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    Expression of pro-apoptotic and anti-apoptotic proteins in granulosa cells of women with diminished ovarian reserve. Journal of assisted reproduction and genetics PURPOSE:To evaluate the expressions of caspase-3 and cytochrome c and heat shock protein 70 (Hsp70) in granulosa cells (GCs) from women with normal ovarian reserve (NOR) and diminished ovarian reserve (DOR) undergoing intracytoplasmic sperm injection (ICSI). METHODS:GCs were collected from 117 infertile women during oocyte retrieval. Patients were classified into four groups as follows: DOR-COC score of 0, DOR-COC score of I, NOR-COC score of 0, and NOR-COC score of I. The caspase-3, cytochrome c, and Hsp70 analyses were performed immunohistochemically in GCs. The ICSI outcomes were evaluated prospectively. RESULTS:The clinical pregnancy and live birth rates were higher in DOR-COC score of I (15, 30.6%; 14, 38.9%) and NOR-COC score of I (19, 38.77%; 19, 52.7%) groups, compared with DOR-COC score of 0 (12, 24.4%; 3, 6.1%) and NOR-COC score of 0 (3, 6.1%; 0%) groups (p = 0.0001; 0.00002), respectively. Caspase-3 and cytochrome c expression levels were higher in DOR-COC score of 0 (23, 65.7%; 25, 71.4%) and NOR-COC score of 0 groups (19, 61.3%; 20, 64.5%), compared with DOR-COC score of I (8, 32%; 9, 36%) and NOR-COC score of I groups (7, 26.9%; 8, 30.8%) (p = 0.00297; p = 0.002), respectively. Lower expression levels of Hsp70 were found in DOR-COC score of 0 (11, 31.4%) and NOR-COC score of 0 groups (10, