielli A,Genovese P,Garófalo E G Theriogenology The aim was to investigate the histologic distribution of estrogen receptor α (ERα), oxytocin receptor (OxR), LH receptor (LHR), and cyclooxygenase-2 (COX-2) in the cervix of the ewe during the estrous cycle. Immunohistochemistry was performed in the cranial and caudal cervix of Corriedale ewes on Day 1 (n = 6), 6 (n = 5), or 13 (n = 6) after estrous detection (Day 0). The ERα proportional score (%ERα nuclei) was lower in the cranial cervix than in the caudal cervix, whereas the OxR and COX-2 immunostaining areas (%areas) were greater in the cranial cervix than in the caudal cervix (P < 0.04). The %ERα nuclei decreased from Days 1 to 13 in luminal epithelia, but increased from Days 1 to 6 or remained unchanged in stromata (P < 0.003). The %OxR area was higher on Day 6 than on Days 1 and 13 in the superficial glandular epithelium, and increased from Days 1 to 13 in the deep glandular epithelium (P < 0.04). The %LHR area increased during the estrous cycle in luminal epithelia and fold stroma (P < 0.004). The %COX-2 area was restricted to epithelia, and it was lower on Day 1 than on Days 6 and 13 in luminal epithelia (P < 0.05). Differences in ERα, OxR, LHR, and COX-2 between cranial and caudal cervical zones indicate different physiological functions, and their cyclic variations in the cervical epithelia, in contrast to little or no variations in the stroma, suggest a hormone-responsive driving role of epithelia in cervical function. 10.1016/j.theriogenology.2013.12.019
    Intrauterine coadministration of ERK1/2 inhibitor U0126 inhibits interferon TAU action in the endometrium and restores luteolytic PGF2alpha pulses in sheep. Lee JeHoon,Stanley Jone A,McCracken John A,Banu Sakhila K,Arosh Joe A Biology of reproduction In ruminants, prostaglandin F2 alpha (PGF2alpha) is synthesized and released in a pulsatile pattern from the endometrial luminal epithelial (LE) cells during the process of luteolysis. Interferon tau (IFNT) is a Type 1 IFN secreted by the trophoblast cells of the developing conceptus. IFNT acts locally on endometrial LE cells to inhibit pulsatile releases of PGF2alpha and thus establish an endocrine environment for recognition of pregnancy. Cell signaling pathways through which IFNT stimulates expression of multiple genes or proteins in endometrial LE are largely unknown. Results of the present investigation indicate that intrauterine administration of IFNT inhibits pulsatile release of PGF2alpha, while coadministration IFNT and ERK 1/2 inhibitor U0126 restores luteolytic PGF2alpha pulses in sheep. IFNT increases phosphorylation of ERK1/2 proteins and increases its interaction with PGT proteins in endometrial LE. Blockade of ERK1/2 pathways inhibits IFNT action, decreases pERK1/2 and PGT protein interactions, and re-establishes the spatial expression of the oxytocin receptor protein completely and the estrogen receptor protein partially without modulating the expression of interferon regulatory factor-2 (IRF-2) protein in endometrial LE. IFNT does not decrease expression of COX-2, PGDH, or PGT protein in endometrial LE. Our results provide important new insights into IFNT signaling and the molecular endocrine control of PGF2alpha release at the time of establishment of pregnancy in ruminants. This novel IFNT-ERK1/2 signaling module needs to be explored in future studies to understand molecular and cellular mechanisms of IFNT action in endometrial LE in ruminants. 10.1095/biolreprod.113.111872
    Biological roles of uterine glands in pregnancy. Spencer Thomas E Seminars in reproductive medicine All mammalian uteri contain glands in the endometrium that synthesize or transport and secrete substances essential for survival and development of the conceptus (embryo/fetus and associated extraembryonic membranes). This review summarizes information related to the biological roles of uterine glands and their secretions in blastocyst/conceptus survival and implantation, uterine receptivity, and stromal cell decidualization in humans and animal models. The infertility and recurrent pregnancy loss observed in the ovine uterine gland knockout (UGKO) model unequivocally supports a primary role for uterine glands and, by inference, their secretions present in uterine luminal fluid in survival and development of the conceptus. Further, studies with mutant and progesterone-induced UGKO mice found that uterine glands and their secretions are required for establishment of uterine receptivity and blastocyst implantation as well as stromal cell decidualization. Similarly in humans, uterine glands and their secretory products are likely critical regulators of blastocyst implantation, uterine receptivity, and conceptus growth and development during the first trimester. Circumstantial evidence suggests that deficient glandular activity may be a causative factor in pregnancy failure and complications in humans. Thus, an increased understanding of uterine gland biology is important for diagnosis, prevention, and treatment of fertility and pregnancy problems in mammals. 10.1055/s-0034-1376354
    Local over-expression of VEGF-DΔNΔC in the uterine arteries of pregnant sheep results in long-term changes in uterine artery contractility and angiogenesis. Mehta Vedanta,Abi-Nader Khalil N,Shangaris Panicos,Shaw S W Steven,Filippi Elisa,Benjamin Elizabeth,Boyd Michael,Peebles Donald M,Martin John,Zachary Ian,David Anna L PloS one BACKGROUND:The normal development of the uteroplacental circulation in pregnancy depends on angiogenic and vasodilatory factors such as vascular endothelial growth factor (VEGF). Reduced uterine artery blood flow (UABF) is a common cause of fetal growth restriction; abnormalities in angiogenic factors are implicated. Previously we showed that adenovirus (Ad)-mediated VEGF-A165 expression in the pregnant sheep uterine artery (UtA) increased nitric oxide synthase (NOS) expression, altered vascular reactivity and increased UABF. VEGF-D is a VEGF family member that promotes angiogenesis and vasodilatation but, in contrast to VEGF-A, does not increase vascular permeability. Here we examined the effect of Ad.VEGF-DΔNΔC vector encoding a fully processed form of VEGF-D, on the uteroplacental circulation. METHODS:UtA transit-time flow probes and carotid artery catheters were implanted in mid-gestation pregnant sheep (n = 5) to measure baseline UABF and maternal haemodynamics respectively. 7-14 days later, after injection of Ad.VEGF-DΔNΔC vector (5×10(11) particles) into one UtA and an Ad vector encoding β-galactosidase (Ad.LacZ) contralaterally, UABF was measured daily until scheduled post-mortem examination at term. UtAs were assessed for vascular reactivity, NOS expression and endothelial cell proliferation; NOS expression was studied in ex vivo transduced UtA endothelial cells (UAECs). RESULTS:At 4 weeks post-injection, Ad.VEGF-DΔNΔC treated UtAs showed significantly lesser vasoconstriction (Emax144.0 v/s 184.2, p = 0.002). There was a tendency to higher UABF in Ad.VEGF-DΔNΔC compared to Ad.LacZ transduced UtAs (50.58% v/s 26.94%, p = 0.152). There was no significant effect on maternal haemodynamics. An increased number of proliferating endothelial cells and adventitial blood vessels were observed in immunohistochemistry. Ad.VEGF-DΔNΔC expression in cultured UAECs upregulated eNOS and iNOS expression. CONCLUSIONS:Local over-expression of VEGF-DΔNΔC in the UtAs of pregnant mid-gestation sheep reduced vasoconstriction, promoted endothelial cell proliferation and showed a trend towards increased UABF. Studies in cultured UAECs indicate that VEGF-DΔNΔC may act in part through upregulation of eNOS and iNOS. 10.1371/journal.pone.0100021
    Uterine histotroph and conceptus development. I. cooperative effects of arginine and secreted phosphoprotein 1 on proliferation of ovine trophectoderm cells via activation of the PDK1-Akt/PKB-TSC2-MTORC1 signaling cascade. Wang Xiaoqiu,Johnson Greg A,Burghardt Robert C,Wu Guoyao,Bazer Fuller W Biology of reproduction The greatest limitation to reproductive performance in most mammals, including humans, is embryonic mortality, which, in general, claims 20%-40% of the embryos during the peri-implantation period of pregnancy. Both arginine and secreted phosphoprotein 1 (SPP1) are multifunctional molecules that increase significantly in ovine uterine histotroph during early pregnancy. However, little is known about the relationship and underlying mechanisms for synergistic effects of arginine and SPP1, if any, on conceptus (embryo/fetus and associated extraembryonic membranes) development. Therefore, we conducted in vitro experiments using our established ovine trophectoderm cell line (oTr1) isolated from Day 15 ovine conceptuses to determine their proliferative response to individual and synergistic effects of arginine and recombinant SPP1 (rSPP1) that contains an RGD binding sequence. At physiological concentrations, arginine (0.2 mM) stimulated oTr1 cell proliferation 1.7-fold (P < 0.05) at 48 h, whereas rSPP1 (10 ng/ml) had no such effect. However, an additive effect on oTr1 cell proliferation was induced by combination of arginine and SPP1 as compared to the control (2.1-fold increase; P < 0.01), arginine alone (1.3-fold increase; P < 0.05), and rSPP1 alone (1.5-fold increase; P < 0.01). This additive effect was mediated through cooperative activation of the PDK1-Akt/PKB-TSC2-MTORC1 cell signaling cascade. Collectively, results suggest that arginine and SPP1 in histotroph act cooperatively to enhance survival, growth, and development of ovine conceptuses. 10.1095/biolreprod.114.125971
    An in vitro investigation of the actions of reproductive hormones on the cervix of the ewe in the follicular stage: the effects of 17β-estradiol, oxytocin, FSH, and arachidonic acid on the cervical pathway for the synthesis of prostaglandin E2. Falchi L,Scaramuzzi R J Theriogenology During the periovulatory period, the cervix of the ewe relaxes and this mechanism is thought to be mediated by oxytocin and prostaglandin E2 (PGE2) in response to increased concentrations of 17β-estradiol and perhaps FSH. The aim of the study was to determine the in vitro effects of 17β-estradiol, FSH, oxytocin, and arachidonic acid (AA) on the synthesis of PGE2 and on the expression of oxytocin receptor (OTR), cytoplasmic phospholipase A2 (cPLA2), and cyclooxygenase 2 (COX-2) in explants of cervical tissue collected from ewes in the periovulatory phase of the estrous cycle. Cervical minces from ewes in the follicular phase of the estrous cycle were cultured in supplemented Eagle's Minimum Essential Medium for 48 hours with 17β-estradiol, FSH, oxytocin, or AA. After incubation, the tissue was stored at -80 °C and the media at -20 °C. Western immunoblotting was used to determine relative levels of OTR, cPLA2, and COX-2 in cervical tissue, and the media was analyzed by RIA, to determine the concentration of PGE2. The addition of 17β-estradiol increased the concentration of PGE2 in the media (P = 0.001), the levels of COX-2 (P = 0.02) and OTR (P = 0.006) but not those of cPLA2 (P = 0.15). The addition of FSH increased the levels of COX-2 (P = 0.01) but, it had no effect on the concentration of PGE2 (P = 0.08) or on the levels of OTR (P = 0.07) and cPLA2 (P = 0.15). Oxytocin did not increase the levels of COX-2 (P = 0.38) but increased those of OTR (P = 0.001) and cPLA2 (P = 0.01) but not on the concentration of PGE2 in the media. Arachidonic acid increased the levels of cPLA2 (P = 0.01) and those of COX-2 (P = 0.02) but not the concentration of PGE2 in the media. Our findings suggest that the PGE2-mediated mechanisms of cervical relaxation in the ewe during the follicular phase are stimulated by FSH, 17β-estradiol, oxytocin, and AA. They all appear to act by inducing receptors and enzymes along the synthetic pathway for PGE2. 10.1016/j.theriogenology.2014.12.003
    Osteopontin: a leading candidate adhesion molecule for implantation in pigs and sheep. Johnson Greg A,Burghardt Robert C,Bazer Fuller W Journal of animal science and biotechnology Osteopontin (OPN; also known as Secreted Phosphoprotein 1, SPP1) is a secreted extra-cellular matrix (ECM) protein that binds to a variety of cell surface integrins to stimulate cell-cell and cell-ECM adhesion and communication. It is generally accepted that OPN interacts with apically expressed integrin receptors on the uterine luminal epithelium (LE) and conceptus trophectoderm to attach the conceptus to the uterus for implantation. Research conducted with pigs and sheep has significantly advanced understanding of the role(s) of OPN during implantation through exploitation of the prolonged peri-implantation period of pregnancy when elongating conceptuses are free within the uterine lumen requiring extensive paracrine signaling between conceptus and endometrium. This is followed by a protracted and incremental attachment cascade of trophectoderm to uterine LE during implantation, and development of a true epitheliochorial or synepitheliochorial placenta exhibited by pigs and sheep, respectively. In pigs, implanting conceptuses secrete estrogens which induce the synthesis and secretion of OPN in adjacent uterine LE. OPN then binds to αvβ6 integrin receptors on trophectoderm, and the αvβ3 integrin receptors on uterine LE to bridge conceptus attachment to uterine LE for implantation. In sheep, implanting conceptuses secrete interferon tau that prolongs the lifespan of CL. Progesterone released by CL then induces OPN synthesis and secretion from the endometrial GE into the uterine lumen where OPN binds integrins expressed on trophectoderm (αvβ3) and uterine LE (identity of specific integrins unknown) to adhere the conceptus to the uterus for implantation. OPN binding to the αvβ3 integrin receptor on ovine trophectoderm cells induces in vitro focal adhesion assembly, a prerequisite for adhesion and migration of trophectoderm, through activation of: 1) P70S6K via crosstalk between FRAP1/MTOR and MAPK pathways; 2) MTOR, PI3K, MAPK3/MAPK1 (Erk1/2) and MAPK14 (p38) signaling to stimulate trohectoderm cell migration; and 3) focal adhesion assembly and myosin II motor activity to induce migration of trophectoderm cells. Further large in vivo focal adhesions assemble at the uterine-placental interface of both pigs and sheep and identify the involvement of sizable mechanical forces at this interface during discrete periods of trophoblast migration, attachment and placentation in both species. 10.1186/2049-1891-5-56
    [Molecular cloning and tissue expression of the CCNG1 gene in sheep]. Lei Gao,Min Shen,Shangquan Gan,Jingquan Yang,Yiyuan Zhang Yi chuan = Hereditas The CCNG1 gene encodes cyclin G1, which is an important cell cycle regulator and has been reported to be involved in reproductive biological processes, such as oocyte maturation and granule cell proliferation in mammals. But the study of CCNG1 in sheep has been rarely reported. To examine the effects of CCNG1 on estrous control and seasonal breeding in sheep, we first cloned and characterized the expression level of the sheep CCNG1 gene. Then by Real-time PCR, we detected and analyzed the expressions of CCNG1 gene at mRNA levels in the hypothalamus-pituitary-ovary (HPO) axis in different stages of an estrous cycle in Duo Lang sheep (non-seasonal breeding) and Merino sheep (seasonal breeding). The results showed that the open reading frame of the sheep CCNG1 gene is 885 bp in length and encodes 294 amino acids. Bioinformatic analysis indicated that the secondary structure of the sheep CCNG1 protein contained multiple phosphorylation sites and some Protein Kinase C phosphorylation sites. CCNG1 mRNA was identified in all tissues tested, with the levels in ovary and kidney higher than others. The expression profiles of CCNG1 in the HPO axis in different stages of an estrous cycle were similar in different sheep breeds: the expression levels of CCNG1 in the ovary, uterus, pineal gland and pituitary gland all peaked in the estrus phase. But there were significant differences for expression change extent of CCNG1 in ovaries in the oestrus and metestrus phase between different sheep breeds. The results suggested that CCNG1 probably participated in the regulation of estrous behavior and seasonal reproduction through controling the growth and development of follicles in sheep. 10.16288/j.yczz.14-436
    Placental development during early pregnancy in sheep: estrogen and progesterone receptor messenger RNA expression in pregnancies derived from in vivo-produced and in vitro-produced embryos. Reynolds L P,Haring J S,Johnson M L,Ashley R L,Redmer D A,Borowicz P P,Grazul-Bilska A T Domestic animal endocrinology Sex steroids are important regulators of angiogenesis and growth in reproductive tissues, including the placenta. In experiment (exp.) 1, to examine the expression of a suite of sex steroid receptors throughout early pregnancy, maternal (caruncular [CAR]) and fetal (fetal membranes [FM]) placental tissues were collected on days 14 to 30 after mating and on day 10 after estrus (nonpregnant controls). In exp. 2, to examine the hypothesis that assisted reproductive technology would affect the expression of the same suite of sex steroid receptors, pregnancies were achieved through natural mating (NAT) or transfer of embryos from natural mating (NAT-ET), in vitro fertilization (IVF), or in vitro activation (IVA), and CAR and FM were collected on day 22. In exp. 1, for CAR messenger RNA (mRNA) expression of estrogen receptors (ESR) 1 and 2, nuclear (n) progesterone receptors (PGR) and membrane (m) PGRα, β, and γ were affected (P < 0.02) by pregnancy stage, as were ESR1, nPGR, and mPGRα, β, and γ for FM (P < 0.03). In exp. 2, for CAR, mRNA expression of ESR1 and nPGR was decreased (P < 0.001) in NAT-ET, IVF, and IVA groups compared with NAT. For FM, mRNA expression of ESR1 tended to be greater (P = 0.10) in the IVA group compared with NAT and NAT-ET, and GPER1 was greater (P < 0.05) in NAT-ET and IVF compared with NAT. These data establish the normal pattern of sex steroid receptor mRNA expression in maternal and fetal placenta during early pregnancy in sheep, and in addition, suggest that altered expression of placental sex steroid receptors may be an early event leading to poor placental vascularization and growth after assisted reproductive technology. 10.1016/j.domaniend.2015.05.003
    The Receptive Endometrial Transcriptomic Signature Indicates an Earlier Shift from Proliferation to Metabolism at Early Diestrus in the Cow. Mesquita F S,Ramos R S,Pugliesi G,Andrade S C S,Van Hoeck V,Langbeen A,Oliveira M L,Gonella-Diaza A M,Gasparin G,Fukumasu H,Pulz L H,Membrive C M,Coutinho L L,Binelli M Biology of reproduction This study aimed to characterize the endometrial transcriptome and functional pathways overrepresented in the endometrium of cows treated to ovulate larger (≥13 mm) versus smaller (≤12 mm) follicles. Nelore cows were presynchronized prior to receiving cloprostenol (large follicle [LF] group) or not (small follicle [SF] group), along with a progesterone (P4) device on Day (D) -10. Devices were withdrawn and cloprostenol administered 42-60 h (LF) or 30-36 h (SF) before GnRH agonist treatment (D0). Tissues were collected on D4 (experiment [Exp.] 1; n = 24) or D7 (Exp. 2; n = 60). Endometrial transcriptome was obtained by RNA-Seq, whereas proliferation and apoptosis were assessed by immunohistochemistry. Overall, LF cows developed larger follicles and corpora lutea, and produced greater amounts of estradiol (D-1, Exp. 1, SF: 0.7 ± 0.2; LF: 2.4 ± 0.2 pg/ml; D-1, Exp. 2, SF: 0.5 ± 0.1; LF: 2.3 ± 0.6 pg/ml) and P4 (D4, Exp. 1, SF: 0.8 ± 0.1; LF: 1.4 ± 0.2 ng/ml; D7, Exp. 2, SF: 2.5 ± 0.4; LF: 3.7 ± 0.4 ng/ml). Functional enrichment indicated that biosynthetic and metabolic processes were enriched in LF endometrium, whereas SF endometrium transcriptome was biased toward cell proliferation. Data also suggested reorganization of the extracellular matrix toward a proliferation-permissive phenotype in SF endometrium. LF endometrium showed an earlier onset of proliferative activity, whereas SF endometrium expressed a delayed increase in glandular epithelium proliferation. In conclusion, the periovulatory endocrine milieu regulates bovine endometrial transcriptome and seems to determine the transition from a proliferation-permissive to a biosynthetic and metabolically active endometrial phenotype, which may be associated with the preparation of an optimally receptive uterine environment. 10.1095/biolreprod.115.129031
    Insights into conceptus elongation and establishment of pregnancy in ruminants. Spencer T E,Forde N,Lonergan P Reproduction, fertility, and development This review integrates established and new information on the factors and pathways regulating conceptus-endometrial interactions, conceptus elongation and establishment of pregnancy in sheep and cattle. Establishment of pregnancy in domestic ruminants begins at the conceptus stage (embryo or fetus and associated extra-embryonic membranes) and includes pregnancy recognition signalling, implantation and the onset of placentation. Survival and growth of the preimplantation blastocyst and elongating conceptus require embryotrophic factors (amino acids, carbohydrates, proteins, lipids and other substances) provided by the uterus. The coordinated and interactive actions of ovarian progesterone and conceptus-derived factors (interferon-τ and prostaglandins) regulate expression of elongation- and implantation-related genes in the endometrial epithelia that alter the uterine luminal milieu and affect trophectoderm proliferation, migration, attachment, differentiation and function. A comparison of sheep and cattle finds both conserved and non-conserved embryotrophic factors in the uterus; however, the overall biological pathways governing conceptus elongation and establishment of pregnancy are likely conserved. Given that most pregnancy losses in ruminants occur during the first month of pregnancy, increased knowledge is necessary to understand why and provide a basis for new strategies to improve pregnancy outcome and reproductive efficiency. 10.1071/RD16359
    Activin A and follistatin during the oestrous cycle and early pregnancy in ewes. O'Connell Anne R,McNatty Kenneth P,Hurst Peter R,Spencer Thomas E,Bazer Fuller W,Reader Karen L,Johnstone Peter D,Davis George H,Juengel Jennifer L The Journal of endocrinology The activin pathway has been postulated to be involved in regulation of multiple reproductive processes important for survival of the conceptus. These processes include luteinisation of the follicular cells and thus function of the corpus luteum, early embryo development and uterine function including implantation of the conceptus. Therefore, the aim of the current study was to determine whether the concentrations of activin A and follistatin (FST), an activin-binding protein, differed between ewes with a lifetime history of enhanced or reduced embryonic survival (ES). The mRNAs encoding FST and activin A (inhibin beta A subunit; INHBA) were present in the uterus and abundant in the uterine luminal or glandular epithelia by day 18 of gestation. A peak of activin A was observed in the systemic circulation around the time of oestrus, and activin A concentrations were elevated in animals with reduced ES during the oestrous cycle and early gestation. Concentrations of activin A in uterine fluid were approximately twofold greater on day 16 of gestation in ewes with reduced ES compared to those with enhanced ES. No consistent differences in FST were observed between these groups. Treatment of luteinising ovine granulosa cells with activin A in vitro suppressed progesterone secretion providing evidence of a potential pathway whereby increased concentrations of activin A may decrease ES. 10.1530/JOE-15-0367
    Characterization and comparative analyses of transcriptomes for in vivo and in vitro produced peri-implantation conceptuses and endometria from sheep. Wei Xia,Xiaoling Zhang,Kai Miao,Rui Wang,Jing Xu,Min Guo,Zhonghong Wu,Jianhui Tian,Xinyu Zhang,Lei An The Journal of reproduction and development An increasing number of reports indicate that in vitro fertilization (IVF) is highly associated with long‑term side effects on embryonic and postnatal development, and can sometimes result in embryonic implant failure. While high‑throughput gene expression analysis has been used to explore the mechanisms underlying IVF-induced side effects on embryonic development, little is known about the effects of IVF on conceptus-endometrial interactions during the peri-implantation period. Using sheep as a model, we performed a comparative transcriptome analysis between in vivo (IVO; in vivo fertilized followed by further development in the uterus) and in vitro produced (IVP; IVF with further culture in the incubator) conceptuses, and the caruncular and intercaruncular areas of the ovine endometrium. We identified several genes that were differentially expressed between the IVO and IVP groups on day 17, when adhesion between the trophoblast and the uterine luminal epithelium begins in sheep. By performing Gene Ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, we found that, in the conceptus, differentially expressed genes (DEGs) were associated mainly with functions relating to cell binding and the cell cycle. In the endometrial caruncular area, DEGs were involved in cell adhesion/migration and apoptosis, and in the intercaruncular area, they were significantly enriched in pathways of signal transduction and transport. Thus, these DEGs are potential candidates for further exploring the mechanism underlying IVF/IVP-induced embryonic implant failure that occurs due to a loss of interaction between the conceptus and endometrium during the peri-implantation period. 10.1262/jrd.2015-064
    Gestation-related gene expression and protein localization in endometrial tissue of Suffolk and Cheviot ewes at gestation Day 19, after transfer of Suffolk or Cheviot embryos. Sequeira M,Pain S J,de Brun V,Meikle A,Kenyon P R,Blair H T Theriogenology The objective of this study was to investigate the gene expression of progesterone and estrogen receptor α (PR, ERα), insulin-like growth factor (IGF) 1, IGF-2, their receptor (IGFR1), IGF-binding proteins (BP) 1 to 6, insulin receptor, adiponectin receptors (AdipoR1/2), cyclooxygenase 2 (PTGS2), mucin 1 and to localize PR, ERα, IGF-1, IGFR1, PTGS2, and proliferating cellular nuclear antigen (PCNA) in the endometrium of pregnant (Day 19) Suffolk and Cheviot ewes carrying Suffolk and Cheviot embryos transferred within and reciprocally between breeds. Gene expression was determined by real-time quantitative polymerase chain reaction (RT-qPCR), and antigen determination was measured by immunohistochemistry in the luminal epithelium (LE), superficial and deep glands (SG, DG, respectively) and superficial and deep stroma. Gene expression of PR, IGF-1, IGFBP2, and IGFBP5 was higher in Suffolk than that in Cheviot ewes (P < 0.05). Greater abundance of IGF-2 and IGBP3 expression was found in Cheviot ewes carrying Cheviot embryos than Cheviot ewes carrying Suffolk embryos (P < 0.05). No staining for PR and ERα was observed in the LE, very scarce staining in SG and DG, whereas positive staining was observed in both superficial and deep stroma. No differences were found for PR staining, but Cheviot ewes had higher ERα staining intensity than Suffolk ewes (P < 0.05). Positive staining for IGF-1 was observed in all cell types except DG, and staining of IGFR1 was observed in all cell types. No differences among groups in staining were found for IGF-1 or IGFR1 in any cell type. Positive staining of PTGS2 was observed in LE and SG in all groups. An interaction between ewe and embryo breed affected PTGS2 staining (P < 0.05), whereby Cheviot ewes carrying Suffolk embryos had a lower PTGS2 staining than Suffolk ewes carrying Suffolk embryos. Positive staining of PCNA was found in LE and SG. Suffolk ewes carrying Suffolk embryos showed lower PCNA immunostaining than Cheviot ewes carrying Suffolk embryos (P < 0.05), whereas no differences were observed in ewes carrying Cheviot embryos. This study showed that gestation-related protein expression in the endometrium of Suffolk and Cheviot ewes is affected by both ewe and embryo breed at Day 19 of pregnancy. 10.1016/j.theriogenology.2016.05.015
    The effect of the intracervical administration of FSH or LH on the levels of hyaluronan, COX2, and COX2 mRNA in the cervix of the nonpregnant ewe. Leethongdee Sukanya,Khalid Muhammad,Scaramuzzi Rex J Theriogenology During the periovulatory period, the cervix relaxes in response to changes in circulating concentrations of reproductive hormones. The present study investigated the role of gonadotrophins in cervical function by examining the expression of cyclooxygenase-2 (COX2) and COX2 mRNA and the concentration of hyaluronan (HA) in the cervix, after intracervical treatment with either FSH or LH. Eighteen ewes were assigned to four groups. They were then treated with commercial intravaginal progestagen sponges and eCG to synchronize their estrous cycles. Intracervical treatments were given 24 hours after removal of the sponges as follows: group 1: FSH, 2 mg; group 2: LH, 2 mg; group 3: vehicle; and group 4: control. Cervices were collected 54 hours after sponge removal and then divided into three regions. The expression of COX2 and COX2 mRNA was determined by immunohistochemistry and in situ hybridization and those of HA by ELISA. The levels of expression of COX2, COX2 mRNA, and HA were compared in six tissue layers (luminal epithelium, subepithelial stroma, circular, longitudinal and transverse muscle, and serosa) and in three cervical regions (vaginal, mid, and uterine). The results showed that both FSH and LH significantly increased the levels the COX2 mRNA and COX2 in the cervix, but the effects of the gonadotrophins were selective. The effects of both FSH and LH were most evident at the vaginal end of the cervix and least at the uterine end of the cervix. Furthermore, their effects were confined to the stroma and smooth muscle layers of the cervix in the case of FSH and to smooth muscle only in the case of LH. Neither FSH nor LH affected the concentration of HA in the cervix although FSH but not LH reduced the concentration of HA in cervical mucus. These findings suggest that the gonadotrophins regulate the expression of COX2 in the cervix and that they may have a role facilitating relaxation of the cervix during estrus in the ewe. 10.1016/j.theriogenology.2016.07.014
    Analysis of the Uterine Epithelial and Conceptus Transcriptome and Luminal Fluid Proteome During the Peri-Implantation Period of Pregnancy in Sheep. Brooks Kelsey,Burns Gregory W,Moraes Joao G N,Spencer Thomas E Biology of reproduction Studies support the idea that uterine epithelia and their secretions have important biological roles in conceptus survival, elongation, and implantation in sheep. The present study evaluated the transcriptome of the uterine luminal epithelium (LE) and glandular epithelium (GE) and the conceptus and proteome of uterine luminal fluid (ULF) during the peri-implantation period of pregnancy. Transcriptome (RNA-sequencing) analysis was conducted in LE and GE isolated from uteri of Day 10, 12, 14, 16, and 20 pregnant sheep by laser capture microdissection. In the LE, the total number of expressed genes increased between Days 10 and 20, whereas expressed genes in the GE increased from Days 10 to 14 and then decreased to Day 20. Most of the expressed genes in LE and GE from Days 10 to 14 are involved in cell survival and growth, whereas genes involved in cell organization and protein synthesis were most abundant on Days 16 and 20. Total expressed genes in the conceptus was greatest on Day 12, decreased to Day 16, and then increased to Day 20. Genes abundantly expressed in the elongating conceptus included IFNT, PTGS2, MGST1, FADS1, and FADS2, whereas SERPINA1, CSH1, and PLET1 were most abundant in the Day 20 conceptus. Proteins, identified by mass spectrometry, increased in the ULF from Days 10 to 16 and are involved in cellular reorganization or are proteases or chaperone proteins. These results support the idea that conceptus elongation and implantation is regulated by both extrinsic and intrinsic factors. This study provides critical information that serves as a foundation to discover new regulatory pathways governing uterine receptivity, conceptus elongation, trophectoderm differentiation, conceptus-endometrial interactions, and pregnancy establishment in ruminants. 10.1095/biolreprod.116.141945
    The sheep conceptus modulates proteome profiles in caruncular endometrium during early pregnancy. Arianmanesh Mitra,Fowler Paul A,Al-Gubory Kaïs H Animal reproduction science The stage-specific expression of functional proteins within the endometrium, and their regulation by conceptus-derived signals, are crucial for conceptus development and successful establishment of pregnancy. Accurate knowledge of endometrium-conceptus interactions is key for the development of effective strategies to improve conceptus implantation rates both following natural conception and/or assisted reproductive technologies. The unilateral pregnant ewe provides a powerful experimental model for the study of endometrial function in the presence or absence of conceptuses during the peri-implantation period. Two-dimensional gel electrophoresis and mass spectrometry-based proteomics were used to compare and identify differentially expressed proteins in caruncular endometrium collected from the gravid uterine horns and the non-gravid uterine horns at the time of conceptus attachment (day 16 of pregnancy) and early post-implantation period (day 20 of pregnancy). Fifty seven protein spots were up-regulated in the gravid horn at day 16 of pregnancy and twenty seven protein spots were up-regulated in the gravid horn at day 20 of pregnancy. Sixteen proteins with different functions such as protein metabolism, cholesterol and ion transport and cell adhesion were identified. In conclusion, the use of the unilaterally pregnant ewe model provides evidence that the early implantation and post-implanting conceptus-derived signals up-regulate caruncle endometrial proteins, including carbonic anhydrase 2 (CA-II) and apolipoprotein A-1 (APOA1) and down-regulate caruncle endometrial proteins, including adenosylhomocysteinase (AHCY) and heat shock 60kDa protein 1 (HSP60). These regulated proteins are likely involved in providing a suitable intra-uterine environment required for conceptus attachment, implantation, early post-implantation development and the successful establishment of pregnancy in sheep. 10.1016/j.anireprosci.2016.11.001
    Gap junctional connexin messenger RNA expression in the ovine uterus and placenta: effects of estradiol-17β-treatment, early pregnancy stages, and embryo origin. Johnson M L,Redmer D A,Reynolds L P,Grazul-Bilska A T Domestic animal endocrinology Gap junctions play a major role in direct, contact-dependent cell-cell communication, and they have been implicated in the regulation of cellular metabolism and the coordination of cellular functions during growth and differentiation of organs and tissues. Gap junctional channels, composed of connexin (Cx) proteins, have been detected and shown to be influenced by hormones (eg, estrogen and progesterone) in uterine and placental tissues in several species. We hypothesized that (1) the messenger RNA (mRNA) for Cx26, Cx32, Cx37, and Cx43 is expressed in the uterus of ovariectomized sheep treated with estradiol-17β (E2) and in ovine placenta during early pregnancy, (2) E2-treatment of ovariectomized ewes would cause time-specific changes in Cx26, Cx32, Cx37, and Cx43 mRNA expression (experiment 1), and (3) expression of these 4 Cx would vary across the days of early pregnancy (experiment 2) and will be affected by embryo origin (ie, after application of assisted reproductive technologies [ARTs]; experiment 3). Thus, we collected uterine tissues at 0 to 24 h after E2 treatments (experiment 1), and placental tissues during days 14 to 30 of early pregnancy after natural (NAT) breeding (experiment 2) and on day 22 of early pregnancy established after transfer of embryos generated through natural breeding (NAT-ET), in vitro fertilization (IVF), or in vitro activation (IVA, parthenotes; experiment 3). In experiment 1, the expression of Cx26, Cx37, and Cx43 mRNA increased (P < 0.05) and Cx32 mRNA decreased (P < 0.06) in both caruncular and intercaruncular tissues after E2 treatment. In experiment 2, during early pregnancy, there were significant changes (P < 0.01) across days in the expression of Cx26, Cx37, and Cx43 mRNA in the maternal placenta, accompanied by changes (P < 0.001) in Cx37 and Cx43 mRNA in the fetal placenta. In experiment 3, in maternal placenta, Cx32 mRNA expression was decreased (P < 0.001) in NAT-ET, IVF, and IVA groups compared to the NAT group; but in fetal placenta, Cx32 mRNA expression was increased (P < 0.05) in NAT-ET, IVF and IVF groups, and Cx26 mRNA expression was increased (P < 0.05) in IVA compared to NAT group. These data suggest that Cx26, Cx32, Cx37, and Cx43 play specific roles in E2-regulated uterine function and in placental development during early gestation both after natural mating and with application of ART. 10.1016/j.domaniend.2016.09.004
    Preattachment Embryos of Domestic Animals: Insights into Development and Paracrine Secretions. Sandra Olivier,Charpigny Gilles,Galio Laurent,Hue Isabelle Annual review of animal biosciences In mammalian species, endometrial receptivity is driven by maternal factors independently of embryo signals. When pregnancy initiates, paracrine secretions of the preattachment embryo are essential both for maternal recognition and endometrium preparation for implantation and for coordinating development of embryonic and extraembryonic tissues of the conceptus. This review mainly focuses on domestic large animal species. We first illustrate the major steps of preattachment embryo development, including elongation in bovine, ovine, porcine, and equine species. We next highlight conceptus secretions that are involved in the communication between extraembryonic and embryonic tissues, as well as between the conceptus and the endometrium. Finally, we introduce experimental data demonstrating the intimate connection between conceptus secretions and endometrial activity and how adverse events perturbing this interplay may affect the progression of implantation that will subsequently impact pregnancy outcome, postnatal health, and expression of production traits in livestock offspring. 10.1146/annurev-animal-022516-022900
    Citrullination regulates the expression of insulin-like growth factor-binding protein 1 (IGFBP1) in ovine uterine luminal epithelial cells. Young Coleman H,Rothfuss Heather M,Gard Philip F,Muth Aaron,Thompson Paul R,Ashley Ryan L,Cherrington Brian D Reproduction (Cambridge, England) There are five peptidylarginine deiminase (PAD) isozymes designated as PADs 1, 2, 3, 4 and 6, and many are expressed in female reproductive tissues. These enzymes post-translationally convert positively charged arginine amino acids into neutral citrulline residues. Targets for PAD-catalyzed citrullination include arginine residues on histone tails, which results in chromatin decondensation and changes in gene expression. Some of the first studies examining PADs found that they are localized to rodent uterine epithelial cells. Despite these findings, the function of PAD-catalyzed citrullination in uterine epithelial cells is still unknown. To address this, we first examined PAD expression in uterine cross-sections from pregnant ewes on gestation day 25 (d25). Immunohistochemistry revealed that the levels of PADs 2 and 4 are robust in luminal and glandular epithelia compared with those of PADs 1 and 3. As PADs 2 and 4 have well-characterized roles in histone citrullination, we next hypothesized that PADs citrullinate histones in these uterine cells. Examination of caruncle lysates from pregnant ewes on gestation d25 and an ovine luminal epithelial (OLE) cell line shows that histone H3 arginine residues 2, 8, 17 and 26 are citrullinated, but histone H4 arginine 3 is not. Using a pan-PAD inhibitor, we next attenuated histone citrullination in OLE cells, which resulted in a significant decrease in the expression of insulin-like growth factor-binding protein 1 () mRNA. As IGFBP1 is important for the migration and attachment of the trophectoderm to uterine endometrium, our results suggest that PAD-catalyzed citrullination may be an important post-translational mechanism for the establishment of pregnancy in ewes. 10.1530/REP-16-0494
    Prolactin modulates luteal activity in the short-nosed fruit bat, Cynopterus sphinx during delayed embryonic development. Anuradha ,Krishna Amitabh General and comparative endocrinology The aim of this study was to evaluate the role of prolactin as a modulator of luteal steroidogenesis during the period of delayed embryonic development in Cynopterus sphinx. A marked decline in circulating prolactin levels was noted during the months of November through December coinciding with the period of decreased serum progesterone and delayed embryonic development. The seasonal changes in serum prolactin levels correlated positively with circulating progesterone (P) level, but inversely with circulating melatonin level during first pregnancy showing delayed development in Cynopterus sphinx. The results also showed decreased expression of prolactin receptor-short form (PRL-RS) both in the corpus luteum and in the utero-embryonic unit during the period of delayed embryonic development. Bats treated in vivo with prolactin during the period of delayed development showed significant increase in serum progesterone and estradiol levels together with significant increase in the expression of PRL-RS, luteinizing hormone receptor (LH-R), steroidogenic acute receptor protein (STAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD) in the ovary. Prolactin stimulated ovarian angiogenesis (vascular endothelial growth factor) and cell survival (B-cell lymphoma 2) in vivo. Significant increases in ovarian progesterone production and the expression of prolactin-receptor, LH-R, STAR and 3β-HSD proteins were noted following the exposure of LH or prolactin in vitro during the delayed period. In conclusion, short-day associated increased melatonin level may be responsible for decreased prolactin release during November-December. The decline in prolactin level might play a role in suppressing P and estradiol-17β (E2) estradiol levels thereby causing delayed embryonic development in C. sphinx. 10.1016/j.ygcen.2017.04.008
    Effects of LPA2R, LPA3R, or EP4R agonists on luteal or endometrial function in vivo or in vitro and sirtuin or EP1R, EP2R, EP3R or EP4R agonists on endometrial secretion of PGE and PGFin vitro. LaPorte Magen E,Weems Yoshie S,Arreguin-Arevalo Alejandro,Nett Terry M,Tsutahara Nicole,Sy Tracy,Haberman Jade,Chon Michel,Randel Ronald D,Weems Charles W Theriogenology In previous work, an EP2 prostanoid receptor (EP2R) agonist in vivo increased mRNA expression of luteal LH receptors (LHR), unoccupied and occupied luteal; LHR, and circulating progesterone, while an EP3R or FPR agonist decreased; mRNA expression of luteal LHR, unoccupied and occupied luteal LHR, and; circulating progesterone. An EP4R and lysophosphatidic acid (LPA) LPA2R and LPA3R agonists were reported to inhibit luteal function and sirtuins have been proposed to increase prostaglandin synthesis. The objectives were to determine; whether an EP4R, LPA2R, or LPA3R agonist affect ovine luteal function in vivo or; in vitro. In addition, whether sirtuin (SIRT)-1, 2, or 3; LPA2R or LPA3R; or EP1R, EP2R, EP3R, or EP4R agonists affect caruncular endometrial PGF or PGE (PGE1+PGE2) secretion in vitro. Day-10 nonpregnant ewes received a single injection of Vehicle (N = 5); an LPA2R (N = 5); LPA3R (N = 6); or EP4R (N = 5) agonist given into the interstitial tissue of the ovarian vascular pedicle adjacent to the luteal-containing ovary to determine effects on circulating progesterone, mRNA expression of luteal LHR, and luteal unoccupied and occupied LHR. In addition, agonists for LPA2R, LPA3R, EP1R, EP2R, EP3R, or EP4R or SIRT-1, SIRT-2, or SIRT-3 activators were incubated with caruncular endometrial slices in vitro to determine their effect on caruncular endometrial PGF, or PGE secretion. LPA2R, LPA3R, or an EP4R agonist in vivo did not affect (P ≥ 0.05) luteal weight, circulating progesterone, or occupied luteal LHR. However, an LPA2R or EP4R agonist, but; not LPA3R agonist, in vivo increased (P ≤ 0.05) mRNA expression of luteal LHR. An; LPA2R, LPA3R, or EP4R agonist increased (P ≤ 0.05) luteal unoccupied LHR, but; not occupied LHR. An LPA2R, LPA3R, or an EP4R agonist did not affect (P ≥ 0.05); luteal progesterone secretion in vitro. An LPA2R or LPA3R agonist did not affect (P ≥ 0.05) luteal PGF, or PGE secretion in vitro. However, an EP4R agonist tended to decrease (P < 0.066) luteal PGF secretion and increased (P ≤ 0.05) luteal PGE; secretion in vitro. EP1R, EP2R, EP3R, or an EP4R agonist did not affect (P ≥ 0.05); caruncular endometrial PGF secretion in vitro. However, EP1R, EP3R, or an EP4R agonist increased caruncular endometrial PGE secretion in vitro, while two different EP2R agonists did not affect (P ≥ 0.05) caruncular endometrial PGE; secretion. A SIRT-1 activator, but not SIRT-2 or SIRT-3 activators, increased (P ≤ 0.05) caruncular endometrial PGE secretion, while sirtuin 1, 2, or 3 activators did not affect (P ≥ 0.05) caruncular endometrial PGF secretion. In conclusion, receptors for EP4, LPA2, and LPA3 do not appear to be involved; in luteolysis, but EP4R and LPA2R might participate in preventing luteolysis by maintaining luteal mRNA expression for LHR and preventing loss of unoccupied luteal LHR. In addition, SIRT-1, EP1R, EP3R, and EP4R might be involved in; regulating caruncular endometrial PGE secretion, but not PGF secretion. 10.1016/j.theriogenology.2017.02.017
    Molecular characterization and expression of the GDF9 gene in New Zealand white rabbits. Sun Caixia,Xie Shuyu,Huang Tao,Zhang Wei,Wang Ansi,Wang Dan,Li Ming,Sun Guirong Journal of genetics Growth differentiation factor 9 (GDF9) has been shown to be involved in regulating follicular development and reproduction in many mammalian species. However, related information about the effect of the GDF9 gene on reproductive traits of New Zealand white rabbits was rarely reported. In this study, rabbits were distributed into two groups (poor and prolific offspring productions) and cloning and quantitative real-time PCR (qPCR) were employed to characterize the rabbit GDF9 gene. By cloning, 2515-bp genomic DNA and 1359-bp cDNA sequences were obtained. Comparing the two cDNA sequences, three potential mutation sites (C.539C>T,C.562G>C and C.718C>G) in exon 2 of the GDF9 gene were found, and the corresponding amino acids changed (P.183T>M, P.188E>Q and P.240L>V). The qPCR results revealed that GDF9 was not tissue-specific, but rather expressed in all collected tissues. The expression level of the GDF9 gene was highest in the ovary, and was significantly increased (P< 0.05) compared with the other tissues. The liver had the second highest expression, and the heart and spleen had the least expression in New Zealand white rabbits. In the prolific group, the expression quantity of the GDF9 gene significantly increased (P < 0.05) in the heart, spleen, ovary, liver and uterus (P < 0.01) than the other groups. The amino acid sequence identities of human, sheep, goat, mouse, cattle, pig, cat, donkey, Nancy Ma's night monkey and olive baboon were 72, 68, 69, 66, 69, 71, 67, 73, 75 and 73%, respectively. Bioinformatics analysis was executed, and a random coil was determined to be the primary secondary structure.
    Different enzymatic antioxidative pathways operate within the sheep caruncular and intercaruncular endometrium throughout the estrous cycle and early pregnancy. Al-Gubory K H,Faure P,Garrel C Theriogenology There has been a growing interest in the role played by antioxidant enzymes in the regulation of endometrial function in mammals. However, little is known about enzymatic antioxidative pathways involved in conditioning the cyclic and early pregnant endometrium for conceptus attachment and implantation in domestic ruminants. We aimed to investigate changes in activities of superoxide dismutase 1 and 2 (SOD1, SOD2), glutathione peroxidase (GPX), glutathione reductase (GR) and catalase (CAT) in sheep caruncles (CAR) and intercaruncles (ICAR) endometrial tissues of cyclic and early pregnant ewes. Irrespective of day of cycle or pregnancy, CAR demonstrated higher activities of SOD1 and SOD2 than in ICAR. On day 12 of the estrous cycle, ICAR demonstrated higher activity of GPX and GR than in CAR tissues. On days 12 and 16 the estrous cycle, ICAR demonstrated higher activity of CAT than in CAR. CAR demonstrated higher activity of GPX on day 18 than on days 4, 8, 12 and 16 of the estrous cycle. CAR demonstrated higher activity of CAT on day 18 than on days 4, 8, 12 and 16 of the estrous cycle. ICAR demonstrated higher activity of CAT on day 18 than on days 4, 8, and 16 of the estrous cycle. The activity of CAT in ICAR increased from days 4 and 8 to day 12 of the estrous cycle. The activity of SOD2 in CAR increased from day 16 to day 18 of pregnancy. On day 12 of pregnancy, CAR demonstrated higher activity of GPX than in ICAR. On day 16 of pregnancy, ICAR demonstrated higher activity of GPX than in CAR. The activity of GPX in ICAR increased from day 12 to day 16 of pregnancy. The activity of GPX in CAR increased from day 16 to day 18 of pregnancy. The activity of GR in CAR and ICAR increased from days 12 and 16 to day 18 of pregnancy. On days 16 and 18 of pregnancy, ICAR demonstrated higher activity of CAT than in CAR. The activity of CAT in CAR decreased from day 12 to days 16 and 18 of pregnancy. The activity of CAT in ICAR decreased from day 12 to day 16 of pregnancy and then increased from day 16 to day 18 of pregnancy. In conclusion, different antioxidant mechanisms operate within CAR and ICAR endometrium throughout the estrous cycle and during early pregnancy. This might be related to the different but important roles of CAR and ICAR endometrial tissues for the establishment of pregnancy. 10.1016/j.theriogenology.2017.05.017
    Paracrine and endocrine actions of interferon tau (IFNT). Hansen Thomas R,Sinedino Leticia D P,Spencer Thomas E Reproduction (Cambridge, England) This review focuses on the paracrine and endocrine actions of interferon tau (IFNT) during pregnancy recognition and establishment in ruminants. Pregnancy recognition involves the suppression of the endometrial luteolytic mechanism by the conceptus to maintain progesterone production by the corpus luteum (CL). The paracrine antiluteolytic effects of conceptus-derived IFNT inhibit upregulation of oxytocin receptors in the endometrial epithelia of the uterus, thereby preventing the production of luteolytic prostaglandin F2 alpha (PGF2α) pulses. In the endometrium, IFNT induces or upregulates a large number of classical IFN-stimulated genes (ISGs) and regulates expression of many other genes in a cell-specific manner that are likely important for conceptus elongation, implantation and establishment of pregnancy. Further, IFNT has endocrine effects on extrauterine cells and tissues. In sheep, IFNT induces luteal resistance to PGF2α, thereby ensuring survival of the CL for maintenance of pregnancy. The ISGs induced in circulating peripheral blood mononuclear cells by IFNT may also be useful as an indicator of pregnancy status in cattle. An increased knowledge of IFNT and ISGs is important to improve the reproductive efficiency in ruminants. 10.1530/REP-17-0315
    Effect of exogenous progesterone on embryo size and ewe uterine gene expression in an ovine 'dam size' model of maternal constraint. Fermin Lisanne M,Pain Sarah J,Morel Patrick C H,Gedye Kristene R,Kenyon Paul R,Blair Hugh T Reproduction, fertility, and development Progesterone (P4), acting via its receptor, regulates uterine function and histotroph production, which are crucial to embryo growth. This study aimed to examine exogenous P4 effects on embryo size and differential endometrial gene expression at Day 19 of gestation using a 'dam size' sheep model of maternal constraint. Purebred Suffolk (S, genotypically large) embryos were transferred into recipient groups of Cheviot (C, genotypically small) or Suffolk ewes that had, or had not, been pre-treated with P4 from Days 0 to 6 of pregnancy. At Day 19S embryos were collected from four experimental groups: P4 pretreated S ewes (SP4; n=5), untreated S ewes (SnP4; n=15), P4 pretreated C ewes (CP4; n=7) and untreated C ewes (CnP4; n=21). Day-19 embryos from CP4 ewes were larger (P<0.05) than those from CnP4 ewes and similar in size (P>0.05) to embryos from SnP4 and SP4 ewes. Expression of mucin 1 (MUC1) and prostaglandin-endoperoxide synthase 2 (PTGS2) was upregulated in uterine horns ipsilateral to the corpus luteum from CP4 ewes. Prostaglandin receptor (PGR), MUC1 and PTGS2 expression was upregulated, whilst cathepsin L (CTSL) and radical S-adenosyl methionine domain-containing 2 (RSAD2) expression was downregulated in the ipsilateral horn of SP4 ewes. This suggests that pretreating ewes with exogenous P4 may alleviate early pregnancy maternal constraint via mechanisms that alter uterine function. However, further research is required to investigate the timing of P4 administration and its impact on conception rates. 10.1071/RD17096
    Expression and function of MUC1 in uterine tissues during early pregnancy in sheep after natural oestrous or artificially-induced oestrous. Wang Xiangguo,Zhu Boyang,Xiong Shanhui,Sheng Xihui,Qi Xiaolong,Huang Qizhen,Chen Chaolei,Guo Yong,Ni Hemin Theriogenology Mucin 1 (MUC1), a cell surface glycoprotein, is expressed mainly in the endometrial luminal epithelium (LE) and glandular epithelium (GE) of the endometrium in many mammalian species including mice, rats, pigs, sheep, horses and humans during various stages of a menstrual or oestrous cycle, where it plays an important role in embryo implantation and placentation. However, the expression and function of MUC1 in uterine tissues during early pregnancy in sheep after artificially-induced oestrous is not known. Therefore, we investigated the expression and function of MUC1 in the early pregnant and non-pregnant uterine tissues of sheep with natural oestrous or artificially-induced oestrous on days 10, 12, 14, 16 and 18 of the cycle by in situ hybridization, quantitative real-time polymerase chain reaction, immunohistochemical staining and western blotting methods. According to our results, MUC1 mRNA and protein expression increased initially but then decreased from days 10-18, peaking on day 14 in the uterine tissues of non-pregnant ewes after both natural and artificially-induced oestrous. MUC1 protein localisation was observed in the LE on days 10, 12 and 14 and in the GE on days 16 and 18. In contrast, MUC1 mRNA and protein expression increased on days 10 and 12, decreased on day 14, but increased again on days 16 and 18 in the uterine tissues of pregnant ewes both in natural oestrous and in artificially-induced oestrous. Additionally, the MUC1 mRNA and protein expression levels in the uterine tissues of the early pregnant sheep were significantly lower than those in the non-pregnant sheep on days 10, 14, and 18, except on day 16(P < 0.01). Enhancing MUC1 protein expression with oestrogen or/and progesterone decreased the blastocyst adhesion rate when blastocysts were co-cultured with endometrial epithelial cells (EECs), while inhibiting MUC1 protein expression with IFN-τ increased the blastocyst adhesion rate when the blastocysts were co-cultured with EECs. Compared with the ewes undergoing natural oestrus, the expression trend and regulation of MUC1 did not change, and the MUC1 expression levels only increased under artificial oestrus conditions. Our data provide important information for improving the conception rate in sheep undergoing artificially-induced oestrus and offer some reference points relating to embryo transfer, oestrus synchronisation and superovulation. 10.1016/j.theriogenology.2017.12.030
    Progesterone effects on extracellular vesicles in the sheep uterus. Burns Gregory W,Brooks Kelsey E,O'Neil Eleanore V,Hagen Darren E,Behura Susanta K,Spencer Thomas E Biology of reproduction Progesterone (P4) acts via the endometrium to promote conceptus growth and implantation for pregnancy establishment. Many cells release extracellular vesicles (EVs) that are membrane-bound vesicles of endosomal and plasma membrane origin. In sheep, endometrial-derived EVs were found to traffic to the conceptus trophectoderm. Thus, EVs are hypothesized to be an important mode of intercellular communication by transferring select RNAs, proteins, and lipids between the endometrium and conceptus. Electron microscopy analysis found that the endometrial luminal and glandular epithelia were the primary source of EVs in the uterus of cyclic sheep. Size exclusion chromatography and nanoparticle tracking analysis (NTA) found that total EV number in the uterine lumen increased from day 10 to 14 in cyclic sheep. Next, ewes were ovariectomized and hormone replaced to determine effects of P4 on the endometrium and EVs in the uterine lumen. Transcriptome analyses found that P4 regulated 1611 genes and nine miRNAs in the endometrium. Total EV number in the uterine lumen was increased by P4 treatment. Small RNA sequencing of EVs detected expression of 768 miRNAs and determined that P4 regulated seven of those miRNAs. These studies provide fundamental new information on how P4 influences endometrial function to regulate conceptus growth for pregnancy establishment in sheep. 10.1093/biolre/ioy011
    Follicle stimulating hormone receptor protein is expressed in ovine uterus during the estrous cycle and utero-placenta during early pregnancy: An immunohistochemical study. Grazul-Bilska Anna T,Reyaz Arshi,Valkov Veselina,Dorsam Sheri T,Redmer Dale A Acta histochemica Follicle stimulating hormone (FSH) is a well characterized gonadotropin that controls primarily development and functions of ovarian follicles in mammalian species. FSH binds to a specific G protein-coupled receptor (FSHR) belonging to the glycoprotein hormone receptor family that plays an essential role in reproduction. Although the primary location of FSHR is in the gonads (mainly in ovarian follicles), FSHR protein and/or mRNA have also been detected in extragonadal female reproductive tissues including embryo, placenta, endometrium, cervix, ovarian cancer tissues, and/or endometriotic lesions in several species. To determine the pattern of FSHR expression in the uterus and placenta, uterine tissues were collected at the early, mid- and/or late luteal phases of the estrous cycle from non-treated or FSH-treated ewes, and utero-placental tissues were collected during early pregnancy followed by immunohistochemistry and image generation. FSHR was immunolocalized to several uterine and utero-placental compartments including luminal epithelium, endometrial glands and surrounding stroma, myometrium, and endothelium and vascular smooth muscle cells in endometrium, myometrium and mesometrium. Intensity of staining and distribution of FSHR in selected compartments differed and seems to depend on the stage of the estrous cycle or pregnancy, and FSH-treatment. These novel data demonstrate differential expression of FSHR protein indicating that FSH plays a specific role in regulation of uterine and utero-placenta functions in sheep. 10.1016/j.acthis.2018.05.005
    Changes in expression of ISG15, progesterone receptor and progesterone-induced blocking factor in ovine thymus during early pregnancy. Zhang Leying,Xue Jie,Wang Qingkai,Lv Wan,Mi Hao,Liu Yong,Yang Ling Theriogenology Interferon-tau (IFNT) is the main signal for the maternal recognition of pregnancy in ruminants, and exerts its effects by stimulating the expression of interferon-stimulated genes, including the expression of interferon-stimulated gene15 kDa protein (ISG15). Progesterone (P4) exerts significant immune effects on the uterus during early pregnancy in ruminants that are partly mediated by progesterone-induced blocking factor (PIBF). The thymus is necessary for the normal development of immunologic function. In this study, thymuses were obtained on day 16 of the estrous cycle and on days 13, 16 and 25 of pregnancy (n = 6 for each group) from ewes. Our results showed that the expression of ISG15, P4 receptor (PGR) and PIBF mRNA and the expression of ISG15 and ISG15-conjugated proteins were upregulated in the thymuses during early pregnancy, and the 89-kDa PGR isoform and the 80-kDa PIBF variant were expressed constantly in the thymuses. However, there was no expression of the 60-kDa PGR isoform and the 62-kDa PIBF variant on day 16 of the estrous cycle. ISG15 and ISG15-conjugated proteins were limited to the epithelial reticular cells, capillaries and thymic corpuscles. This paper reports for the first time that early pregnancy exerts its effects on the thymus through IFNT and P4 in sheep. 10.1016/j.theriogenology.2018.08.018
    Development and Function of Uterine Glands in Domestic Animals. Spencer Thomas E,Kelleher Andrew M,Bartol Frank F Annual review of animal biosciences All mammalian uteri contain glands that synthesize or transport and secrete substances into the uterine lumen. Uterine gland development, or adenogenesis, is uniquely a postnatal event in sheep and pigs and involves differentiation of glandular epithelium from luminal epithelium, followed by invagination and coiling morphogenesis throughout the stroma. Intrinsic transcription factors and extrinsic factors from the ovary and pituitary as well as the mammary gland (lactocrine) regulate uterine adenogenesis. Recurrent pregnancy loss is observed in the ovine uterine gland knockout sheep, providing unequivocal evidence that glands and their products are essential for fertility. Uterine gland hyperplasia and hypertrophy during pregnancy are controlled by sequential actions of hormones from the ovary and/or pituitary as well as the placenta. Gland-derived histotroph is transported by placental areolae for fetal growth. Increased knowledge of uterine gland biology is expected to improve pregnancy outcomes, as well as the health and productivity of mothers and their offspring. 10.1146/annurev-animal-020518-115321
    Expression profiles of interferon-stimulated gene 15 and prostaglandin synthases in the ovine lymph nodes during early pregnancy. Yang Ling,Wang Qingkai,Liu Yong,Zhang Leying,Lv Wan,Liu Baoliang Molecular reproduction and development Lymph nodes are distributed all over the body and are part of the lymphatic system. The interferon-stimulated gene 15 kDa protein (ISG15) and prostaglandins (PGs) are involved in the establishment of pregnancy and are expressed in the uterus during early pregnancy in sheep. In this study, the ovine lymph nodes were obtained on Day 16 of the estrous cycle, and Days 13, 16, and 25 of pregnancy, and the expression of ISG15 and PG synthases, including cyclooxygenase 1 (COX-1), COX-2, prostaglandin E (PGE) synthase (PTGES), and a PGF synthase (aldo-keto reductase family 1, member B1, AKR1B1) were detected by quantitative real-time polymerase chain reaction, western blot analysis, and immunohistochemistry analysis. Our results showed that there were peaks in the expression of mRNAs and the proteins of ISG15, COX-1, COX-2, PTGES, and AKR1B1 in the lymph nodes during early pregnancy and that the COX-2 and AKR1B1 proteins were limited to the subcapsular sinus and lymph sinuses. In conclusion, the ISG15, COX-1, COX-2, PTGES, and AKR1B1 were upregulated in the maternal lymph nodes, which may be beneficial for the development of conceptus, maternal systemic immunoregulation, and anti-luteolysis during early pregnancy in sheep. 10.1002/mrd.23085
    New functions for old factors: the role of polyamines during the establishment of pregnancy. Fenelon Jane C,Murphy Bruce D Reproduction, fertility, and development Implantation is essential for the establishment of a successful pregnancy, and the preimplantation period plays a significant role in ensuring implantation occurs in a timely and coordinated manner. This requires effective maternal-embryonic signalling, established during the preimplantation period, to synchronise development. Although multiple factors have been identified as present during this time, the exact molecular mechanisms involved are unknown. Polyamines are small cationic molecules that are ubiquitously expressed from prokaryotes to eukaryotes. Despite being first identified over 300 years ago, their essential roles in cell proliferation and growth, including cancer, have only been recently recognised, with new technologies and interest resulting in rapid expansion of the polyamine field. This review provides a summary of our current understanding of polyamine synthesis, regulation and function with a focus on recent developments demonstrating the requirements for polyamines during the establishment of pregnancy up to the implantation stage, in particular the role of polyamines in the control of embryonic diapause and the identification of an alternative pathway for their synthesis in sheep pregnancy. This, along with other novel discoveries, provides new insights into the control of the peri-implantation period in mammals and highlights the complexities that exist in regulating this critical period of pregnancy. 10.1071/RD18235
    Comparative Transcriptomics Reveal Key Sheep (Ovis aries) Hypothalamus LncRNAs that Affect Reproduction. Zhang Zhuangbiao,Tang Jishun,Di Ran,Liu Qiuyue,Wang Xiangyu,Gan Shangquan,Zhang Xiaosheng,Zhang Jinlong,Hu Wenping,Chu Mingxing Animals : an open access journal from MDPI The diverse functions of long noncoding RNAs (lncRNAs), which execute their functions mainly through modulating the activities of their target genes, have been have been widely studied for many years (including a number of studies involving lncRNAs in the ovary and uterus). Herein, for the first time, we detect lncRNAs in sheep hypothalami with FecB++ through RNA Sequencing (RNA-Seq) and identify a number of known and novel lncRNAs, with 622 and 809 found to be differentially expressed in polytocous sheep in the follicular phase (PF) vs. monotocous sheep in the follicular phase (MF) and polytocous sheep in the luteal phase (PL) vs. monotocous sheep in the luteal phase (ML), respectively. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed based on the predicted target genes. The most highly enriched GO terms (at the molecular function level) included carbonyl reductase (NADPH), 15-hydroxyprostaglandin dehydrogenase (NADP+), and prostaglandin-E2 9-reductase activity in PF vs. MF, and phosphatidylinositol-3,5-bisphosphate binding in PL vs. ML was associated with sheep fecundity. Interestingly, the phenomena of valine, leucine, and isoleucine degradation in PL vs. ML, and valine, leucine, and isoleucine biosynthesis in PF vs. MF, were present. In addition, the interactome of lncRNA and its targets showed that MSTRG.26777 and its targets ENSOARG00000013744, ENSOARG00000013700, and ENSOARG00000013777, and MSTRG.105228 and its target WNT7A may participate in the sheep reproductive process at the hypothalamus level. Significantly, MSTRG.95128 and its target Forkhead box L1 (FOXG1) were shown to be upregulated in PF vs. MF but downregulated in PL vs. ML. All of these results may be attributed to discoveries of new candidate genes and pathways related to sheep reproduction, and they may provide new views for understanding sheep reproduction without the effects of the mutation. 10.3390/ani9040152
    CXCR4 signaling at the ovine fetal-maternal interface regulates vascularization, CD34+ cell presence, and autophagy in the endometrium†. Biology of reproduction Placenta development is characterized by extensive angiogenesis and vascularization but if these processes are compromised placental dysfunction occurs, which is the underlying cause of pregnancy complications such as preeclampsia and intrauterine growth restriction. Dysregulation of placental angiogenesis has emerged as one of the main pathophysiological features in the development of placental insufficiency and its clinical consequences. The signaling axis initiated by chemokine ligand 12 (CXCL12) and its receptor CXCR4 stimulates angiogenesis in other tissues, and may be central to placental vascularization. We hypothesized that CXCL12-CXCR4 signaling governs the pro-angiogenic placental microenvironment by coordinating production of central angiogenic factors and receptors and regulates endometrial cell survival essential for placental function and subsequent fetal longevity. The CXCR4 antagonist, AMD3100, was used to elucidate the role of CXCL12-CXCR4 signaling regarding uteroplacental vascular remodeling at the fetal-maternal interface. On day 12 postbreeding, osmotic pumps were surgically installed and delivered either AMD3100 or PBS into the uterine lumen ipsilateral to the corpus luteum. On day 20, endometrial tissues were collected, snap-frozen in liquid nitrogen, and uterine horn cross sections preserved for immunofluorescent analysis. In endometrium from ewes receiving AMD3100 infusion, the abundance of select angiogenic factors was diminished, while presence of CD34+ cells increased compared to control ewes. Ewes receiving AMD3100 infusion also exhibited less activation of Akt/mTOR signaling, and elevated LC3B-II, a marker of cellular autophagy in endometrium. This study suggests that CXCL12-CXCR4 signaling governs placental homeostasis by serving as a critical upstream mediator of vascularization and cell viability, thereby ensuring appropriate placental development. 10.1093/biolre/ioz073
    Uterine Glands: Developmental Biology and Functional Roles in Pregnancy. Kelleher Andrew M,DeMayo Francesco J,Spencer Thomas E Endocrine reviews All mammalian uteri contain glands in the endometrium that develop only or primarily after birth. Gland development or adenogenesis in the postnatal uterus is intrinsically regulated by proliferation, cell-cell interactions, growth factors and their inhibitors, as well as transcription factors, including forkhead box A2 (FOXA2) and estrogen receptor α (ESR1). Extrinsic factors regulating adenogenesis originate from other organs, including the ovary, pituitary, and mammary gland. The infertility and recurrent pregnancy loss observed in uterine gland knockout sheep and mouse models support a primary role for secretions and products of the glands in pregnancy success. Recent studies in mice revealed that uterine glandular epithelia govern postimplantation pregnancy establishment through effects on stromal cell decidualization and placental development. In humans, uterine glands and, by inference, their secretions and products are hypothesized to be critical for blastocyst survival and implantation as well as embryo and placental development during the first trimester before the onset of fetal-maternal circulation. A variety of hormones and other factors from the ovary, placenta, and stromal cells impact secretory function of the uterine glands during pregnancy. This review summarizes new information related to the developmental biology of uterine glands and discusses novel perspectives on their functional roles in pregnancy establishment and success. 10.1210/er.2018-00281
    Immunohistochemical identification of resistin in the uterus of ewes subjected to different diets: Preliminary results. Dall'Aglio Cecilia,Scocco Paola,Maranesi Margherita,Petrucci Linda,Acuti Gabriele,De Felice Elena,Mercati Francesca European journal of histochemistry : EJH Resistin is a polypeptide hormone of the adipokine-family, primarily, but not exclusively, produced by the adipose tissue. Recent studies suggested that resistin may affect the male and female reproductive activity. The study aim was to immunohistochemically evaluate the presence and distribution of resistin in the ovine uterus. Uterine samples were collected from two groups of ewes at the end of an experimental trial during which the animals of the first group (CTRL) were fed only by grazing while those of the second one (EXP) were supplemented with barley and corn. Using a monoclonal antibody against resistin, tested by Western Blot, the immunopositive reaction was identified in the cytoplasm of epithelial lining cells and uterine glands. The endogenous production of resistin seemed to be affected by different diet, as evidenced by staining differences between the CTRL and EXP groups. Our findings support the existence of a peripheral resistin system in the sheep uterus. It is possible that this system is involved in the functionality of the uterus, which is also affected by the animal's nutritional status. 10.4081/ejh.2019.3020
    Expression of oestrogen receptor, androgen receptor and progesterone nuclear receptor in sheep uterus during the oestrous cycle. Duan Hongwei,Xiao Longfei,Hu Junjie,Zhang Yong,Zhao Xingxu,Ge Wenbo,Jiang Yuting,Song Liangli,Yang Shanshan,Luo Wenze Reproduction in domestic animals = Zuchthygiene Oestrogen, androgen and progesterone are involved in the regulation of uterine physiological functions, with the participation of the following proteins: oestrogen receptor (ER), androgen receptor (AR) and progesterone nuclear receptor (PGR). In this study, we used immunohistochemistry to detect the localization of ERα, ERβ, AR and PGR in sheep uterus. Additionally, we used real-time polymerase chain reaction (RT-qPCR) and Western blot technique to analyse their expression profiles at different stages of sheep oestrous cycle in the endometrium and myometrium. Immunohistochemical analysis showed that ERα, ERβ, AR and PGR were present in sheep uterus in oestrus, mainly in the uterine luminal epithelium, stroma, gland and myometrium. Real-time polymerase chain reaction results showed that in the endometrium, ERα expression level was highest in oestrus. ERβ and PGR, instead, were highly expressed in pro-oestrus. In the myometrium, ERα was highly expressed in both oestrus and pro-oestrus, and ERβ was highly expressed in oestrus and dioestrus. Progesterone nuclear receptor expression was highest in oestrus, followed by metoestrus. In the endometrium, both receptors ERα and ERβ were abundant in pro-oestrus, while the maximum AR protein content was found in oestrus. At this stage of the oestrous cycle, PGR protein concentration in the myometrium was significantly lower than those observed in other stages. These results suggest that these receptors are important for sheep reproductive function, as their expression at mRNA and protein levels exhibits particular time- and tissue-specific profiles along the oestrous cycle. 10.1111/rda.13489
    Identification and characterization of mRNAs and lncRNAs in the uterus of polytocous and monotocous Small Tail Han sheep (). PeerJ BACKGROUND:Long non-coding RNAs (lncRNAs) regulate endometrial secretion and uterine volume. However, there is little research on the role of lncRNAs in the uterus of Small Tail Han sheep (++). Herein, RNA-seq was used to comparatively analyze gene expression profiles of uterine tissue between polytocous and monotocous sheep (++) in follicular and luteal phases. METHODS:To identify lncRNA and mRNA expressed in the uterus, the expression of lncRNA and mRNA in the uterus of Small Tail Han sheep (++) from the polytocous group ( = 6) and the monotocous group ( = 6) using RNA-sequencing and real-time polymerase chain reaction (RT-PCR). Identification of differentially expressed lncRNAs and mRNAs were performed between the two groups and two phases . Gene ontology (GO) and pathway enrichment analyses were performed to analyze the biological functions and pathways for the differentially expressed mRNAs. LncRNA-mRNA co-expression network was constructed to further analyses the function of related genes. RESULTS:In the follicular phase, 473 lncRNAs and 166 mRNAs were differentially expressed in polytocous and monotocous sheep; in the luteal phase, 967 lncRNAs and 505 mRNAs were differentially expressed in polytocous and monotocous sheep. GO and KEGG enrichment analysis showed that the differentially expressed lncRNAs and their target genes are mainly involved in ovarian steroidogenesis, retinol metabolism, the oxytocin signaling pathway, steroid hormone biosynthesis, and the Foxo signaling pathway. Key lncRNAs may regulate reproduction by regulating genes involved in these signaling pathways and biological processes. Specifically, , , , , and , which are targeted by ., , , , and , may play key regulatory roles. These results offer insight into molecular mechanisms underlying sheep prolificacy. 10.7717/peerj.6938
    Effects of l-arginine on endometrial estrogen receptor α/β and progesterone receptor expression in nutrient-restricted sheep. Gao Xiao-Xiao,Zhang Qi-Fan,Zhu Ming,Li Xiao-He,Guo Yi-Xuan,Pang Jing,Liu Zi-Fei,You Pei-Hua,Zhang Guo-Min,Wang Feng Theriogenology This study aimed to determine the effects of l-arginine (L-Arg) supplementation on steroid hormone receptors in non-pregnant ovine endometrium. All experimental ewes were randomly assigned to either a control group (n = 6), a nutrient-restricted group (n = 6), or an L-Arg supplemented nutrient-restricted group (n = 6). The effects of L-Arg on estrogen receptor α/β (ERα/β) and progesterone receptor (PGR) expression in the ovine endometrium were assessed. Our results showed that levels of ERβ and PGR expression were significantly increased by nutrient restriction, but L-Arg counteracted the effect of nutrient restriction on ERβ and PGR expression (p < 0.05). Also, expression of endometrial ERα was substantially increased (p < 0.05) by L-Arg supplementation. Furthermore, ERα/β and PGR were mainly detected in the endometrial luminal epithelium and glandular epithelium. Therefore, we isolated and identified endometrial epithelial cells (EECs) from sheep. Different concentrations of L-Arg were added to investigate the effects on ERα/β and PGR in EECs. The expression levels of endothelial nitric oxide synthase, ERβ, and PGR were significantly increased in response to low-concentration (200 μmol) L-Arg supplementation, which subsequently decreased with a high concentration (800 μmol) (p < 0.05). Otherwise, ERα expression was remarkably increased at both L-Arg concentrations in EECs (p < 0.05). Overall, the results indicated that L-Arg performed crucial roles in the regulation of ovine steroid hormone receptor expression in the endometrium. The results of this study provide a theoretical basis and technical means for the normal function of endometrium in response to low nutrient levels. 10.1016/j.theriogenology.2019.07.018
    Intrauterine inhibition of chemokine receptor 4 signaling modulates local and systemic inflammation in ovine pregnancy. American journal of reproductive immunology (New York, N.Y. : 1989) PROBLEM:Chemokines help coordinate inflammation within the fetal-maternal microenvironment during gestation. The chemokine CXCL12 signaling through its receptor CXCR4 regulates inflammatory activity, but this phenomenon is not well understood during pregnancy, and there are no reports exploring the role of this pair in peripheral immune tolerance during gestation. Herein, we hypothesize that intrauterine CXCL12-CXCR4 signaling governs local and systemic immunomodulatory dynamics during early gestation in ewes. METHOD OF STUDY:Osmotic pumps were surgically installed for intrauterine infusion of a CXCR4 inhibitor, AMD3100, beginning on day 12 post-breeding in sheep. Endometrial tissues were collected on day 35 of gestation and evaluated for inflammatory potential, Akt pathway activation, and autophagy induction. Demonstrative of peripheral immune activity, levels of select cytokines were assessed in daily blood samples collected throughout the study, as well as in corpus luteum and spleen on day 35. RESULTS:Anti-inflammatory IL10 was primarily localized to endometrial glandular epithelium with lower abundance when CXCR4 was antagonized. Inhibition of CXCR4 at the fetal-maternal interface resulted in less activation of Akt in endometrium, while evidence of autophagy induction was greater. Corpora lutea from ewes receiving intrauterine AMD3100 exhibited lower interferon-gamma (IFNG) expression. Blood inflammatory potential was differentially altered in a temporal fashion throughout infusion. IL10 abundance in spleen was greater following CXCR4 inhibition at the fetal-maternal interface, while IFNG was less. CONCLUSION:Intrauterine CXCL12-CXCR4 signaling governs endometrial and systemic inflammation; disruption of this axis may have detrimental impacts on offspring and maternal health. 10.1111/aji.13181
    Cloning and expression analysis of the follicle-stimulating hormone receptor (FSHR) gene in the reproductive axis of female yaks (Bos grunniens). Xia Y,Wang Q,He X D,Chen Y,JiGe M T,Zi X D Domestic animal endocrinology Follicle-stimulating hormone receptor (FSHR) plays a central role in promoting follicle maturation through the follicle-stimulating hormone (FSH)-mediated cAMP pathway in animals. The objectives of the present study were to clone the FSHR gene of yaks (Bos grunniens) and compare differences in FSHR mRNA expression in the reproductive axis between yaks and cattle. Hypothalamus, anterior pituitary, oviduct, ovary, and uterus tissue samples were collected from adult female yaks (n = 5) and cattle (n = 5) during the follicular phase. Using reverse transcriptase-polymerase chain reaction (RT-PCR), we found that the FSHR coding region of the yak is 2088 bp and encodes 695 amino acids. Its amino acid sequence showed 99.38%-72.22% similarity to the homologous genes of cattle, goats, sheep, cats, donkeys, horses, humans, chickens, monkeys, mice, rats, and wild boar. Real-time PCR analysis revealed that the FSHR gene was expressed in all tissues examined. Expression of the FSHR gene in the yak was higher in the uterus than other tissues (P < 0.05) but, in cattle, was higher in the ovary than other tissues (P < 0.05). The FSHR gene expression level in the cattle ovary was significantly higher than that in the yak ovary (P < 0.01). These results indicate that the FSHR gene is relatively conserved in the course of animal evolution. The variation in sequence and expression level of FSHR between the two species might be associated with the difference in their reproduction. 10.1016/j.domaniend.2019.07.011
    Interferon-Tau Exerts Direct Prosurvival and Antiapoptotic Actions in Luteinized Bovine Granulosa Cells. Basavaraja Raghavendra,Madusanka Senasige Thilina,Drum Jessica N,Shrestha Ketan,Farberov Svetlana,Wiltbank Milo C,Sartori Roberto,Meidan Rina Scientific reports Interferon-tau (IFNT), serves as a signal to maintain the corpus luteum (CL) during early pregnancy in domestic ruminants. We investigated here whether IFNT directly affects the function of luteinized bovine granulosa cells (LGCs), a model for large-luteal cells. Recombinant ovine IFNT (roIFNT) induced the IFN-stimulated genes (ISGs; MX2, ISG15, and OAS1Y). IFNT induced a rapid and transient (15-45 min) phosphorylation of STAT1, while total STAT1 protein was higher only after 24 h. IFNT treatment elevated viable LGCs numbers and decreased dead/apoptotic cell counts. Consistent with these effects on cell viability, IFNT upregulated cell survival proteins (MCL1, BCL-xL, and XIAP) and reduced the levels of gamma-H2AX, cleaved caspase-3, and thrombospondin-2 (THBS2) implicated in apoptosis. Notably, IFNT reversed the actions of THBS1 on cell viability, XIAP, and cleaved caspase-3. Furthermore, roIFNT stimulated proangiogenic genes, including FGF2, PDGFB, and PDGFAR. Corroborating the in vitro observations, CL collected from day 18 pregnant cows comprised higher ISGs together with elevated FGF2, PDGFB, and XIAP, compared with CL derived from day 18 cyclic cows. This study reveals that IFNT activates diverse pathways in LGCs, promoting survival and blood vessel stabilization while suppressing cell death signals. These mechanisms might contribute to CL maintenance during early pregnancy. 10.1038/s41598-019-51152-6
    Proteomic analysis of sheep uterus reveals its role in prolificacy. La Yongfu,Tang Jishun,Guo Xiaofei,Zhang Liping,Gan Shangquan,Zhang Xiaosheng,Zhang Jinlong,Hu Wenping,Chu Mingxing Journal of proteomics Small Tail Han sheep have attracted attention for their high fecundity and year-round estrus. However, the molecular mechanisms of this fecundity are unknown. Polymorphism of the FecB gene has been shown to be associated with the ovulation rate and litter size in sheep. In this study, we used tandem mass tag quantitative proteomic techniques to identify the differentially abundant proteins in polytocous and monotocous Small Tail Han sheep (FecB++) uterine tissues in the follicular and luteal phases. In total, 41 and 43 differentially abundant proteins were identified in the follicular and luteal phases, respectively. Correlation analysis between the transcriptome and proteome revealed a positive correlation at the two omics levels of prolificacy. GO and KEGG pathway analyses indicated that the mRNAs and proteins upregulated in the polytocous group relative to the monotocous group are involved in sphingolipid metabolism and amino acid metabolism, and may be important in maintaining uterine functions and increasing the embryo survival rate during the estrus cycle of polytocous sheep. In conclusion, our work provides a prospective understanding of the molecular mechanism underlying the high prolificacy of Small Tail Han sheep. SIGNIFICANCE: Fecundity critically affects the profitability of sheep production, but the genetic mechanism of high-prolificacy is still unclear in sheep. We identified potential signaling pathways and differentially abundant proteins associated with reproductive performance through a combination of sheep uterus tissues proteome and transcriptome analyses. These findings will facilitate a better revealing the mechanism and provide possible targets for molecular design breeding for the formation of polytocous traits in sheep. 10.1016/j.jprot.2019.103526
    Differential Expression of Circular RNAs in Polytocous and Monotocous Uterus during the Reproductive Cycle of Sheep. La Yongfu,Tang Jishun,Di Ran,Wang Xiangyu,Liu Qiuyue,Zhang Liping,Zhang Xiaosheng,Zhang Jinlong,Hu Wenping,Chu Mingxing Animals : an open access journal from MDPI CircRNA plays important roles in cell proliferation, differentiation, autophagy and apoptosis during development. However, there are few reports on circRNAs related to livestock reproduction. In this study, we identified circRNAs by deep sequencing and analyzed their expression in the uteri of polytocous and monotocous sheep (FecB++) during follicular and luteal phases. There were 147 and 364 circRNAs with differential expression in the follicular and luteal phases, respectively. GO and KEGG enrichment analysis was performed for the host genes of the circRNAs to predict the functions of differentially expressed circRNAs. These source genes were mainly involved in the estrogen signaling pathway, TGFβ signaling pathway, GnRH signaling pathway, oxytocin signaling pathway, pentose phosphate pathway, and starch and sucrose metabolism related to reproduction and energy metabolism. CircRNA expression patterns were validated by RT-qPCR. Our findings provide a solid foundation for the identification and characterization of key important circRNAs involved in reproduction. 10.3390/ani9100797
    Molecular characterization and expression profile of interferon-stimulated gene 15 (ISG15) in the endometrium of goat (Capra hircus). Chandrakar K,Jain A,Khan J R,Jain T,Singh M,Mishra O P Theriogenology Interferon-stimulated gene 15 (ISG15), a ubiquitin-like protein, is responsible for uterine receptivity, implantation and conceptus development in different ruminant species, but in goat (Capra hircus) its role is yet to be explicated. In the present study, the ISG15 gene was cloned, characterized and its temporal expression profile was examined in the endometrium of caprine (cp). A fragment of cpISG15 gene, 1033 bp in length, was amplified, cloned and sequenced from genomic DNA covering the coding region. Sequence analysis of cpISG15 gene revealed that it was comprised of two exons of 59 bp and 496 bp encoding a peptide of 157 amino acids. Complementary DNA (cDNA) and deduced amino acid sequences of cpISG15 exhibited 99 and 98, 93 and 88, 94 and 89, 76 and 66, and 73 and 62% identity with that of sheep, cattle, buffalo, human and mice, respectively. Further, relative expression of cpISG15 mRNA and protein was determined by quantitative real-time PCR (qPCR) and Western blot, respectively, in the endometrium of pregnant and cyclic does. Both cpISG15 mRNA and protein were expressed maximally (P < 0.05) in the endometrium during early stage of pregnancy (16-24 d) as compared to cyclic does, but no significant difference was observed in cpISG15 mRNA and protein expression in the endometrium between the later stage of pregnancy (25-40 d) and cyclic does. It is concluded that cpISG15 is almost similar in structure and probably in function also to other species as it has been found significantly upregulated during early pregnancy. 10.1016/j.theriogenology.2019.10.013
    Prostaglandin-endoperoxide synthase 2 is not required for preimplantation ovine conceptus development in sheep. O'Neil Eleanore V,Brooks Kelsey,Burns Gregory W,Ortega Martha S,Denicol Anna C,Aguiar Luis H,Pedroza Gabriela H,Benne Joshua,Spencer Thomas E Molecular reproduction and development Conceptus development and elongation is required for successful pregnancy establishment in ruminants and is coincident with the production of interferon τ (IFNT) and prostaglandins (PGs). In both the conceptus trophectoderm and endometrium, PGs are primarily synthesized through a prostaglandin-endoperoxide synthase 2 (PTGS2) pathway and modify endometrial gene expression and thus histotroph composition in the uterine lumen to promote conceptus growth and survival. Chemical inhibition of PG production by both the endometrium and the conceptus prevented elongation in sheep. However, the contributions of conceptus-derived PGs to preimplantation conceptus development remain unclear. In this study, CRISPR-Cas9 genome editing was used to inactivate PTGS2 in ovine embryos to determine the role of PTGS2-derived PGs in conceptus development and elongation. PTGS2 edited conceptuses produced fewer PGs, but secreted similar amounts of IFNT to their Cas9 control counterparts and elongated normally. Expression of PTGS1 was lower in PTGS2 edited conceptuses, but PPARG expression and IFNT secretion were unaffected. Content of PGs in the uterine lumen was similar as was gene expression in the endometrium of ewes who received either Cas9 control or PTGS2 edited conceptuses. These results support the idea that intrinsic PTGS2-derived PGs are not required for preimplantation embryo or conceptus survival and development in sheep. 10.1002/mrd.23300
    Contribution of the immune system to follicle differentiation, ovulation and early corpus luteum formation. Abdulrahman Noof,Fair Trudee Animal reproduction Much of what we know about the involvement of the immune system in periovulatory follicle differentiation, ovulation and subsequent formation of the corpus luteum in cattle is drawn from the findings of studies in several mammalian livestock species. By integrating published histological data from cattle, sheep and pigs and referring back to the more comprehensive knowledge bank that exists for mouse and humans we can sketch out the key cells of the immune system and the cytokines and growth factors that they produce that are involved in follicle differentiation and luteinization, ovulation and early follicle development. These contributions are reviewed and the key findings, discussed. 10.21451/1984-3143-AR2019-0087
    Lineage Differentiation Markers as a Proxy for Embryo Viability in Farm Ungulates. Pérez-Gómez Alba,González-Brusi Leopoldo,Bermejo-Álvarez Pablo,Ramos-Ibeas Priscila Frontiers in veterinary science Embryonic losses constitute a major burden for reproductive efficiency of farm animals. Pregnancy losses in ungulate species, which include cattle, pigs, sheep and goats, majorly occur during the second week of gestation, when the embryo experiences a series of cell differentiation, proliferation, and migration processes encompassed under the term conceptus elongation. Conceptus elongation takes place following blastocyst hatching and involves a massive proliferation of the extraembryonic membranes trophoblast and hypoblast, and the formation of flat embryonic disc derived from the epiblast, which ultimately gastrulates generating the three germ layers. This process occurs prior to implantation and it is exclusive from ungulates, as embryos from other mammalian species such as rodents or humans implant right after hatching. The critical differences in embryo development between ungulates and mice, the most studied mammalian model, have precluded the identification of the genes governing lineage differentiation in livestock species. Furthermore, conceptus elongation has not been recapitulated , hindering the study of these cellular events. Luckily, recent advances on transcriptomics, genome modification and post-hatching culture are shedding light into this largely unknown developmental window, uncovering possible molecular markers to determine embryo quality. In this review, we summarize the events occurring during ungulate pre-implantation development, highlighting recent findings which reveal that several dogmas in Developmental Biology established by knock-out murine models do not hold true for other mammals, including humans and farm animals. The developmental failures associated to produced embryos in farm animals are also discussed together with Developmental Biology tools to assess embryo quality, including molecular markers to assess proper lineage commitment and a post-hatching culture system able to directly determine developmental potential circumventing the need of experimental animals. 10.3389/fvets.2021.680539
    Maternal organism and embryo biosensoring: insights from ruminants. Sandra Olivier,Constant Fabienne,Vitorino Carvalho Anais,Eozénou Caroline,Valour Damien,Mauffré Vincent,Hue Isabelle,Charpigny Gilles Journal of reproductive immunology In terms of contribution to pregnancy, the mother not only produces gametes, but also hosts gestation, whose progression in the uterus is conditioned by early events during implantation. In ruminants, this period is associated with elongation of the extra-embryonic tissues, gastrulation of the embryonic disk and cross-talk with the endometrium. Recent data have prompted the need for accurate staging of the bovine conceptus and shown that asynchrony between elongation and gastrulation processes may account for pregnancy failure. Data mining of endometrial gene signatures has allowed the identification of molecular pathways and new factors regulated by the conceptus (e.g. FOXL2, SOCS6). Interferon-tau has been recognised to be the major signal of pregnancy recognition, but prostaglandins and lysophospholipids have also been demonstrated to be critical players at the conceptus-endometrium interface. Interestingly, up-regulation of interferon-regulated gene expression has been identified in circulating immune cells during implantation, making these factors a potential source of non-invasive biomarkers for early pregnancy. Distinct endometrial responses have been shown to be elicited by embryos produced by artificial insemination, in vitro fertilisation or somatic cell nuclear transfer. These findings have led to the concept that endometrium is an early biosensor of embryo quality. This biological property first demonstrated in cattle has been recently extended and associated with embryo selection in humans. Hence, compromised or suboptimal endometrial quality can subtly or deeply affect embryo development, with visible and sometimes severe consequences for placentation, foetal development, pregnancy outcome and the long-term health of the offspring. 10.1016/j.jri.2014.12.005
    A review of uterine structural modifications that influence conceptus implantation and development in sheep and goats. Igwebuike U M Animal reproduction science Evolution of the placenta and viviparity in eutherian animals underscores the need for an intimate relationship between the developing conceptus (embryo/foetus and associated extra-embryonic membranes) and the dam throughout the period of pregnancy. Thus, maternal support is unequivocally important for conceptus survival and development in utero. Under the influence of several pregnancy-associated hormones, the maternal uterine architecture undergoes rapid growth and substantial remodeling early in gestation. These changes are necessary preparations to accommodate and support rapid conceptus development and growth in the later two-thirds of pregnancy. There are species variations in the nature and extent of uterine remodeling during pregnancy. The regulatory influence of these uterine wall modifications on conceptus survival, implantation and placentation in sheep and goats are discussed in this review. 10.1016/j.anireprosci.2008.12.010
    Zearalenone Blocks Autophagy Flow and Induces Cell Apoptosis During Embryo Implantation in Gilts. Wu Lihang,Duan Qianni,Gao Dengying,Wang Yueying,Xue Songyi,Li Wenchao,Lei Minggang Toxicological sciences : an official journal of the Society of Toxicology Zearalenone (ZEA) has been proved to be toxic, particularly to the reproductive system of gilts. The effect of ZEA on gilts during embryo implantation window period is of particular interests. Here, we observed window stage dysontogenesis of gilts treated with ZEA. In endometrial tissues and cells, autophagosomes increased significantly and mitochondria were damaged with increasing ZEA concentration. Addition of autophagy inhibitor confirmed that ZEA blocks the autophagic flow in the fusion of autophagosomes and lysosomes. In conclusion, ZEA exposure during embryo implantation results in endometrium inflammation by activating autophagy while blocking autophagy flow at the same time, leading to the significant accumulation of autophagosomes. The aforementioned effects of ZEA induce the apoptosis of primary endometrial cells through the caspase3 pathway, which would break the uterus environment balance and finally lead to embryo implantation failure and dysontogenesis in gilts. 10.1093/toxsci/kfaa018
    MicroRNAs in Small Extracellular Vesicles Indicate Successful Embryo Implantation during Early Pregnancy. Tan Qiang,Shi Shuang,Liang Jingjie,Zhang Xiaowei,Cao Dingren,Wang Zhengguang Cells Synchronous communication between the developing embryo and the receptive endometrium is crucial for embryo implantation. Thus, uterine receptivity evaluation is vital in managing recurrent implantation failure (RIF). The potential roles of small extracellular vesicle (sEV) miRNAs in pregnancy have been widely studied. However, the systematic study of sEVs derived from endometrium and its cargos during the implantation stage have not yet been reported. In this study, we isolated endometrium-derived sEVs from the mouse endometrium on D2 (pre-receptive phase), D4 (receptive phase), and D5 (implantation) of pregnancy. Herein, we reveal that multivesicular bodies (MVBs) in the endometrium increase in number during the window of implantation (WOI). Moreover, our findings indicate that CD63, a well-known sEV marker, is expressed in the luminal and glandular epithelium of mouse endometrium. The sEV miRNA expression profiles indicated that miR-34c-5p, miR-210, miR-369-5p, miR-30b, and miR-582-5p are enriched during WOI. Further, we integrated the RIF's database analysis results and found out that miR-34c-5p regulates growth arrest specific 1 (GAS1) for normal embryo implantation. Notably, miR-34c-5p is downregulated during implantation but upregulated in sEVs. An implication of this is the possibility that sEVs miR-34c-5p could be used to evaluate uterine states. In conclusion, these findings suggest that the endometrium derived-sEV miRNAs are potential biomarkers in determining the appropriate period for embryo implantation. This study also has several important implications for future practice, including therapy of infertility. 10.3390/cells9030645
    Endometrial receptivity and implantation require uterine BMP signaling through an ACVR2A-SMAD1/SMAD5 axis. Monsivais Diana,Nagashima Takashi,Prunskaite-Hyyryläinen Renata,Nozawa Kaori,Shimada Keisuke,Tang Suni,Hamor Clark,Agno Julio E,Chen Fengju,Masand Ramya P,Young Steven L,Creighton Chad J,DeMayo Francesco J,Ikawa Masahito,Lee Se-Jin,Matzuk Martin M Nature communications During early pregnancy in the mouse, nidatory estrogen (E2) stimulates endometrial receptivity by activating a network of signaling pathways that is not yet fully characterized. Here, we report that bone morphogenetic proteins (BMPs) control endometrial receptivity via a conserved activin receptor type 2 A (ACVR2A) and SMAD1/5 signaling pathway. Mice were generated to contain single or double conditional deletion of SMAD1/5 and ACVR2A/ACVR2B receptors using progesterone receptor (PR)-cre. Female mice with SMAD1/5 deletion display endometrial defects that result in the development of cystic endometrial glands, a hyperproliferative endometrial epithelium during the window of implantation, and impaired apicobasal transformation that prevents embryo implantation and leads to infertility. Analysis of Acvr2a-PRcre and Acvr2b-PRcre pregnant mice determined that BMP signaling occurs via ACVR2A and that ACVR2B is dispensable during embryo implantation. Therefore, BMPs signal through a conserved endometrial ACVR2A/SMAD1/5 pathway that promotes endometrial receptivity during embryo implantation. 10.1038/s41467-021-23571-5
    FOXO1 regulates uterine epithelial integrity and progesterone receptor expression critical for embryo implantation. Vasquez Yasmin M,Wang Xiaoqiu,Wetendorf Margeaux,Franco Heather L,Mo Qianxing,Wang Tianyuan,Lanz Rainer B,Young Steven L,Lessey Bruce A,Spencer Thomas E,Lydon John P,DeMayo Francesco J PLoS genetics Successful embryo implantation requires a receptive endometrium. Poor uterine receptivity can account for implantation failure in women who experience recurrent pregnancy loss or multiple rounds of unsuccessful in vitro fertilization cycles. Here, we demonstrate that the transcription factor Forkhead Box O1 (FOXO1) is a critical regulator of endometrial receptivity in vivo. Uterine ablation of Foxo1 using the progesterone receptor Cre (PgrCre) mouse model resulted in infertility due to altered epithelial cell polarity and apoptosis, preventing the embryo from penetrating the luminal epithelium. Analysis of the uterine transcriptome after Foxo1 ablation identified alterations in gene expression for transcripts involved in the activation of cell invasion, molecular transport, apoptosis, β-catenin (CTNNB1) signaling pathway, and an increase in PGR signaling. The increase of PGR signaling was due to PGR expression being retained in the uterine epithelium during the window of receptivity. Constitutive expression of epithelial PGR during this receptive period inhibited expression of FOXO1 in the nucleus of the uterine epithelium. The reciprocal expression of PGR and FOXO1 was conserved in human endometrial samples during the proliferative and secretory phase. This demonstrates that expression of FOXO1 and the loss of PGR during the window of receptivity are interrelated and critical for embryo implantation. 10.1371/journal.pgen.1007787
    MicroRNAs activate gene transcription epigenetically as an enhancer trigger. Xiao Min,Li Jin,Li Wei,Wang Yu,Wu Feizhen,Xi Yanping,Zhang Lan,Ding Chao,Luo Huaibing,Li Yan,Peng Lina,Zhao Liping,Peng Shaoliang,Xiao Yao,Dong Shihua,Cao Jie,Yu Wenqiang RNA biology MicroRNAs (miRNAs) are small non-coding RNAs that function as negative gene expression regulators. Emerging evidence shows that, except for function in the cytoplasm, miRNAs are also present in the nucleus. However, the functional significance of nuclear miRNAs remains largely undetermined. By screening miRNA database, we have identified a subset of miRNA that functions as enhancer regulators. Here, we found a set of miRNAs show gene-activation function. We focused on miR-24-1 and found that this miRNA unconventionally activates gene transcription by targeting enhancers. Consistently, the activation was completely abolished when the enhancer sequence was deleted by TALEN. Furthermore, we found that miR-24-1 activates enhancer RNA (eRNA) expression, alters histone modification, and increases the enrichment of p300 and RNA Pol II at the enhancer locus. Our results demonstrate a novel mechanism of miRNA as an enhancer trigger. 10.1080/15476286.2015.1112487
    Steering Against Wind: A New Network of NamiRNAs and Enhancers. Liang Ying,Zou Qingping,Yu Wenqiang Genomics, proteomics & bioinformatics MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs with regulatory functions. Traditionally, miRNAs are thought to play a negative regulatory role in the cytoplasm by binding to the 3'UTR of target genes to degrade mRNA or inhibit translation. However, it remains a challenge to interpret the potential function of many miRNAs located in the nucleus. Recently, we reported a new type of miRNAs present in the nucleus, which can activate gene expression by binding to the enhancer, and named them nuclear activating miRNAs (NamiRNAs). The discovery of NamiRNAs showcases a complementary regulatory mechanism of miRNA, demonstrating their differential roles in the nucleus and cytoplasm. Here, we reviewed miRNAs in nucleus to better understand the function of NamiRNAs in their interactions with the enhancers. Accordingly, we propose a NamiRNA-enhancer-target gene activation network model to better understand the crosstalk between NamiRNAs and enhancers in regulating gene transcription. Moreover, we hypothesize that NamiRNAs may be involved in cell identity or cell fate determination during development, although further study is needed to elucidate the underlying mechanisms in detail. 10.1016/j.gpb.2017.05.001
    Reactivation of tumour suppressor in breast cancer by enhancer switching through NamiRNA network. Liang Ying,Lu Qi,Li Wei,Zhang Dapeng,Zhang Fanglin,Zou Qingping,Chen Lu,Tong Ying,Liu Mengxing,Wang Shaoxuan,Li Wenxuan,Ren Xiaoguang,Xu Peng,Yang Zhicong,Dong Shihua,Zhang Baolong,Huang Yanni,Li Daqiang,Wang Hailin,Yu Wenqiang Nucleic acids research Dysfunction of Tumour Suppressor Genes (TSGs) is a common feature in carcinogenesis. Epigenetic abnormalities including DNA hypermethylation or aberrant histone modifications in promoter regions have been described for interpreting TSG inactivation. However, in many instances, how TSGs are silenced in tumours are largely unknown. Given that miRNA with low expression in tumours is another recognized signature, we hypothesize that low expression of miRNA may reduce the activity of TSG related enhancers and further lead to inactivation of TSG during cancer development. Here, we reported that low expression of miRNA in cancer as a recognized signature leads to loss of function of TSGs in breast cancer. In 157 paired breast cancer and adjacent normal samples, tumour suppressor gene GPER1 and miR-339 are both downregulated in Luminal A/B and Triple Negative Breast Cancer subtypes. Mechanistic investigations revealed that miR-339 upregulates GPER1 expression in breast cancer cells by switching on the GPER1 enhancer, which can be blocked by enhancer deletion through the CRISPR/Cas9 system. Collectively, our findings reveal novel mechanistic insights into TSG dysfunction in cancer development, and provide evidence that reactivation of TSG by enhancer switching may be a promising alternative strategy for clinical breast cancer treatment. 10.1093/nar/gkab626
    FoxO1 is a cell-specific core transcription factor for endometrial remodeling and homeostasis during menstrual cycle and early pregnancy. Human reproduction update BACKGROUND:Endometrium is a vital multicellular tissue for progression of pregnancy. Forkhead box O1 (FoxO1) transcription factor plays an important role in the endometrium as it regulates various cellular processes with its unique expression in different cell types. OBJECTIVE AND RATIONALE:This review focuses on the role of FoxO1 in endometrium with a particular emphasis on FoxO1 signaling in individual endometrial cell types during the menstrual cycle and early pregnancy. SEARCH METHODS:A literature search was conducted in PubMed, Web of Science and Scopus to select studies reporting the role of FoxO1 in endometrium using the keywords: FoxO1, endometrium, menstrual cycle, early pregnancy, endometrial receptivity, implantation, decidualization, angiogenesis and neoplasia. Papers published before October 2020 were selected. Drawing on advances in laboratory science and preclinical studies, we performed a narrative review of the scientific literature to provide a timely update on the roles of FoxO1 during the menstrual cycle and early pregnancy. OUTCOMES:FoxO1 is considered to be a decidualization marker in endometrial stromal cells, mainly because it regulates the transcription of decidual prolactin and insulin-like growth factor-binding protein 1 genes. Importantly, 507 of 3405 genes that are specifically regulated during decidualization of human endometrial stromal cells are expressed abnormally as a result of FOXO1 reduction. Epithelial FoxO1 is currently accepted as a novel endometrial receptivity marker for humans and mice owing to its timely and specific expression at the window of implantation. On the other hand, FOXO1 is essential in endometrial epithelial cell proliferation and differentiation to achieve endometrial homeostasis since loss of function of FOXO1 causes endometrial neoplasia. Last but not least, FoxO1 seems to act like a navigator molecule for embryo homing owing to its notably decreased nuclear expression in endometrial luminal epithelial cells, specifically at the blastocyst attachment region, which results in differentiation, entosis and apoptosis of endometrial epithelial cells during the peri-implantation period. In endothelium, FoxO1 expression coincides with the timing of increased vascular permeabilization during early pregnancy. There are limited data regarding the importance of FoxO1 upregulation in endometrial endothelial cells, therefore, it is time to investigate the role of endothelial FoxO1, which is the missing piece in the puzzle of the enigmatic endometrium. Another missing piece in the puzzle for the role of FoxO1 is on embryo development. WIDER IMPLICATIONS:FoxO1 is a cell-specific core transcription factor involved in efficient endometrial remodeling during the menstrual cycle and early pregnancy. A better understanding of the role of FoxO1 as a decidualization marker, as an emerging marker of endometrial receptivity, and as a therapeutic target to prevent endometrial neoplasia could help us to make sense of endometrial biology and thus to improve the outcomes of ART in the clinic. 10.1093/humupd/dmaa060
    LncRNA TTN-AS1 regulates osteosarcoma cell apoptosis and drug resistance via the miR-134-5p/MBTD1 axis. Fu Dong,Lu Chunwen,Qu Xingzhou,Li Peng,Chen Kai,Shan Liancheng,Zhu Xiaodong Aging AIM:To explore the mechanism by which long non-coding RNA (lncRNA) TTN-AS1 regulates osteosarcoma cell apoptosis and drug resistance via the microRNA miR-134-5p/malignant brain tumour domain containing 1 (MBTD1) axis. RESULTS:The lncRNA TTN-AS1 was highly expressed in osteosarcoma and was associated with poor prognosis. The lncRNA TTN-AS1 promoted cell viability and inhibited apoptosis. MiR-134-5p targeted MBTD1, which was regulated by lncRNA TTN-AS1. MBTD1 was highly expressed in osteosarcoma and was associated with poor prognosis. MBTD1 promoted cell viability and inhibited apoptosis, and knockdown of MBTD1 reversed the cancer-promoting effects of lncRNA TTN-AS1. Downregulation of lncRNA TTN-AS1 reduced drug resistance. CONCLUSION:In osteosarcoma, lncRNA TTN-AS1 promoted the expression of MBTD1 by targeting miR-134-5p and regulated cell growth, apoptosis and drug resistance. METHODS:The expression characteristics of genes in osteosarcoma patients were analysed using bioinformatics. Plasmid transfection technology was applied to silence or overexpress lncRNA TTN-AS1, miR-134-5p and MBTD1. Western blotting and quantitative polymerase chain reaction (qPCR) were used to detect protein and RNA, respectively. A cell counting kit 8 (CCK-8) and flow cytometry were used to detect cell viability and apoptosis. The effects of lncRNA TTN-AS1 and MBTD1 on osteosarcoma in vivo were studied by using a tumour burden assay. 10.18632/aging.102325
    Reduced homeobox protein MSX1 in human endometrial tissue is linked to infertility. Bolnick Alan D,Bolnick Jay M,Kilburn Brian A,Stewart Tamika,Oakes Jonathan,Rodriguez-Kovacs Javier,Kohan-Ghadr Hamid-Reza,Dai Jing,Diamond Michael P,Hirota Yasushi,Drewlo Sascha,Dey Sudhansu K,Armant D Randall, Human reproduction (Oxford, England) STUDY QUESTION:Is protein expression of the muscle segment homeobox gene family member MSX1 altered in the human secretory endometrium by cell type, developmental stage or fertility? SUMMARY ANSWER:MSX1 protein levels, normally elevated in the secretory phase endometrium, were significantly reduced in endometrial biopsies obtained from women of infertile couples. WHAT IS KNOWN ALREADY:Molecular changes in the endometrium are important for fertility in both animals and humans. Msx1 is expressed in the preimplantation mouse uterus and regulates uterine receptivity for implantation. The MSX protein persists a short time, after its message has been down-regulated. Microarray analysis of the human endometrium reveals a similar pattern of MSX1 mRNA expression that peaks before the receptive period, with depressed expression at implantation. Targeted deletion of uterine Msx1 and Msx2 in mice prevents the loss of epithelial cell polarity during implantation and causes infertility. STUDY DESIGN, SIZE DURATION:MSX1 mRNA and cell type-specific levels of MSX1 protein were quantified from two retrospective cohorts during the human endometrial cycle. MSX1 protein expression patterns were compared between fertile and infertile couples. Selected samples were dual-labeled by immunofluorescence microscopy to localize E-cadherin and β-catenin in epithelial cells. PARTICIPANTS/MATERIALS, SETTING METHODS:MSX1 mRNA was quantified by PCR in endometrium from hysterectomies (n = 14) determined by endometrial dating to be in the late-proliferative (cycle days 10-13), early-secretory (cycle days 14-19) or mid-secretory (cycle days 20-24) phase. MSX1 protein was localized using high-throughput, semi-quantitative immunohistochemistry with sectioned endometrial biopsy tissues from fertile (n = 89) and infertile (n = 89) couples. Image analysis measured stain intensity specifically within the luminal epithelium, glands and stroma during the early-, mid- and late- (cycle days 25-28) secretory phases. MAIN RESULTS AND THE ROLE OF CHANCE:MSX1 transcript increased 5-fold (P < 0.05) between the late-proliferative and early secretory phase and was then down-regulated (P < 0.05) prior to receptivity for implantation. In fertile patients, MSX1 protein displayed strong nuclear localization in the luminal epithelium and glands, while it was weakly expressed in nuclei of the stroma. MSX1 protein levels accumulated throughout the secretory phase in all endometrial cellular compartments. MSX1 protein decreased (P < 0.05) in the glands between mid- and late-secretory phases. However, infertile patients demonstrated a broad reduction (P < 0.001) of MSX1 accumulation in all cell types throughout the secretory phase that was most pronounced (∼3-fold) in stroma and glands. Infertility was associated with persistent co-localization of E-cadherin and β-catenin in epithelial cell junctions in the mid- and late-secretory phases. LIMITATIONS, REASONS FOR CAUTION:Details of the infertility diagnoses and other patient demographic data were not available. Therefore, patients with uterine abnormalities (Mullerian) could not be distinguished from other sources of infertility. Antibody against human MSX2 is not available, limiting the study to MSX1. However, both RNAs in the human endometrium are similarly regulated. In mice, Msx1 and Msx2 are imperative for murine embryo implantation, with Msx2 compensating for genetic ablation of Msx1 through its up-regulation in a knockout model. WIDER IMPLICATIONS OF THE FINDINGS:This investigation establishes that the MSX1 homeobox protein accumulation is associated with the secretory phase in endometrium of fertile couples, and is widely disrupted in infertile patients. It is the first study to examine MSX1 protein localization in the human endometrium, and supported by genetic findings in mice, suggests that genes regulated by MSX1 are linked to the loss of epithelial cell polarity required for uterine receptivity during implantation. STUDY FUNDING/COMPETING INTERESTS:This research was supported by the NICHD National Cooperative Reproductive Medicine Network grant HD039005 (M.P.D.), NIH grants HD068524 (S.K.D.), HD071408 (D.R.A., M.P.D.), and HL128628 (S.D.), the Intramural Research Program of the NICHD, March of Dimes (S.K.D., S.D.) and JSPS KAKENHI grant 26112506 (Y.H.). There were no conflicts or competing interests. 10.1093/humrep/dew143
    Uterine inactivation of muscle segment homeobox (Msx) genes alters epithelial cell junction proteins during embryo implantation. Sun Xiaofei,Park Craig B,Deng Wenbo,Potter S Steven,Dey Sudhansu K FASEB journal : official publication of the Federation of American Societies for Experimental Biology Embryo implantation requires that the uterus differentiate into the receptive state. Failure to attain uterine receptivity will impede blastocyst attachment and result in a compromised pregnancy. The molecular mechanism by which the uterus transitions from the prereceptive to the receptive stage is complex, involving an intricate interplay of various molecules. We recently found that mice with uterine deletion ofMsxgenes (Msx1(d/d)/Msx2(d/d)) are infertile because of implantation failure associated with heightened apicobasal polarity of luminal epithelial cells during the receptive period. However, information on Msx's roles in regulating epithelial polarity remains limited. To gain further insight, we analyzed cell-type-specific gene expression by RNA sequencing of separated luminal epithelial and stromal cells by laser capture microdissection fromMsx1(d/d)/Msx2(d/d)and floxed mouse uteri on d 4 of pseudopregnancy. We found that claudin-1, a tight junction protein, and small proline-rich (Sprr2) protein, a major component of cornified envelopes in keratinized epidermis, were substantially up-regulated inMsx1(d/d)/Msx2(d/d)uterine epithelia. These factors also exhibited unique epithelial expression patterns at the implantation chamber (crypt) inMsx1(f/f)/Msx2(f/f)females; the patterns were lost inMsx1(d/d)/Msx2(d/d)epithelia on d 5, suggesting important roles during implantation. The results suggest thatMsxgenes play important roles during uterine receptivity including modulation of epithelial junctional activity.-Sun, X., Park, C. B., Deng, W., Potter, S. S., Dey, S. K. Uterine inactivation of muscle segment homeobox (Msx) genes alters epithelial cell junction proteins during embryo implantation. 10.1096/fj.15-282798
    Estrogen induces EGR1 to fine-tune its actions on uterine epithelium by controlling PR signaling for successful embryo implantation. Kim Hye-Ryun,Kim Yeon Sun,Yoon Jung Ah,Yang Seung Chel,Park Mira,Seol Dong-Won,Lyu Sang Woo,Jun Jin Hyun,Lim Hyunjung Jade,Lee Dong Ryul,Song Haengseok FASEB journal : official publication of the Federation of American Societies for Experimental Biology The harmonized actions of ovarian E and progesterone (P) regulate the proliferation and differentiation of uterine cells in a spatiotemporal manner. Imbalances between these hormones often lead to infertility and gynecologic diseases. Whereas numerous factors that are involved in P signaling have been identified, few local factors that mediate E actions in the uterus have been revealed. Here, we demonstrate that estrogen induces the transcription factor, early growth response 1 ( Egr1), to fine-tune its actions in uterine epithelial cells (ECs) that are responsible for uterine receptivity for embryo implantation. In the presence of exogenous gonadotrophins, ovulation, fertilization, and embryonic development normally occur in Egr1 mice, but these animals experience the complete failure of embryo implantation with reduced artificial decidualization. Although serum levels of E and P were comparable between Egr1 and Egr1 mice on d 4 of pregnancy, aberrantly reduced levels of progesterone receptor in Egr1 uterine ECs caused enhanced E activity and impaired P response. Ultrastructural analyses revealed that Egr1 ECs are not fully able to provide proper uterine receptivity. Uterine mRNA landscapes in Egr1 mice revealed that EGR1 controls the expression of a subset of E-regulated genes. In addition, P signaling was unable to modulate estrogen actions, including those that are involved in cell-cycle progression, in ECs that were deficient in EGR1. Furthermore, primary coculture of Egr1 ECs with Egr1 stromal cells, and vice versa, supported the notion that Egr1 is required to modulate E actions on ECs to prepare the uterine environment for embryo implantation. In contrast to its role in ECs, loss of Egr1 in stroma significantly reduced stromal cell proliferation. Collectively, our results demonstrate that E induces EGR1 to streamline its actions for the preparation of uterine receptivity for embryo implantation in mice.-Kim, H.-R., Kim, Y. S., Yoon, J. A., Yang, S. C., Park, M., Seol, D.-W., Lyu, S. W., Jun, J. H., Lim, H. J., Lee, D. R., Song, H. Estrogen induces EGR1 to fine-tune its actions on uterine epithelium by controlling PR signaling for successful embryo implantation. 10.1096/fj.201700854RR
    EHD1 impairs decidualization by regulating the Wnt4/β-catenin signaling pathway in recurrent implantation failure. Zhou Quan,Yan Guijun,Ding Lijun,Liu Jingyu,Yu Xiaoying,Kong Shuangbo,Zhang Mei,Wang Zhilong,Liu Yang,Jiang Yue,Kong Na,Sun Jianxin,Sun Haixiang EBioMedicine BACKGROUND:Recurrent implantation failure (RIF) remains a critical and challenging problem in assisted reproductive technology mainly due to impaired decidualization. The endocytic and transcytotic activity in the endometrium are crucial for decidualization. The most representative endocytic gene is the C-terminal Eps15 homology domain-containing 1 (EHD1), but whether EHD1-mediated endocytic function is responsible for embryo implantation during decidualization remains unclear. METHODS:A transcriptomic analysis was performed to evaluate the differentially expressed genes between the fertile control and RIF group. The expression and location of EHD1 in endometrial tissues were further examined by IHC, qRT-PCR and Western blotting. The transduction of an EHD1 recombinant adenovirus into human endometrial stromal cells was performed to investigate relevant decidualization marker genes. Additionally, a microarray analysis following the adenovirus-mediated overexpression of EHD1 was conducted to identify EHD1-related changes in HESCs, and the potential molecular mechanisms were further confirmed through immunofluorescence and coimmunoprecipitation analyses. FINDINGS:An RNA-seq analysis demonstrated that EHD1 expression was significantly higher in the mid-secretory endometrium of the RIF group than in that of the fertile control group. The analysis of the menstrual cycle showed that expression of EHD1 increased in the mid-proliferative phase and showed a gradual decrease in the mid-secretory and decidual phases. Furthermore, EHD1 overexpression impaired decidualization by suppressing the expression of prolactin and insulin-like growth factor binding protein-1 and the formation of the cytoskeleton. The mechanistic analysis revealed the EHD1 regulated LRP5/6 protein function through the endocytic pathway, and subsequently suppressed the Wnt4/β-catenin pathway during decidualization. In addition, a Wnt4 agonist improved an impaired decidualization process. INTERPRETATION:Regulation of the EHD1-Wnt4 pathway might serve as a promising therapeutic strategy for improving endometrial receptivity in RIF women. 10.1016/j.ebiom.2019.10.018
    Temporal and spatial expression of adrenomedullin and its receptors in the porcine uterus and peri-implantation conceptuses. Biology of reproduction Adrenomedullin (ADM) is an evolutionarily conserved multifunctional peptide hormone that regulates implantation, embryo spacing, and placentation in humans and rodents. However, the potential roles of ADM in implantation and placentation in pigs, as a litter-bearing species, are not known. This study determined abundances of ADM in uterine luminal fluid, and the patterns of expression of ADM and its receptor components (CALCRL, RAMP2, RAMP3, and ACKR3) in uteri from cyclic and pregnant gilts, as well as conceptuses (embryonic/fetus and its extra-embryonic membranes) during the peri-implantation period of pregnancy. Total recoverable ADM was greater in the uterine fluid of pregnant compared with cyclic gilts between Days 10 and 16 post-estrus and was from uterine luminal epithelial (LE) and conceptus trophectoderm (Tr) cells. Uterine expression of CALCRL, RAMP2, and ACKR3 were affected by day (P < 0.05), pregnant status (P < 0.01) and/or day x status (P < 0.05). Within porcine conceptuses, the expression of CALCRL, RAMP2, and ACKR3 increased between Days 10 and 16 of pregnancy. Using an established porcine trophectoderm (pTr1) cell line, it was determined that 10-7 M ADM stimulated proliferation of pTr1 cells (P < 0.05) at 48 h, and increased phosphorylated mechanistic target of rapamycin (p-MTOR) and 4E binding protein 1 (p-4EBP1) by 6.1- and 4.9-fold (P < 0.0001), respectively. These novel results indicate a significant role for ADM in uterine receptivity for implantation and conceptus growth and development in pigs. They also provide a framework for future studies of ADM signaling to affect proliferation and migration of Tr cells, spacing of blastocysts, implantation, and placentation in pigs. 10.1093/biolre/ioab110
    Rictor/mTORC2 is involved in endometrial receptivity by regulating epithelial remodeling. Zhang Yue,Du Xinman,Chen Xuemei,Tang Hongyu,Zhou Qin,He Junlin,Ding Yubin,Wang Yingxiong,Liu Xueqing,Geng Yanqing FASEB journal : official publication of the Federation of American Societies for Experimental Biology Successful embryo implantation requires well-functioning endometrial luminal epithelial cells to establish uterine receptivity. Inadequate uterine receptivity is responsible for approximately two thirds of implantation failures in humans. However, the regulatory mechanism governing this functional process remains largely unexplored. A previous study revealed that the expression of Rictor, the main member of mTORC2, in mouse epithelial cells is increased on the fourth day of gestation (D4). Here, we provide the first report of the involvement of Rictor in the regulation of endometrial receptivity. Rictor was conditionally ablated in the mouse endometrium using a progesterone receptor cre (PR ) mouse model. Loss of Rictor altered polarity remodeling and the Na channel protein of endometrial cells by mediating Rac-1/PAK1(pPAK1)/ERM(pERM) and Sgk1/pSgk1 signaling, respectively, ultimately resulting in impaired fertility. In the endometrium of women with infertility, the expression of Rictor was changed, along with the morphological transformation and Na channel protein of epithelial cells. Our findings demonstrate that Rictor is crucial for the establishment of uterine receptivity in both mice and humans. The present study may help improve the molecular regulatory network of endometrial receptivity and provide new diagnostic and treatment strategies for infertility. 10.1096/fj.202100529RR
    GLUT4 in Mouse Endometrial Epithelium: Roles in Embryonic Development and Implantation. Long Yun,Wang Yi-Cheng,Yuan Dong-Zhi,Dai Xin-Hua,Liao Lin-Chuan,Zhang Xue-Qin,Zhang Li-Xue,Ma Yong-Dan,Lei Yi,Cui Zhi-Hui,Zhang Jin-Hu,Nie Li,Yue Li-Min Frontiers in physiology GLUT4 is involved in rapid glucose uptake among various kinds of cells to contribute to glucose homeostasis. Prior data have reported that aberrant glucose metabolism by GLUT4 dysfunction in the uterus could be responsible for infertility and increased miscarriage. However, the expression and precise functions of GLUT4 in the endometrium under physiological conditions remain unknown or controversial. In this study, we observed that GLUT4 exhibits a spatiotemporal expression in mouse uterus on pregnant days 1-4; its expression especially increased on pregnant day 4 during the window of implantation. We also determined that estrogen, in conjunction with progesterone, promotes the expression of GLUT4 in the endometrial epithelium or . GLUT4 is an important transporter that mediates glucose transport in endometrial epithelial cells (EECs) or . , glucose uptake decreased in mouse EECs when the cells were treated with GLUT4 small interfering RNA (siRNA). , the injection of GLUT4-siRNA into one side of the mouse uterine horns resulted in an increased glucose concentration in the uterine fluid on pregnant day 4, although it was still lower than in blood, and impaired endometrial receptivity by inhibiting pinopode formation and the expressions of leukemia inhibitory factor (LIF) and integrin ανβ3, finally affecting embryonic development and implantation. Overall, the obtained results indicate that GLUT4 in the endometrial epithelium affects embryo development by altering glucose concentration in the uterine fluid. It can also affect implantation by impairing endometrial receptivity due to dysfunction of GLUT4. 10.3389/fphys.2021.674924
    Adult bone marrow progenitors become decidual cells and contribute to embryo implantation and pregnancy. Tal Reshef,Shaikh Shafiq,Pallavi Pallavi,Tal Aya,López-Giráldez Francesc,Lyu Fang,Fang Yuan-Yuan,Chinchanikar Shruti,Liu Ying,Kliman Harvey J,Alderman Myles,Pluchino Nicola,Kayani Jehanzeb,Mamillapalli Ramanaiah,Krause Diane S,Taylor Hugh S PLoS biology Decidua is a transient uterine tissue shared by mammals with hemochorial placenta and is essential for pregnancy. The decidua is infiltrated by many immune cells promoting pregnancy. Adult bone marrow (BM)-derived cells (BMDCs) differentiate into rare populations of nonhematopoietic endometrial cells in the uterus. However, whether adult BMDCs become nonhematopoietic decidual cells and contribute functionally to pregnancy is unknown. Here, we show that pregnancy mobilizes mesenchymal stem cells (MSCs) to the circulation and that pregnancy induces considerable adult BMDCs recruitment to decidua, where some differentiate into nonhematopoietic prolactin-expressing decidual cells. To explore the functional importance of nonhematopoietic BMDCs to pregnancy, we used Homeobox a11 (Hoxa11)-deficient mice, having endometrial stromal-specific defects precluding decidualization and successful pregnancy. Hoxa11 expression in BM is restricted to nonhematopoietic cells. BM transplant (BMT) from wild-type (WT) to Hoxa11-/- mice results in stromal expansion, gland formation, and marked decidualization otherwise absent in Hoxa11-/- mice. Moreover, in Hoxa11+/- mice, which have increased pregnancy losses, BMT from WT donors leads to normalized uterine expression of numerous decidualization-related genes and rescue of pregnancy loss. Collectively, these findings reveal that adult BMDCs have a previously unrecognized nonhematopoietic physiologic contribution to decidual stroma, thereby playing important roles in decidualization and pregnancy. 10.1371/journal.pbio.3000421
    A hypoxia-induced Rab pathway regulates embryo implantation by controlled trafficking of secretory granules. Bhurke Arpita,Kannan Athilakshmi,Neff Alison,Ma Qiuyan,Laws Mary J,Taylor Robert N,Bagchi Milan K,Bagchi Indrani C Proceedings of the National Academy of Sciences of the United States of America Implantation is initiated when an embryo attaches to the uterine luminal epithelium and subsequently penetrates into the underlying stroma to firmly embed in the endometrium. These events are followed by the formation of an extensive vascular network in the stroma that supports embryonic growth and ensures successful implantation. Interestingly, in many mammalian species, these processes of early pregnancy occur in a hypoxic environment. However, the mechanisms underlying maternal adaptation to hypoxia during early pregnancy remain unclear. In this study, using a knockout mouse model, we show that the transcription factor hypoxia-inducible factor 2 alpha (Hif2α), which is induced in subluminal stromal cells at the time of implantation, plays a crucial role during early pregnancy. Indeed, when preimplantation endometrial stromal cells are exposed to hypoxic conditions in vitro, we observed a striking enhancement in HIF2α expression. Further studies revealed that HIF2α regulates the expression of several metabolic and protein trafficking factors, including RAB27B, at the onset of implantation. RAB27B is a member of the Rab family of GTPases that allows controlled release of secretory granules. These granules are involved in trafficking MMP-9 from the stroma to the epithelium to promote luminal epithelial remodeling during embryo invasion. As pregnancy progresses, the HIF2α-RAB27B pathway additionally mediates crosstalk between stromal and endothelial cells via VEGF granules, developing the vascular network critical for establishing pregnancy. Collectively, our study provides insights into the intercellular communication mechanisms that operate during adaptation to hypoxia, which is essential for embryo implantation and establishment of pregnancy. 10.1073/pnas.2000810117
    Suppression of autophagy and HCK signaling promotes PTGS2 FCGR3 NK cell differentiation triggered by ectopic endometrial stromal cells. Mei Jie,Zhou Wen-Jie,Zhu Xiao-Yong,Lu Han,Wu Ke,Yang Hui-Li,Fu Qiang,Wei Chun-Yan,Chang Kai-Kai,Jin Li-Ping,Wang Jian,Wang Yong-Ming,Li Da-Jin,Li Ming-Qing Autophagy Impaired NK cell cytotoxic activity contributes to the local dysfunctional immune environment in endometriosis (EMS), which is an estrogen-dependent gynecological disease that affects the function of ectopic endometrial tissue clearance. The reason for the impaired cytotoxic activity of NK cells in an ectopic lesion microenvironment (ELM) is largely unknown. In this study, we show that the macroautophagy/autophagy level of endometrial stromal cells (ESCs) from EMS decreased under negative regulation of estrogen. The ratio of peritoneal FCGR3 NK to FCGR3 NK cells increases as EMS progresses. Moreover, the autophagy suppression results in the downregulation of HCK (hematopoietic cellular kinase) by inactivating STAT3 (signal transducer and activator of transcription 3), as well as the increased secretion of the downstream molecules CXCL8/IL8 and IL23A by ESCs, and this increase induced the upregulation of FCGR3 NK cells and decline of cytotoxic activity in ELM. This process is mediated through the depression of microRNA MIR1185-1-3p, which is associated with the activation of the target gene PTGS2 in NK cells. FCGR3 NK with a phenotype of PTGS2/COX2 IFNG PRF1 GZMB induced by hck knockout (hck) or 3-methyladenine (3-MA, an autophagy inhibitor)-stimulated ESCs accelerates ESC's growth both in vitro and in vivo. These results suggest that the estrogen-autophagy-STAT3-HCK axis participates in the differentiation of PTGS2 IFNG PRF1 GZMB FCGR3 NK cells in ELM and contributes to the development of EMS. This result provides a scientific basis for potential therapeutic strategies to treat diseases related to impaired NK cell cytotoxic activity. ABBREVIATIONS:anti-FCGR3: anti-FCGR3 with neutralizing antibody; Ctrl-ESC: untreated ESCs; CXCL8: C-X-C motif chemokine ligand 8; ectoESC: ESCs from ectopic lesion; ELM: ectopic lesion microenvironment; EMS: endometriosis; ESCs: endometrial stromal cells; eutoESC:eutopic ESCs; HCK: hematopoietic cellular kinase; HCK(OE): overexpression of HCK; IFNG: interferon gamma; IL23A (OE): overexpression of IL23A; KLRK1: Killer cell lectin like receptor K1; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; 3 -MA: 3-methyladenine; 3-MA-ESC: 3-MA-treated ESCs; MIR1185-1-3p: overexpression of HsMIR1185-1-3p; NK: natural killer; normESCs: normal ESCs; Rap-ESC:rapamycin-treated ESCs; PCNA: proliferating cell nuclear antigen; PF: peritoneal fluid; SFKs: SRC family of cytoplasmic tyrosine kinases; si-HCK: silencing of HCK; siIL23A: silencing of IL23A; USCs: uterus stromal cells. 10.1080/15548627.2018.1476809
    ETV4 Is Necessary for Estrogen Signaling and Growth in Endometrial Cancer Cells. Rodriguez Adriana C,Vahrenkamp Jeffery M,Berrett Kristofer C,Clark Kathleen A,Guillen Katrin P,Scherer Sandra D,Yang Chieh-Hsiang,Welm Bryan E,Janát-Amsbury Margit M,Graves Barbara J,Gertz Jason Cancer research Estrogen signaling through estrogen receptor alpha (ER) plays a major role in endometrial cancer risk and progression, however, the molecular mechanisms underlying ER's regulatory role in endometrial cancer are poorly understood. In breast cancer cells, ER genomic binding is enabled by FOXA1 and GATA3, but the transcription factors that control ER genomic binding in endometrial cancer cells remain unknown. We previously identified ETV4 as a candidate factor controlling ER genomic binding in endometrial cancer cells, and here we explore the functional importance of ETV4. Homozygous deletion of ETV4, using CRISPR/Cas9, led to greatly reduced ER binding at the majority of loci normally bound by ER. Consistent with the dramatic loss of ER binding, the gene expression response to estradiol was dampened for most genes. ETV4 contributes to estrogen signaling in two distinct ways. ETV4 loss affects chromatin accessibility at some ER bound loci and impairs ER nuclear translocation. The diminished estrogen signaling upon ETV4 deletion led to decreased growth, particularly in 3D culture, where hollow organoids were formed and in the context of estrogen-dependent growth. These results show that ETV4 plays an important role in estrogen signaling in endometrial cancer cells. SIGNIFICANCE: Estrogen receptor alpha (ER) is a key oncogene in endometrial cancer. This study uncovers ETV4 as an important factor in controlling the activity of ER and the growth of endometrial cancer cells. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/6/1234/F1.large.jpg. 10.1158/0008-5472.CAN-19-1382
    CD34KLF4 Stromal Stem Cells Contribute to Endometrial Regeneration and Repair. Yin Mingzhu,Zhou Huanjiao Jenny,Lin Caixia,Long Lingli,Yang Xiaolei,Zhang Haifeng,Taylor Hugh,Min Wang Cell reports The regenerative capacity of the human endometrium requires a population of local stem cells. However, the phenotypes, locations, and origin of these cells are still unknown. In a mouse menstruation model, uterine stromal SM22α-derived CD34KLF4 stem cells are activated and integrate into the regeneration area, where they differentiate and incorporate into the endometrial epithelium; this process is correlated with enhanced protein SUMOylation in CD34KLF4 cells. Mice with a stromal SM22α-specific SENP1 deletion (SENP1smKO) exhibit accelerated endometrial repair in the regeneration model and develop spontaneous uterine hyperplasia. Mechanistic studies suggest that SENP1 deletion induces SUMOylation of ERα, which augments ERα transcriptional activity and proliferative signaling in SM22αCD34KLF4 cells. These cells then transdifferentiate to the endometrial epithelium. Our study reveals that CD34KLF4 stromal-resident stem cells directly contribute to endometrial regeneration, which is regulated through SENP1-mediated ERα suppression. 10.1016/j.celrep.2019.04.088
    TRIM65 Promotes Invasion of Endometrial Stromal Cells by Activating ERK1/2/C-myc Signaling via Ubiquitination of DUSP6. Wu Ying-Ting,Ma Si-Yu,Sun Wen-Qin,Shen Wei-Wei,Zhu Hui-Ting,Zhang Qin,Chen Hui-Fen The Journal of clinical endocrinology and metabolism BACKGROUND:Endometriosis (EM) is a benign gynecological disease that shares some characteristics with malignancy, such as proliferation and invasion. So far, the pathogenesis of EM is still unclear. In this study, we investigated whether TRIM65 can play a role in the development of EM. METHODS:TRIM65 expression levels in eutopic, ectopic, and normal endometrium were detected by quantitative real-time PCR and Western blot. Cell proliferation and invasion of primary endometrial stromal (EMS) cells were detected by CCK-8 and Transwell analysis. The interaction between TRIM65 and DUSP6 or C-myc was measured by coimmunoprecipitation, ubiquitylation, dual luciferase, and chromatin immunoprecipitation analysis. RESULTS:We found that TRIM65 was identified as an up-regulated gene in ectopic endometrial tissues and EMS cells compared with control groups without EM. TRIM65 expression was positively correlated with the levels of p-ERK1/2, C-myc, matrix metalloproteinase-2, and integrin β1 in ectopic endometrial tissues in patients and mice. TRIM65 promoted the cell proliferation and invasion of EMS cells via the ERK1/2/C-myc pathway through ubiquitination of DUSP6. C-myc promoted TRIM65 expression through inducing TRIM65 promoter activity. Additionally, the increased expression of TRIM65, C-myc, matrix metalloproteinase-2, integrin β1, and p-ERK1/2 and the decreased expression of DUSP6 in ectopic endometrial tissues were significantly suppressed by inhibition of ERK1/2 signaling pathway in ectopic endometrial tissues in experimental mice model. CONCLUSION:In conclusion, TRIM65 promotes invasion of ectopic EMS cells by activating a feedback loop with the ERK1/2/C-myc signaling pathway and may be a potential therapeutic target for EM. 10.1210/clinem/dgaa804
    ALK3-SMAD1/5 Signaling Mediates the BMP2-Induced Decrease in PGE2 Production in Human Endometrial Stromal Cells and Decidual Stromal Cells. Zhang Yu,Zhu Hua,Chang Hsun-Ming,Leung Peter C K Frontiers in cell and developmental biology BMP2 is a critical factor that is involved in the processes of embryo implantation and uterine decidualization. The expression of cyclooxygenase (COX) and subsequent prostaglandin E2 (PGE2) production are critical for successful pregnancy. However, it is not clear whether BMP2 can regulate the production of PG during endometrial decidualization. The aim of this study was to investigate the effects of BMP2 on COX-1 expression and PGE2 production as well as the underlying molecular mechanisms in the human endometrium. Immortalized human endometrial stromal cells (HESCs) and human decidual stromal cells (HDSCs) were used as the study model to investigate the effects of BMP2-induced cellular activities. Our results showed that BMP2 treatment significantly decreased PGE2 production by downregulating COX-1 expression in both human endometrial stromal and decidual stromal cells. Additionally, BMP2 induced an increase in the levels of phosphorylated SMAD1/5/8, and this effect was completely abolished by the addition of the inhibitors DMH-1 and dorsomorphin, but not by SB431542. Knocking down ALK3 completely reversed the BMP2-induced downregulation of COX-1. Moreover, concomitantly knocking down SMAD1 and SMAD5 completely reversed the BMP2-induced downregulation of COX-1. Our results indicated that BMP2 decreased PGE2 production by downregulating COX-1 expression, most likely through the ALK3/SMAD1-SMAD5 signaling pathway in human endometrial stromal and human decidual stromal cells. These findings deepen our understanding of the functional role of BMP2 in the regulation of endometrial decidualization in humans. 10.3389/fcell.2020.573028
    LncRNA MALAT1 inhibits apoptosis of endometrial stromal cells through miR-126-5p-CREB1 axis by activating PI3K-AKT pathway. Feng Ying,Tan Bu-Zhen Molecular and cellular biochemistry Endometriosis is a common, chronic and painful disease in women, whose pathogenesis remains not entirely clear. Long non-coding RNA (lncRNA) MALAT1 participates in the development of endometriosis. This study further investigated the regulation of MALAT1-miR-126-5p-CREB1 axis in the pathological process of endometriosis. MALAT1, miR-126-5p, and CREB1 levels in human endometrial stromal cells (HESCs) were detected by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Protein levels were determined by Western blotting. Cell viability and apoptosis was assessed by MTT assay and annexin V-FITC staining, respectively. The interactivity between miR-126-5p and MALAT1 (or CREB1) was assessed by dual luciferase reporter system. Knockdown of MALAT1 or CREB1 restrained proliferation and induced apoptosis as confirmed by upregulating cleaved caspase-3 and Bax, and down-regulating Bcl-2 in HESCs, while inhibition of miR-126-5p presented the opposite results. Moreover, silencing of MALAT1 triggered apoptosis of HESCs via targeting miR-126-5p. In addition, miR-126-5p directly regulated CREB1 expression via binding to its 3' non-coding region. Finally, miR-126-5p inhibitor-mediated apoptosis inhibition was restrained by CREB1 silencing via inactivation of PI3K-AKT pathway in HESCs. Taken together, our study firstly demonstrates that MALAT1 regulates apoptosis of HESCs through miR-126-5p/CREB1 axis mediated PI3K/AKT pathway. Our findings explained the pathogenesis of endometriosis and offered promising therapeutic option for endometriosis. 10.1007/s11010-020-03871-y
    MiR-370-3p inhibits the development of human endometriosis by downregulating EDN1 expression in endometrial stromal cells. Zhou Shun,Huang Chengyi,Wang Wei,Liu Juan Cell biology international MiR-370-3p has been demonstrated to be downregulated in patients with endometriosis (EM). However, its role and molecular mechanisms in the progression of EM remain unclear. Real-time polymerase chain reaction was used to measure the expression of miR-370-3p and endothelin-1 (EDN1) in patients with or without EM. After miR-370-3p overexpression or knockdown in ectopic endometrial hEM15A cells, the changes in the proliferation, apoptosis, and migration and invasion capacities were detected by using cell counting kit-8, flow cytometry, and transwell methods. The interplay between miR-370-3p and EDN1 was confirmed by a luciferase reporter assay. Patients with EM showed adverse expression of EDN1 and miR-370-3p, especially in eutopic endometrium and ectopic endometrium. MiR-370-3p inhibited the proliferation, metastasis, and invasion capacities of hEM15A cells and promoted apoptosis. Investigation of its molecular mechanism revealed that miR-370-3p targeted EDN1 to influence the biological functions of hEM15A cells. MiR-370-3p represented as a therapeutic target for EM treatment. 10.1002/cbin.11552
    Loss of HDAC3 results in nonreceptive endometrium and female infertility. Kim Tae Hoon,Yoo Jung-Yoon,Choi Kyung-Chul,Shin Jung-Ho,Leach Richard E,Fazleabas Asgerally T,Young Steven L,Lessey Bruce A,Yoon Ho-Geun,Jeong Jae-Wook Science translational medicine Endometriosis is a disease in which tissue that normally grows inside the uterus grows outside the uterus and causes chronic pelvic pain and infertility. However, the exact mechanisms of the pathogenesis of endometriosis-associated infertility are unknown. Epigenetic dysregulation has recently been implicated in infertility. Here, we report a reduction of histone deacetylase 3 (HDAC3) protein amounts in eutopic endometrium of infertile women with endometriosis compared to a control group. To investigate the effect of HDAC3 loss in the uterus, we generated mice with conditional ablation of in progesterone receptor (PGR)-positive cells ( ; ). Loss of in the uterus of mice results in infertility due to implantation failure and decidualization defect. Expression microarray and ChIP-seq analyses identified and as direct targets of HDAC3 in both mice and humans. Reduction of abrogated decidualization in a primary culture of human endometrial stromal cells (hESCs) similar to that observed in infertile patients with endometriosis. Whereas attenuation of resulted in p300 recruitment to and genes in the uterus of mice as well as hESCs, inhibition of p300 permitted hESCs to undergo decidualization. Collectively, we found attenuation of HDAC3 and overexpression of collagen type I in the eutopic endometrium of infertile patients with endometriosis. HDAC3 loss caused a defect of decidualization through the aberrant transcriptional activation of and genes in mice and and genes in humans. Our results suggest that HDAC3 is critical for endometrial receptivity and decidualization. 10.1126/scitranslmed.aaf7533
    Endometrial receptivity markers, the journey to successful embryo implantation. Achache Hanna,Revel Ariel Human reproduction update Human embryo implantation is a three-stage process (apposition, adhesion and invasion) involving synchronized crosstalk between a receptive endometrium and a functional blastocyst. This ovarian steroid-dependent phenomenon can only take place during the window of implantation, a self-limited period of endometrial receptivity spanning between days 20 and 24 of the menstrual cycle. Implantation involves a complex sequence of signalling events, consisting in the acquisition of adhesion ligands together with the loss of inhibitory components, which are crucial to the establishment of pregnancy. Histological evaluation, now considered to add little clinically significant information, should be replaced by functional assessment of endometrial receptivity. A large number of molecular mediators have been identified to date, including adhesion molecules, cytokines, growth factors, lipids and others. Thus, endometrial biopsy samples can be used to identify molecules associated with uterine receptivity to obtain a better insight into human implantation. In addition, development of functional in vitro systems to study embryo-uterine interactions will lead to better definition of the interactions existing between the molecules involved in this process. The purpose of this review was not only to describe the different players of the implantation process but also to try to portray the relationship between these factors and their timing in the process of uterine receptivity. 10.1093/humupd/dml004
    The role of the endometrium and embryo in human implantation. Diedrich K,Fauser B C J M,Devroey P,Griesinger G, Human reproduction update Despite many advances in assisted reproductive technologies (ART), implantation rates are still low. The process of implantation requires a reciprocal interaction between blastocyst and endometrium, culminating in a small window of opportunity during which implantation can occur. This interaction involves the embryo, with its inherent molecular programme of cell growth and differentiation, and the temporal differentiation of endometrial cells to attain uterine receptivity. Implantation itself is governed by an array of endocrine, paracrine and autocrine modulators, of embryonic and maternal origin. Implantation failure is thought to occur as a consequence of impairment of embryo developmental potential and/or impairment of uterine receptivity and the embryo-uterine dialogue. Therefore a better comprehension of implantation, and the relative importance of the factors involved, is warranted. New techniques for monitoring changes in the endometrium and/or the embryo at the level of gene regulation and protein expression may lead to the identification of better markers for implantation. Moreover, the use of predictive sets of markers may prove to be more reliable than a single marker. Continuing refinements to ART protocols, such as optimizing ovarian stimulation regimens, the timing of human chorionic gonadotrophin injection, or the timing of embryo transfer, should help to increase implantation rates further. 10.1093/humupd/dmm011
    Estrogen and the endometrium: lessons learned from gene expression profiling in rodents and human. Groothuis P G,Dassen H H N M,Romano A,Punyadeera C Human reproduction update To date, research into the biological processes and molecular mechanisms associated with endometrial receptivity and embryo implantation has been a focus of attention, whereas the complex events that occur in the human endometrium during the menstrual and proliferative phase under the influence of estrogen have received little attention. The objective of this review is to provide an update of our current understanding of the actions of estrogen on both human and rodent endometrium, with special emphasis on the regulation of uterine growth and cell proliferation, and the value of global gene expression analysis, in increasing understanding of these processes. 10.1093/humupd/dmm009
    Implantation failure: molecular mechanisms and clinical treatment. Cakmak Hakan,Taylor Hugh S Human reproduction update BACKGROUND:Implantation is a complex initial step in the establishment of a successful pregnancy. Although embryo quality is an important determinant of implantation, temporally coordinated differentiation of endometrial cells to attain uterine receptivity and a synchronized dialog between maternal and embryonic tissues are crucial. The exact mechanism of implantation failure is still poorly understood. METHODS:This review summarizes the current knowledge about the proposed mechanisms of implantation failure in gynecological diseases, the evaluation of endometrial receptivity and the treatment methods to improve implantation. RESULTS:The absence or suppression of molecules essential for endometrial receptivity results in decreased implantation rates in animal models and gynecological diseases, including endometriosis, hydrosalpinx, leiomyoma and polycystic ovarian syndrome. The mechanisms are diverse and include abnormal cytokine and hormonal signaling as well as epigenetic alterations. CONCLUSIONS:Optimizing endometrial receptivity in fertility treatment will improve success rates. Evaluation of implantation markers may help to predict pregnancy outcome and detect occult implantation deficiency. Treating the underlying gynecological disease with medical or surgical interventions is the optimal current therapy. Manipulating the expression of key endometrial genes with gene or stem cell-based therapies may some day be used to further improve implantation rates. 10.1093/humupd/dmq037
    Polycomb subunit BMI1 determines uterine progesterone responsiveness essential for normal embryo implantation. Xin Qiliang,Kong Shuangbo,Yan Junhao,Qiu Jingtao,He Bo,Zhou Chan,Ni Zhangli,Bao Haili,Huang Lin,Lu Jinhua,Xia Guoliang,Liu Xicheng,Chen Zi-Jiang,Wang Chao,Wang Haibin The Journal of clinical investigation Natural and synthetic progestogens have been commonly used to prevent recurrent pregnancy loss in women with inadequate progesterone secretion or reduced progesterone sensitivity. However, the clinical efficacy of progesterone and its analogs for maintaining pregnancy is variable. Additionally, the underlying cause of impaired endometrial progesterone responsiveness during early pregnancy remains unknown. Here, we demonstrated that uterine-selective depletion of BMI1, a key component of the polycomb repressive complex-1 (PRC1), hampers uterine progesterone responsiveness and derails normal uterine receptivity, resulting in implantation failure in mice. We further uncovered genetic and biochemical evidence that BMI1 interacts with the progesterone receptor (PR) and the E3 ligase E6AP in a polycomb complex-independent manner and regulates the PR ubiquitination that is essential for normal progesterone responsiveness. A close association of aberrantly low endometrial BMI1 expression with restrained PR responsiveness in women who had previously had a miscarriage indicated that the role of BMI1 in endometrial PR function is conserved in mice and in humans. In addition to uncovering a potential regulatory mechanism of BMI1 that ensures normal endometrial progesterone responsiveness during early pregnancy, our findings have the potential to help clarify the underlying causes of spontaneous pregnancy loss in women. 10.1172/JCI92862
    The Multifaceted Actions of Leukaemia Inhibitory Factor in Mediating Uterine Receptivity and Embryo Implantation. Rosario Gracy Xavier,Stewart Colin L American journal of reproductive immunology (New York, N.Y. : 1989) Embryo implantation is mediated by the combined actions of the ovarian hormones E2 and P4 on the uterus. In addition, the pro-inflammatory cytokine, leukaemia inhibitory factor (LIF), plays a pivotal role in regulating uterine receptivity. LIF is expressed in the endometrial glands and has a robust action on the uterine luminal epithelium (LE). In mice, LIF is induced by nidatory E2 and functions to convert the LE from a non-receptive to an embryo-responsive state. LIF mediates its actions by activating the JAK-STAT pathway specifically in the LE. Activation of JAK-STAT pathway results in the induction of many additional pathways, including some 40 +  transcription factors, many of which initiate a cascade of changes affecting epithelial polarity, epithelial-mesenchymal interactions, angiogenesis, stromal cell decidualization, and inhibiting cell proliferation. This review discusses the role of LIF and the recent analysis of its action on the uterine LE in regulating endometrial receptivity and implantation. 10.1111/aji.12474
    Protein expression profiles in Meishan and Duroc sows during mid-gestation reveal differences affecting uterine capacity, endometrial receptivity, and the maternal-fetal Interface. Wang Kejun,Yang Kaijie,Xu Qiao,Liu Yufang,Li Wenting,Bai Ying,Wang Jve,Ding Cui,Liu Ximing,Tang Qiguo,Luo Yabiao,Zheng Jie,Wu Keliang,Fang Meiying BMC genomics BACKGROUND:Embryonic mortality is a major concern in the commercial swine industry and primarily occurs early in gestation, but also during mid-gestation (~ days 50-70). Previous reports demonstrated that the embryonic loss rate was significant lower in Meishan than in commercial breeds (including Duroc). Most studies have focused on embryonic mortality in early gestation, but little is known about embryonic loss during mid-gestation. RESULTS:In this study, protein expression patterns in endometrial tissue from Meishan and Duroc sows were examined during mid-gestation. A total of 2170 proteins were identified in both breeds. After statistical analysis, 70 and 114 differentially expressed proteins (DEPs) were identified in Meishan and Duroc sows, respectively. Between Meishan and Duroc sows, 114 DEPs were detected at day 49, and 98 DEPs were detected at day 72. Functional enrichment analysis revealed differences in protein expression patterns in the two breeds. Around half of DEPs were more highly expressed in Duroc at day 49 (DUD49), relative to DUD72 and Meishan at day 49 (MSD49). Many DEPs appear to be involved in metabolic process such as arginine metabolism. Our results suggest that the differences in expression affect uterine capacity, endometrial matrix remodeling, and maternal-embryo cross-talk, and may be major factors influencing the differences in embryonic loss between Meishan and Duroc sows during mid-gestation. CONCLUSIONS:Our data showed differential protein expression pattern in endometrium between Meishan and Duroc sows and provides insight into the development process of endometrium. These findings could help us further uncover the molecular mechanism involved in prolificacy. 10.1186/s12864-019-6353-2
    Skp2 Deteriorates the Uterine Receptivity by Interacting with HOXA10 and Promoting its Degradation. Han Keping,Zhou Qi Reproductive sciences (Thousand Oaks, Calif.) Receptive endometrium plays a core role in successful embryo implantation, and about one-third of repeated embryo implantation failures are attributed to endometrial receptive defects. S-phase kinase-associated protein 2 (SKP2), a member of the F-box protein family, plays an important role in many cellular processes, including cell proliferation and apoptosis. However, its role in endometrial receptivity is still unclear. Here, we identified SKP2 was obviously upregulated in the patients with infertility. Functional study showed that SKP2 overexpression inhibited endometrial epithelial cell (EEC) proliferation, whereas SKP2 knockdown promoted the proliferation of EECs. In addition, the overexpression of SKP2 also repressed adhesion rate of embryonic cells to EECs. In vivo studies further suggested that the upregulation of SKP2 obviously suppressed endometrium receptivity formation and embryo implantation potential. Mechanistical study clarified that SKP2 directly interacted with HOXA10 and decreased protein stability through promoting the ubiquitin-mediated proteasome degradation of HOXA10. In conclusion, the current study documented that the high expression of SKP2 deteriorates endometrial receptivity formation by decreasing the HOXA10 expression and suggested that SKP2 may be defined as a marker of endometrial receptivity, and as a target for the diagnosis and treatment of infertility. 10.1007/s43032-020-00367-4
    Integrin-linked kinase improves uterine receptivity formation by activating Wnt/β-catenin signaling and up-regulating MMP-3/9 expression. Chen Qian,Ni Ying,Han Mi,Zhou Wen-Jie,Zhu Xiao-Bin,Zhang Ai-Jun American journal of translational research A receptive endometrium is a prerequisite for successful embryo implantation, and about one-third of repeated embryo implantation failure attribute to defective endometrial receptivity. Integrin-linked kinase (ILK), a 59kDa serine/threonine-protein kinase, plays a vital role in multiple cellular processes, including cell proliferation, apoptosis, and invasion. However, its role in endometrial receptivity is still unclear. In the current study, we demonstrated that ILK level was significantly downregulated in the serum of patients with unexplained infertility compared with healthy non-pregnancy. Functionally, ILK knockdown inhibited endometrial epithelial cells (EECs) proliferation and invasion, whereas ILK overexpression promoted endometrial EECs proliferation and invasion. ILK inhibition also repressed the adhesion rate of embryonic cells to EECs. studies further demonstrated that ILK inhibition suppressed endometrium receptivity formation and embryo implantation potential. Mechanistically, the downregulation of ILK inactivated Wnt/β-catenin signaling and thus resulted in the downregulation of MMP-3 and MMP-9 expression. Importantly, activation of Wnt/β-catenin signaling, partially recovered ILK inhibition-caused endometrium receptivity defects, and embryo implantation failure. Considered all the current data, it verified that the low expression of ILK exacerbates endometrial receptivity formation by inactivating Wnt/β-catenin signaling and decreasing the MMP-3/9 expression and indicated that ILK may be applied as an indicator of endometrial receptivity, and as a diagnostic and therapeutic target for infertility.
    Molecular determinants of uterine receptivity. Tu Zhaowei,Ran Hao,Zhang Shuang,Xia Guoliang,Wang Bingyan,Wang Haibin The International journal of developmental biology Uterine receptivity is defined as a limited period when the uterine environment is conducive to blastocyst acceptance and implantation. Any disturbance of this early pregnancy event will compromise pregnancy success. In this review, we first briefly summarize uterine morphological coordination for the attainment of receptivity, then focus on elucidating the molecular complexity in establishing uterine receptivity and hence embryo implantation. A better understanding of the molecular basis governing uterine receptivity will help to improve the outcome of natural pregnancy and pregnancy conceived via assisted reproductive techniques. 10.1387/ijdb.130345wh
    Secrets of Endometrial Receptivity: Some Are Hidden in Uterine Secretome. Bhusane Kashmira,Bhutada Sumit,Chaudhari Uddhav,Savardekar Lalita,Katkam Rajendra,Sachdeva Geetanjali American journal of reproductive immunology (New York, N.Y. : 1989) PROBLEM:Endometrium, the innermost mucosal layer of the uterus, serves as a lodge for the embryo in eutherian mammals. The endometrium is constituted of various cell types, and each cell type executes specific functions to facilitate embryo implantation and development. It is well established that the endometrium, despite being non-permissive to the embryo for the major period of a menstrual cycle, is irreplaceable in the scheme of events essential for procreation. However, the embryo, before initiating physical contact with the endometrium, encounters the uterine cavity that remains bathed in uterine fluid. Uterine fluid is an admixture of endometrial secretions, plasma transudates, and oviductal fluid. Uterine fluid components are believed to play important roles in immunosuppression and embryo development during peri-implantation period. Uterine fluid is also involved in defense against pathogens, sperm migration, and lubrication of endometrium. The advent of high-throughput functional genomics tools has created enormous opportunities to investigate the uterine fluid for its protein repertoire and modulation during the receptive phase of an endometrial cycle in animals and humans. Towards this, few investigations have been conducted in recent years. The data obtained using non-targetted functional genomics approaches need to be assimilated with the existing information on specific components of uterine fluid. METHOD:This review compiles existing information on the composition of uterine fluid and its significance in endometrial functions and dysfunctions. RESULT:Collectively, investigations based on targetted and non-targetted approaches have revealed the presence of several cytokines, growth factors, ions, carbohydrates, and steroids, in human uterine fluid. CONCLUSION:Detailed investigations of human uterine fluid, especially directed towards the elucidation of functional relevance of different proteins in uterine fluid, will help identify novel markers of endometrial receptivity and also gain significant insights into the mechanisms underlying unexplained infertility, recurrent pregnancy losses, and other endometrial pathologies. 10.1111/aji.12472
    The sialyltransferase ST3Gal3 facilitates the receptivity of the uterine endometrium in vitro and in vivo. Yu Ming,Wang Hao,Liu Jianwei,Qin Huamin,Liu Shuai,Yan Qiu FEBS letters The receptive uterine endometrium specifically expresses certain glycosyltransferases, and the corresponding oligosaccharides play important roles in accepting the embryo. The sialyltransferase β-galactoside-α2,3-sialyltransferase III (ST3Gal3) is the key enzyme responsible for sialyl Lewis X (sLeX) oligosaccharide biosynthesis, but the expression and function of ST3Gal3 in the receptive endometrium is still elusive. Here, we found that human endometrial tissues at secretory phase expressed a 4-fold higher ST3Gal3 level relative to the tissues at proliferative phase. Meanwhile, downregulation of ST3Gal3 or sLeX epitope blockage significantly impaired the receptive ability of human endometrial RL95-2 cells to trophoblastic cells in vitro and inhibited implantation in pregnant mice. This study suggests that ST3Gal3 facilitates endometrial receptivity through increasing sLeX oligosaccharide, which gives a better understanding of the glycobiology of implantation. 10.1002/1873-3468.13252
    miR-192-5p suppresses uterine receptivity formation through impeding epithelial transformation during embryo implantation. Theriogenology The establishment of uterine receptivity is a prerequisite for embryo implantation and begins with the transformation of the luminal epithelium. MicroRNAs (miRNAs) have been widely reported to be involved in the regulation of embryo implantation, but their roles in establishing uterine receptivity remain unclear. In this study, through small RNA sequencing analysis, we showed that a low level of miR-192-5p is essential for initiating implantation in mice, and transient upregulation of miR-192-5p led to implantation failure. In situ hybridization results revealed that miR-192-5p was primarily expressed in the endometrial epithelium, and dysregulation of miR-192-5p interfered with the performance of the luminal epithelium, resulting in inadequate receptivity. By manipulating miR-192-5p expression in mouse uterus and an endometrial epithelial cell line, we showed that miR-192-5p maintains cell polarity through stabilizing adherens junction protein E-cadherin, thereby preventing epithelial-mesenchymal transition. Furthermore, miR-192-5p preserved the pattern of microvilli as well as Muc1 expression on the apical membrane of epithelial cells, thereby avoiding embryo adhesion. Moreover, miR-192-5p was found to be regulated by ovarian steroids. Collectively, this study demonstrated that the physiological role of miR-192-5p in mouse uterus is to maintain the nonreceptive state of epithelial cells and prevent their transformation to the receptive state. Thus, a sustained high level of miR-192-5p is detrimental to embryo implantation. These findings help elucidate the mechanisms involved in miRNA-based regulation of uterine physiology in early pregnancy, and may even contribute to the diagnosis and treatment of infertility. 10.1016/j.theriogenology.2020.08.009
    Follistatin is critical for mouse uterine receptivity and decidualization. Fullerton Paul T,Monsivais Diana,Kommagani Ramakrishna,Matzuk Martin M Proceedings of the National Academy of Sciences of the United States of America Embryo implantation remains a significant challenge for assisted reproductive technology, with implantation failure occurring in ∼50% of in vitro fertilization attempts. Understanding the molecular mechanisms underlying uterine receptivity will enable the development of new interventions and biomarkers. TGFβ family signaling in the uterus is critical for establishing and maintaining pregnancy. Follistatin (FST) regulates TGFβ family signaling by selectively binding TGFβ family ligands and sequestering them. In humans, FST is up-regulated in the decidua during early pregnancy, and women with recurrent miscarriage have lower endometrial expression of FST during the luteal phase. Because global knockout of is perinatal lethal in mice, we generated a conditional knockout (cKO) of in the uterus using progesterone receptor-cre to study the roles of uterine during pregnancy. Uterine -cKO mice demonstrate severe fertility defects and deliver only 2% of the number of pups delivered by control females. In -cKO mice, the uterine luminal epithelium does not respond properly to estrogen and progesterone signals and remains unreceptive to embryo attachment by continuing to proliferate and failing to differentiate. The uterine stroma of -cKO mice also responds poorly to artificial decidualization, with lower levels of proliferation and differentiation. In the absence of uterine FST, activin B expression and signaling are up-regulated, and bone morphogenetic protein (BMP) signals are impaired. Our findings support a model in which repression of activin signaling by FST enables uterine receptivity by preserving critical BMP signaling. 10.1073/pnas.1620903114
    Mir-let-7a/g Enhances Uterine Receptivity via Suppressing Wnt/β-Catenin Under the Modulation of Ovarian Hormones. Li Qian,Liu Weimin,Chiu Philip C N,Yeung William S B Reproductive sciences (Thousand Oaks, Calif.) Microarray has indicated a huge number of miRNAs exist in reproductive tissues and cells. Moreover, the expression of miRNA in the reproductive system varies under the strict monitoring of different regulations. To understand the role of miRNA-mediated post-transcriptional gene regulation in female reproduction, we investigated the level and function of a mir-let-7 family member in both mice and human uterine receptivity. As we observed, mir-let-7 a/g had a higher expression in mouse and human receptive uterine epithelium; the level of mir-let-7a was under the inverse regulation of estrogen and progesterone; upregulated mir-let-7a/g in mouse and human uterine epithelium increased uterine receptivity, thus improved implantation-related embryo attachment and outgrowth ability; the let-7a/g enhanced uterine receptivity through suppressing canonical Wnt signaling. In summary, our findings suggest that mir-let-7 a/g increases uterine receptivity via inhibiting Wnt signaling and under the modulation of ovarian hormones. 10.1007/s43032-019-00115-3
    Uterine Epithelial Progesterone Receptor Governs Uterine Receptivity Through Epithelial Cell Differentiation. Gebril Mona,Hirota Yasushi,Aikawa Shizu,Fukui Yamato,Kaku Tetsuaki,Matsuo Mitsunori,Hirata Tomoyuki,Akaeda Shun,Hiraoka Takehiro,Shimizu-Hirota Ryoko,Takeda Norihiko,Taha Tamer,Balah Osama Al,Elnoury Mohamed Amr H,Fujii Tomoyuki,Osuga Yutaka Endocrinology Progesterone receptor (PGR) is indispensable for pregnancy in mammals. Uterine PGR responds to the heightened levels of ovarian progesterone (P4) after ovulation and regulates uterine gene transcription for successful embryo implantation. Although epithelial and stromal P4-PGR signaling may interact with each other to form appropriate endometrial milieu for uterine receptivity and the subsequent embryo attachment, it remains unclear what the specific roles of epithelial P4-PGR signaling in the adult uterus are. Here we generated mice with epithelial deletion of Pgr in the adult uterus (Pgrfl/flLtfCre/+ mice) by crossing Pgr-floxed and Ltf-Cre mice. Pgrfl/flLtfCre/+ mice are infertile due to the impairment of embryo attachment. Pgrfl/flLtfCre/+ uteri did not exhibit epithelial growth arrest, suggesting compromised uterine receptivity. Both epithelial and stromal expressions of P4-responsive genes decreased in Pgrfl/flLtfCre/+ mice during the peri-implantation period, indicating that epithelial Pgr deletion affects not only epithelial but stromal P4 responsiveness. In addition, uterine LIF, an inducer of embryo attachment, was decreased in Pgrfl/flLtfCre/+ mice. The RNA-seq analysis using luminal epithelial specimens dissected out by laser capture microdissection revealed that the signaling pathways related to extracellular matrix, cell adhesion, and cell proliferation are altered in Pgr fl/flLtf Cre/+ mice. These findings suggest that epithelial PGR controls both epithelial and stromal P4 responsiveness and epithelial cell differentiation, which provides normal uterine receptivity and subsequent embryo attachment. 10.1210/endocr/bqaa195
    Estrogen-induced transcription factor EGR1 regulates c-Kit transcription in the mouse uterus to maintain uterine receptivity for embryo implantation. Park Mira,Kim Hye-Ryun,Kim Yeon Sun,Yang Seung Chel,Yoon Jung Ah,Lyu Sang Woo,Lim Hyunjung Jade,Hong Seok-Ho,Song Haengseok Molecular and cellular endocrinology Early growth response 1 (Egr1) is a key transcription factor that mediates the action of estrogen (E) to establish uterine receptivity for embryo implantation. However, few direct target genes of EGR1 have been identified in the uterus. Here, we demonstrated that E induced EGR1-regulated transcription of c-Kit, which plays a crucial role in cell fate decisions. Spatiotemporal expression of c-Kit followed that of EGR1 in uteri of ovariectomized mice at various time points after E treatment. E activated ERK1/2 and p38 to induce EGR1, which then activated c-Kit expression in the uterus. EGR1 transfection produced rapid and transient induction of c-KIT in a time- and dose-dependent manner. Furthermore, luciferase assays to measure c-Kit promoter activity confirmed that a functional EGR1 binding site(s) (EBS) was located within -1 kb of the c-Kit promoter. Site-directed mutagenesis and chromatin immunoprecipitation-PCR for three putative EBS within -1 kb demonstrated that the EBS at -818/-805 was critical for EGR1-dependent c-Kit transcription. c-Kit expression was significantly increased in the uterus on day 4 and administration of Masitinib, a c-Kit inhibitor, effectively interfered with embryo implantation. Collectively, our results showed that estrogen induces transcription factor EGR1 to regulate c-Kit transcription for uterine receptivity for embryo implantation in the mouse uterus. 10.1016/j.mce.2017.09.033
    Dysregulated Pseudogene May Contribute to Preeclampsia as a Competing Endogenous RNA for Hexokinase 2 by Impairing Decidualization. Lv Hong,Tong Jing,Yang Jieqiong,Lv Shijian,Li Wei-Ping,Zhang Cong,Chen Zi-Jiang Hypertension (Dallas, Tex. : 1979) Preeclampsia is a pregnancy-specific hypertensive disorder, which seriously undermines the health of maternity and fetus. However, its cause and pathogenesis remain elusive. Flawed decidualization is considered to be related to preeclampsia. Increasing evidence indicates that long noncoding RNAs are correlated with a variety of diseases, including preeclampsia. In this study, we verified the expression of long noncoding RNA (hexokinase 2 pseudogene 1) and its cognate gene (hexokinase 2), which were found by our previous RNA-sequencing analysis in the decidua of severe preeclampsia patients and matched control subjects. Besides that, we also investigated the function and the mechanism of and during decidualization. HK2 is the crucial enzyme involved in glycolysis. The and genes are homologous to each other. The results demonstrated that , like , stimulated the glucose uptake and lactate production of human endometrial stromal cells. In addition, and are indispensable for endometrial decidualization. Downregulated or inhibited human endometrial stromal cells proliferation and differentiation. Furthermore, there was a significant positive correlation between the expression of and , and regulated the expression via competition for the shared miR-6887-3p. Taken together, our results indicated that the reduced expression of and may have contributed to the occurrence and development of preeclampsia by suppressing glycolysis and impairing decidualization. Our study would be helpful to understand the pathogenesis and the regulatory network of preeclampsia. 10.1161/HYPERTENSIONAHA.117.10084
    microRNA-665 is down-regulated in gastric cancer and inhibits proliferation, invasion, and EMT by targeting PPP2R2A. Zhang Mingjuan,Wang Su,Yi Aiwen,Qiao Yongsheng Cell biochemistry and function Recently, microRNA-665 (miR-665) has been reported to function as both tumour suppressor and oncogene in several cancer types, including gastric cancer, hepatocellular cancer, and lung cancer. However, the biological function of miR-665 and its precise mechanisms in gastric cancer (GC) have not been well clarified. The aim of this study was to study the roles of miR-665/PPP2R2A axis in GC. The levels of PPP2R2A and miR-665 were detected by quantitative PCR assay in GC tissues and cell lines. Moreover, the biological roles of miR-665 and PPP2R2A in GC cells were assessed by cell proliferation, invasion, and epithelial-mesenchymal transition (EMT). The mRNA and protein levels of PPP2R2A were determined by using quantitative PCR and Western blotting assays. Luciferase assays were used to confirm that PPP2R2A was one target of miR-665. In this study, the miR-665 level was dramatically reduced in GC tissues and cell lines, and the PPP2R2A expression was significantly enhanced. What is more, the PPP2R2A expression was negatively related to the miR-665 level in GC tissues. Furthermore, up-regulation of miR-665 obviously restrained GC cells proliferation, invasion, and EMT. We confirmed that miR-665 could directly target PPP2R2A by luciferase reporter assay. Besides, knockdown of PPP2R2A also could markedly inhibit the proliferation, invasion and EMT of GC cells. Finally, overexpression of miR-665 in GC cells partially reversed the promoted effects of PPP2R2A up-regulation. Overexpression of miR-665 restrained GC cells proliferation, invasion and EMT via regulation of PPP2R2A. SIGNIFICANCE OF THE STUDY:  miR-665 has been reported to function as oncogene or tumour suppressor in different cancers. However, the precise roles of miR-665 in GC have not been elucidated. Our study for the first time demonstrated that miR-665 level was significantly down-regulated in GC. Additionally, miR-665 overexpression inhibited cell growth, invasion, and EMT of GC. Moreover, our data suggested a significant negative correlation between miR-665 and PPP2R2A expression in GC. MiR-665 suppressed GC cell proliferation, invasion, and EMT by directly targeting PPP2R2A, which suggested important roles for miR-665/PPP2R2A axis in the GC pathogenesis and its potential application in cancer therapy. 10.1002/cbf.3485
    microRNA-211-mediated targeting of the INHBA-TGF-β axis suppresses prostate tumor formation and growth. Cancer gene therapy Prostate cancer (PCa) stem cells increase the sustainability of tumor growth, resulting in high relapse rates in patients with PCa. This goal of the present study was to elucidate the function of microRNA (miR)-211 in PCa stem cell activities. Based on the initial findings from the GSE26910 dataset, inhibin-β A (INHBA) was used for subsequent experiments, and miR-211 was then predicted as a candidate regulatory miR. Subsequently, INHBA and miR-211 were observed to be highly and poorly expressed in PCa tissues, respectively, and miR-211 negatively target INHBA. CD44CD133 cells were isolated, and both miR-211 and INHBA expression was altered in these cells to assess functional role of miR-211 and INHBA in PCa stem cells. Overexpression of miR-211 decreased expression of TGF-β1, TGF-β2, smad2, smad3, phosphorylated smad2 and smad3, and stem cell markers. miR-211 upregulation or INHBA knockdown resulted in reductions in the proliferation, invasion, colony-forming ability, sphere-forming ability, and stemness of PCa stem cells but enhanced their apoptosis in vitro. Furthermore, miR-211 upregulation or INHBA silencing decreased tumor growth and cell apoptosis in vivo. Taken together, these results indicate that upregulation of miR-211 has tumor-suppressive properties by inhibiting TGF-β pathway activation via INHBA in PCa stem cells. 10.1038/s41417-020-00237-w
    LncRNA INHBA-AS1 promotes colorectal cancer cell proliferation by sponging miR-422a to increase AKT1 axis. Lin H,Hong Y-G,Zhou J-D,Gao X-H,Yuan P-H,Xin C,Huang Z-P,Zhang W,Hao L-Q,Hou K-Z European review for medical and pharmacological sciences OBJECTIVE:In recent years, long non-coding RNAs (lncRNAs) have emerged for regulating the development, as well as progression in colorectal cancer (CRC), which assists in finding new targets for CRC treatment. A previous study indicated that INHBA-AS1 promotes oral squamous cell progression by sponging miR-143-3p. However, the exact function possessed by lncRNA INHBA-AS1 in CRC development remains unclear. PATIENTS AND METHODS:The expression level of INHBA-AS1 in CRC tissues and cell lines was determined by qRT-PCR. The functional role of INHBA-AS1 in CRC was investigated by a series of in vitro assays. RNA immunoprecipitation (RIP), bioinformatics analysis was utilized to explore the potential mechanisms of INHBA-AS1. RESULTS:The present study identified INHBA-AS1 as a kind of lncRNA with high expression in CRC tissues and cells. Functionally, NHBA-AS1 downregulation in CRC cells suppressed CRC cell proliferation as well as colony formability. Mechanistically, INHBA-AS1/miR-422a/AKT1 established the ceRNA network to regulate MMP-2, -7, -9 expressions that participated the modulation of CRC progression. CONCLUSIONS:In summary, LncRNA INHBA-AS1 contributes to CRC progression through AKT1 pathway, and provides a new mechanism to regulate CRC development, as well as a potential target for treating CRC. 10.26355/eurrev_202010_23206
    Zinc is essential for the transcription function of the PGC-1α/Nrf2 signaling pathway in human primary endometrial stromal cells. Lu Xiaodan,Zhang Qiang,Xu Li,Lin Xiuying,Fu Jianhua,Wang Xue,Liu Yinong,Lin Yifan,Li Bing,Wang Ruobing,Liu Lei,Mi Xuguang,Wei Haifeng,Tan Yan,Fang Yanqiu American journal of physiology. Cell physiology Zinc (Zn) has antioxidant effect in different types of organs and is closely associated with human health. Endometrial receptivity is one of the most important factors in the embryo implantation and development. However, the regulatory mechanism of Zn in endometrium tissue is still unclear. In the study, we found that plasma Zn level is significantly associated with female infertility, which severely affects female reproductive health. Primary endometrial stromal cells were isolated from female endometrium and cultured in the laboratory. Zn chelator TPEN treatment reduced the expression of stem cell markers CD73, CD90, and CD105 and generated reactive oxygen species in endometrial stromal cells. However, pretreatment of Zn (zinc sulfate) is able to prevent TPEN-induced oxidative stress in vitro. By transcriptional profiling and gene ontology analysis, we found that Zn increased the cellular pluripotency signaling and extracellular matrix-receptor interaction, but reduced autophagy, endocytosis, and the nitrogen metabolism pathway. We further discovered the antioxidant function of Zn through the peroxisome proliferator-activated receptor gamma coactivator 1α/nuclear factor erythroid-2-related factor signaling pathway in endometrial stromal cells. Zn supplementation may open up an effective therapeutic approach for patients with oxidative stress-related endometrial diseases. 10.1152/ajpcell.00152.2019
    An autophagic deficit in the uterine vessel microenvironment provokes hyperpermeability through deregulated VEGFA, NOS1, and CTNNB1. Lee Bora,Shin Hyejin,Oh Ji-Eun,Park Jaekyoung,Park Mira,Yang Seung Chel,Jun Jin-Hyun,Hong Seok-Ho,Song Haengseok,Lim Hyunjung Jade Autophagy The uterus undergoes vascular changes during the reproductive cycle and pregnancy. Steroid hormone deprivation induces macroautophagy/autophagy in major uterine cell types. Herein, we explored the functions of uterine autophagy using the -driven deletion model. Deletion of was confirmed by functional deficit of autophagy in uterine stromal, myometrial, and vascular smooth muscle cells, but not in endothelial cells. uteri exhibited enhanced stromal edema accompanied by dilation of blood vessels. Ovariectomized uteri showed decreased expression of endothelial junction-related proteins, such as CTNNB1/beta-catenin, with increased vascular permeability, and increased expression of VEGFA and NOS1. Nitric oxide (NO) was shown to mediate VEGFA-induced vascular permeability by targeting CTNNB1. NO involvement in maintaining endothelial junctional stability in uteri was confirmed by the reduction in extravasation following treatment with a NOS inhibitor. We also showed that uterine phenotype improved the fetal weight:placental weight ratio, which is one of the indicators of assessing the status of preeclampsia. We showed that autophagic deficit in the uterine vessel microenvironment provokes hyperpermeability through the deregulation of VEGFA, NOS1, and CTNNB1. ACTA2: actin, alpha 2, smooth muscle, aortic; : anti-Mullerian hormone type 2 receptor; ANGPT1: angiopoietin 1; ATG: autophagy-related; CDH5: cadherin 5; CLDN5: claudin 5; COL1A1: collagen, type I, alpha 1; CSPG4/NG2: chondroitin sulfate proteoglycan 4; CTNNB1: catenin (cadherin associated protein), beta 1; DES: desmin; EDN1: endothelin 1; EDNRB: endothelin receptor type B; F3: coagulation factor III; KDR/FLK1/VEGFR2: kinase insert domain protein receptor; LYVE1: lymphatic vessel endothelial hyaluronan receptor 1; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MCAM/CD146: melanoma cell adhesion molecule; MYL2: myosin, light polypeptide 2, regulatory, cardiac, slow; MYLK: myosin, light polypeptide kinase; NOS1/nNOS: nitric oxide synthase 1, neuronal; NOS2/iNOS: nitric oxide synthase 2, inducible; NOS3/eNOS: nitric oxide synthase 3, endothelial cell; OVX: ovariectomy; PECAM1/CD31: platelet/endothelial cell adhesion molecule 1; POSTN: periostin, osteoblast specific factor; SQSTM1: sequestosome 1; TEK/Tie2: TEK receptor tyrosine kinase; TJP1/ZO-1: tight junction protein 1; TUBB1, tubulin, beta 1 class VI; USC: uterine stromal cell; VEGFA: vascular endothelial growth factor A; VSMC: vascular smooth muscle cell. 10.1080/15548627.2020.1778292
    Decreased Indian hedgehog signaling activates autophagy in endometriosis and adenomyosis. Zhou Yingying,Peng Yangying,Xia Qingqing,Yan Dewen,Zhang Huiping,Zhang Lingmin,Chen Ying,Zhao Xiumin,Li Jie Reproduction (Cambridge, England) Indian hedgehog (Ihh) signaling regulates endometrial receptivity and is an indispensable mediator of embryonic implantation. Hedgehog signaling is known to regulate autophagy, and aberrant regulation of autophagy is critically implicated in the pathogenesis of endometriosis and adenomyosis. However, potential dysregulation of Ihh signaling and its role in autophagy modulation in these diseases remain obscure. In this study, we found that components of Ihh signaling were significantly decreased, whereas the autophagy marker protein, LC3BII, was significantly increased in endometrial tissues of women with endometriosis or adenomyosis. Inhibition of Ihh signaling with the small-molecule inhibitor GANT61 or Gli1 silencing in primary endometrial stromal cells increased autophagic activity, as measured by LC3 turnover assay and tandem mCherry-eGFP-LC3B fluorescence microscopy. Furthermore, we observed that GANT61 treatment significantly attenuated hydrogen peroxide-induced cell death, whereas disruption of autophagy with chloroquine diminished this effect. Collectively, these findings reveal that Ihh signaling is suppressed in endometrial tissues of patients with endometriosis or adenomyosis. This abnormal decrease may contribute to endometrial autophagy activation, which may promote aberrant survival of endometrial cells in ectopic sites in these two gynecological diseases. 10.1530/REP-20-0172
    Estrogen-induced FOS-like 1 regulates matrix metalloproteinase expression and the motility of human endometrial and decidual stromal cells. Chen Chao,Li Congcong,Liu Weichun,Guo Feng,Kou Xi,Sun Si,Ye Taiyang,Li Shanji,Zhao Aimin The Journal of biological chemistry The regulation mechanisms involved in matrix metalloproteinase () expression and the motility of human endometrial and decidual stromal cells (ESCs and DSCs, respectively) during decidualization remain unclear. DSCs show significant increased cell motility and expression of FOS-like 1 () and , , and compared with ESCs, whereas lack of decidualization inducers leads to a rapid decrease in and and expression in DSCs Therefore, we hypothesized that a link exists between decidualization inducers and FOSL1 in up-regulation of motility during decidualization. Based on the response of ESCs/DSCs to different decidualization systems , we found that progesterone (P4) alone had no significant effect and that 17β-estradiol (E2) significantly increased cell motility and and and expression at the mRNA and protein levels, whereas 8-bromo-cAMP significantly decreased cell motility and and expression in the presence of P4. In addition, we showed that E2 triggered phosphorylation of estrogen receptor 1 (ESR1), which could directly bind to the promoter of in ESCs/DSCs. Additionally, we also revealed silencing of expression by siRNA abrogated E2-induced expression at the transcript and protein levels. Moreover, silencing of expression by siRNA was able to block E2-induced and expression and cell motility in ESCs/DSCs. Taken together, our data suggest that, in addition to its enhancement of secretory function, the change in expression and cell motility is another component of the decidualization of ESCs/DSCs, including estrogen-dependent and expression mediated by E2-ESR1-FOSL1 signaling. 10.1074/jbc.RA119.010701
    MicroRNA-136 Promotes Vascular Muscle Cell Proliferation Through the ERK1/2 Pathway by Targeting PPP2R2A in Atherosclerosis. Zhang Chun-feng,Kang Kai,Li Xin-miao,Xie Bao-dong Current vascular pharmacology Aberrant proliferation of vascular smooth muscle cells [VSMCs] is implicated in the pathogenesis of vascular pathologies such as atherosclerosis and restenosis. Accumulating evidences have revealed that microRNAs are involved in cell proliferation in various pathological conditions. In the present study, we showed that miR-136 was up regulated in human coronary atherosclerotic plaques when compared with normal coronary artery tissues. Moreover, miR-136 levels were up regulated in proliferative vascular smooth muscle cells induced by platelet-derived growth factor [PDGF] or serum. In cultured VSMCs, over expression of miR-136 stimulated cell proliferation. PPP2R2A was proved to be the direct target gene of miR-136 and knockdown of PPP2R2A had a proliferative effect on VSMCs. miR-136-induced PPP2R2A down-regulation was accompanied by increased expression of ERK1/2 phosphorylation. Inhibition of ERK1/2 abolished the effect of miR-136 and knockdown of PPP2R2A on VSMCs proliferation. In summary, aberrant miR-136 up regulation in atherosclerosis contributes to abnormal VSMC proliferation through suppressing the ERK1/2 pathway by targeting PPP2R2A. Our study also suggested that specific modulation of miR-136 in human VSMCs may provide a potential approach for the treatment of atherosclerosis. 10.2174/1570161112666141118094612
    An Emerging Role for isomiRs and the microRNA Epitranscriptome in Neovascularization. van der Kwast Reginald V C T,Quax Paul H A,Nossent A Yaël Cells Therapeutic neovascularization can facilitate blood flow recovery in patients with ischemic cardiovascular disease, the leading cause of death worldwide. Neovascularization encompasses both angiogenesis, the sprouting of new capillaries from existing vessels, and arteriogenesis, the maturation of preexisting collateral arterioles into fully functional arteries. Both angiogenesis and arteriogenesis are highly multifactorial processes that require a multifactorial regulator to be stimulated simultaneously. MicroRNAs can regulate both angiogenesis and arteriogenesis due to their ability to modulate expression of many genes simultaneously. Recent studies have revealed that many microRNAs have variants with altered terminal sequences, known as isomiRs. Additionally, endogenous microRNAs have been identified that carry biochemically modified nucleotides, revealing a dynamic microRNA epitranscriptome. Both types of microRNA alterations were shown to be dynamically regulated in response to ischemia and are able to influence neovascularization by affecting the microRNA's biogenesis, or even its silencing activity. Therefore, these novel regulatory layers influence microRNA functioning and could provide new opportunities to stimulate neovascularization. In this review we will highlight the formation and function of isomiRs and various forms of microRNA modifications, and discuss recent findings that demonstrate that both isomiRs and microRNA modifications directly affect neovascularization and vascular remodeling. 10.3390/cells9010061
    Effect of Oxidative Stress on the Estrogen-NOS-NO-K Channel Pathway in Uteroplacental Dysfunction: Its Implication in Pregnancy Complications. Hu Xiang-Qun,Song Rui,Zhang Lubo Oxidative medicine and cellular longevity During pregnancy, the adaptive changes in uterine circulation and the formation of the placenta are essential for the growth of the fetus and the well-being of the mother. The steroid hormone estrogen plays a pivotal role in this adaptive process. An insufficient blood supply to the placenta due to uteroplacental dysfunction has been associated with pregnancy complications including preeclampsia and intrauterine fetal growth restriction (IUGR). Oxidative stress is caused by an imbalance between free radical formation and antioxidant defense. Pregnancy itself presents a mild oxidative stress, which is exaggerated in pregnancy complications. Increasing evidence indicates that oxidative stress plays an important role in the maladaptation of uteroplacental circulation partly by impairing estrogen signaling pathways. This review is aimed at providing both an overview of our current understanding of regulation of the estrogen-NOS-NO-K pathway by reactive oxygen species (ROS) in uteroplacental tissues and a link between oxidative stress and uteroplacental dysfunction in pregnancy complications. A better understanding of the mechanisms will facilitate the development of novel and effective therapeutic interventions. 10.1155/2019/9194269
    Increased expression of neurogenic factors in uterine fibroids. Luddi Alice,Marrocco Camilla,Governini Laura,Semplici Bianca,Pavone Valentina,Capaldo Angela,Tosti Claudia,Greco Stefania,Luisi Stefano,Ciarmela Pasquapina,Petraglia Felice,Piomboni Paola Human reproduction (Oxford, England) STUDY QUESTION:Are selective markers for the neuronal differentiation such as microtubule-associated protein 2 (MAP-2) and synaptophysin (SYP) as well as the nerve growth factor (NGF) expressed by fibroids, myometrium and eutopic endometrium? SUMMARY ANSWER:Neuronal markers NGF, MAP-2 and SYP are highly expressed in fibroids compared with matched myometrium, and this neurogenic pathway is upregulated by tumor necrosis factor (TNF) alpha in cultured smooth muscle cells (SMCs). WHAT IS KNOWN ALREADY:Uterine fibroids or leiomyomas are the most common benign tumors, accounting for approximately one-third of hysterectomies. The present trend is to improve the medical treatment avoiding surgery, also for fertility sparing; hence, the pathogenic mechanisms are investigated, aiming to develop new therapeutic strategy. STUDY DESIGN, SIZE, DURATION:This laboratory-based case-control study is focused on fibroids and myometrial specimens obtained between 2015 and 2017 from 15 women of reproductive age at the proliferative phase of the menstrual cycle. Leiomyomas, matched myometrium and endometrium from each woman were analyzed. Control endometrium was obtained from women undergoing surgery for ovarian cyst (n = 15). PARTICIPANTS/MATERIALS, SETTING, METHODS:qRT-PCR, western blotting and immunostaining were applied to evaluate the expression of neurogenic markers; the effects of TNF on NGF, MAP-2 and SYP expression in cultured SMCs from leiomyomas and matched myometrium were analyzed. MAIN RESULTS AND THE ROLE OF CHANCE:qRT-PCR analyses using tissues from clinical patients showed that the levels of NGF, MAP-2 and SYP mRNA were significantly higher in uterine leiomyomas compared with their matched myometrium (P < 0.05), whereas only NGF was significantly increased in eutopic endometrium compared with healthy endometrium. In primary SMCs, isolated from fibroids or from the adjacent myometrium, NGF, MAP-2 and SYP mRNA expression were significantly increased by TNF treatment (P < 0.05). Finally, human endometrial stromal cells prepared from the endometrium of patients affected by uterine fibroids display higher TNF expression (P < 0.001). LIMITATIONS, REASONS FOR CAUTION:qRT-PCR analysis and immunofluorescence validation are robust methods demonstrating a clear upregulation of neurogenic factors in leiomyomas, even though additional studies are needed to establish a correlation between increased neuronal gene expression and degree of pain, as well as the involvement of inflammation mediators in the development of the neurogenic unhinge. Therefore, more in vivo studies are needed to confirm the results achieved from primary cultured SMCs. WIDER IMPLICATIONS OF THE FINDINGS:The increased expression of neurogenic factors in uterine fibroids and endometrium may contribute to explain the painful stimuli. Accordingly, these neurogenic pathways may represent potential therapeutic avenues to treat the fibroid-related disorders. STUDY FUNDING/COMPETING INTEREST(S):This study was supported by research grants from the University of Siena. The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER:N/A. 10.1093/humrep/dez182
    Epigenetic modifications working in the decidualization and endometrial receptivity. Liu Hong,Huang Xiaobo,Mor Gil,Liao Aihua Cellular and molecular life sciences : CMLS Decidualization is a critical event for the blastocyst implantation, placental development and fetal growth and the normal term. In mice, the embryo implantation to the uterine epithelial would trigger the endometrial stromal cells to differentiate into decidual stromal cells. However, decidualization in women takes place from the secretory phase of each menstrual cycle and continues to early pregnancy if there is conceptus. Deficient decidualization is often associated with pregnancy specific complications and reproductive disorders. Dramatic changes occur in the gene expression profiles during decidualization, which is coordinately regulated by steroid hormones, growth factors, and molecular and epigenetic mechanisms. Recently, emerging evidences showed that epigenetic modifications, mainly including DNA methylation, histone modification, and non-coding RNAs, play an important role in the decidualization process via affecting the target genes' expression. In this review, we will focus on the epigenetic modifications in decidualization and open novel avenues to predict and treat the pregnancy complications caused by abnormal decidualization. 10.1007/s00018-019-03395-9
    A mouse model of endometriosis mimicking the natural spread of invasive endometrium. Wilson Mike R,Holladay Jeanne,Chandler Ronald L Human reproduction (Oxford, England) STUDY QUESTION:Is it possible to establish a genetically engineered mouse model (GEMM) of endometriosis that mimics the natural spread of invasive endometrium? SUMMARY ANSWER:Endometriosis occurs in an ARID1A (AT-rich interactive domain-containing protein 1A) and PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha) mutant GEMM of endometrial dysfunction following salpingectomy. WHAT IS KNOWN ALREADY:Although mouse models of endometriosis have long been established, most models rely on intraperitoneal injection of uterine fragments, steroid hormone treatments or the use of immune-compromised mice. STUDY DESIGN, SIZE, DURATION:Mice harboring the lactotransferrin-Cre (LtfCre0/+), Arid1afl, (Gt)R26Pik3ca*H1047R and (Gt)R26mTmG alleles were subject to unilateral salpingectomies at 6 weeks of age. Control (n = 9), LtfCre0/+; (Gt)R26Pik3ca*H1047R; Arid1afl/+ (n = 8) and LtfCre0/+; (Gt)R26Pik3ca*H1047R; Arid1afl/fl (n = 9) were used for the study. The (Gt)R26mTmG allele was used for the purpose of fluorescent lineage tracing of endometrial epithelium. LtfCre0/+; (Gt)R26mTmG (n = 3) and LtfCre0/+; (Gt)R26Pik3ca*H1047R/mTmG; Arid1afl/fl (n = 4) were used for this purpose. Mice were followed until the endpoint of vaginal bleeding at an average time of 17 weeks of age. PARTICIPANTS/MATERIALS, SETTING, METHODS:At 6 weeks of age, mice were subjected to salpingectomy surgery. Mice were followed until the time point of vaginal bleeding (average 17 weeks), or aged for 1 year in the case of control mice. At time of sacrifice, endometriotic lesions, ovaries and uterus were collected for the purpose of histochemical and immunohistochemical analyses. Samples were analyzed for markers of the endometriotic tissue and other relevant biomarkers. MAIN RESULTS AND THE ROLE OF CHANCE:Following salpingectomy, LtfCre0/+; (Gt)R26Pik3ca*H1047R/mTmG; Arid1afl/fl mice developed endometriotic lesions, including lesions on the ovary, omentum and abdominal wall. Epithelial glands within lesions were negative for ARID1A and positive for phospho-S6 staining, indicating ARID1A-PIK3CA co-mutation status, and expressed EGFP (enhanced green fluorescent protein), indicating endometrial origins. LARGE-SCALE DATA:N/A. LIMITATIONS, REASONS FOR CAUTION:LtfCre0/+; (Gt)R26Pik3ca*H1047R; Arid1afl/fl mice develop vaginal bleeding as a result of endometrial dysfunction at an average age of 17 weeks and must be sacrificed. Furthermore, while this model mimics the natural spread of endometriotic tissue directly from the uterus to the peritoneum, the data presented do not reject current hypotheses on endometriosis pathogenesis. WIDER IMPLICATIONS OF THE FINDINGS:The idea that endometriosis is the result of abnormal endometrial tissue colonizing the peritoneum via retrograde menstruation has gained widespread support over the past century. However, most models of endometriosis take for granted this possibility, relying on the surgical removal of bulk uterine tissue and subsequent transplantation into the peritoneum. Growing evidence suggests that somatic mutations in ARID1A and PIK3CA are present in the endometrial epithelium. The establishment of a GEMM which mimics the natural spread of endometrium and subsequent lesion formation supports the hypothesis that endometriosis is derived from mutant endometrial epithelium with invasive properties. STUDY FUNDING/COMPETING INTEREST(S):This research was supported by the American Cancer Society PF-17-163-02-DDC (M.R.W.), the Mary Kay Foundation 026-16 (R.L.C.) and the Ovarian Cancer Research Fund Alliance 457446 (R.L.C.). The authors declare no competing interests. 10.1093/humrep/dez253
    Assisted reproductive technology strategies in uterus transplantation. de Ziegler Dominique,Pirtea Paul,Carbonnel Marie,Poulain Marine,Ayoubi Jean Marc Fertility and sterility The development of assisted reproductive technology (ART) through four decades has led to offer the ultimate treatment for nearly all forms of infertility. The only remaining factor of childlessness however that still eludes ART and its routine variants are the absolute uterine infertility factors, for which the only option is an experimental approach, uterus transplantation. Progresses has been made over the past few years, and more are underway for simplifying the process notably for simplifying the uterus extraction step performed in the uterus donor. Furthermore, as the technique is being better mastered, the original indications for uterus transplantation, the congenital or acquired absence of the uterus, are now widened to also include incurable uterine fibrosis, or Asherman's syndrome. The ART-related practicalities of uterus transplantation, ovarian stimulation and uterine priming are being discussed in the present review. 10.1016/j.fertnstert.2019.05.028
    Exosome-derived uterine miR-218 isolated from cows with endometritis regulates the release of cytokines and chemokines. Microbial biotechnology As an inflammation of the endometrium, endometritis can affect fertility and lead to serious economic losses in the dairy industry. Widely found in various tissues and body fluids, exosomes and exosome micro (mi)RNAs have been shown to play an important regulatory role in the immune responses. As one of differentially expressed exosome miRNAs, miR-218 is involved in the pathogenesis of bovine endometritis. The mechanisms of miR-218 in regulating the release of cytokines and chemokines in endometritis, however, are poorly understood. Exosomes were isolated from bovine uterine cavity fluid and verified by transmission electron microscopy. An in vitro lipopolysaccharide-treated cell model for bovine endometritis was then established to evaluate the correlation between exosome-derived miR-218 and the immune responses. We demonstrated that exosomes could be used to deliver miR-218 from endometrial epithelial cells (EECs) into the uterine microenvironment and adjacent recipient cells to modulate local immune responses. miR-218 packaged in the exosomes secreted from EECs acts as an inhibitor by blocking immune factors such as interleukin (IL)-6, IL-1β, tumour necrosis factor-α, the chemokines macrophage inflammatory genes (MIP)-1α and MIP-1β to maintain the immune balance in the uterus. However, uterine inflammation altered the immunoregulatory mechanism of exosome miR-218. MiR-218 is a potential biomarker for the detection of endometritis. Our findings also revealed a new mechanism for the development of endometritis in cows. 10.1111/1751-7915.13565
    Physiology of the Endometrium and Regulation of Menstruation. Critchley Hilary O D,Maybin Jacqueline A,Armstrong Gregory M,Williams Alistair R W Physiological reviews The physiological functions of the uterine endometrium (uterine lining) are preparation for implantation, maintenance of pregnancy if implantation occurs, and menstruation in the absence of pregnancy. The endometrium thus plays a pivotal role in reproduction and continuation of our species. Menstruation is a steroid-regulated event, and there are alternatives for a progesterone-primed endometrium, i.e., pregnancy or menstruation. Progesterone withdrawal is the trigger for menstruation. The menstruating endometrium is a physiological example of an injured or "wounded" surface that is required to rapidly repair each month. The physiological events of menstruation and endometrial repair provide an accessible in vivo human model of inflammation and tissue repair. Progress in our understanding of endometrial pathophysiology has been facilitated by modern cellular and molecular discovery tools, along with animal models of simulated menses. Abnormal uterine bleeding (AUB), including heavy menstrual bleeding (HMB), imposes a massive burden on society, affecting one in four women of reproductive age. Understanding structural and nonstructural causes underpinning AUB is essential to optimize and provide precision in patient management. This is facilitated by careful classification of causes of bleeding. We highlight the crucial need for understanding mechanisms underpinning menstruation and its aberrations. The endometrium is a prime target tissue for selective progesterone receptor modulators (SPRMs). This class of compounds has therapeutic potential for the clinical unmet need of HMB. SPRMs reduce menstrual bleeding by mechanisms still largely unknown. Human menstruation remains a taboo topic, and many questions concerning endometrial physiology that pertain to menstrual bleeding are yet to be answered. 10.1152/physrev.00031.2019
    Progesterone modulates integrin {alpha}2 (ITGA2) and {alpha}11 (ITGA11) in the pregnant cervix. Ji Huiling,Long Vit,Briody Victoria,Chien Edward K Reproductive sciences (Thousand Oaks, Calif.) OBJECTIVE:Fibrillar collagen in the cervical extracellular matrix (ECM) is the predominant component providing mechanical support. Cellular integrins contribute to structural integrity by cross-linking ECM components. We investigated the expression of collagen-binding integrins in the normal rat gestation and after treatment with mifepristone to determine whether integrin modulation is involved in changes in tissue resistance. STUDY DESIGN:Cervical tissue was harvested from nonpregnant and timed pregnant Sprague-Dawley rats. Normal gestational expression was evaluated in nonpregnant and timed pregnant tissue on days 12, 16, 18, 20, 21 and 22. Progesterone inhibition was induced with 3 mg mifepristone administered on day 15. Primary rat cervical stromal (RCS) cell cultures were generated from nonpregnant rats using tissue explants. The effects of progesterone environment on RCS cells were evaluated in the presence and absence of various inhibitors. Protein expression and signaling pathways were evaluated by Western blot. RESULTS:Integrin α2 (ITGA2) expression increased over gestation, peaking at the end of gestation (analysis of variance [ANOVA] P < .01). Integrin α11 (ITGA11) expression increased through mid-gestation, peaking on day 18 and decreasing through day 22 (ANOVA P < .001). Progesterone increased the expression of ITGA11 and phosphorylated focal adhesion kinase ([pFAK] P < .002). Mifepristone blocked these effects in vitro. Mifepristone increased ITGA2 and phosphorylated extracellular signal-regulated kinases 1 and 2 (pERK1/2) in vivo and in vitro. Mifepristone-induced upregulation of ITGA2 was abrogated by inhibition of ERK1/2. CONCLUSION:Progesterone/progesterone withdrawal is involved in regulating the expression of collagen-binding integrins. These changes differ among the collagen-binding integrins. Mitogen-activated protein kinase (MAPK) signaling is involved in regulating some of these integrins. 10.1177/1933719110382305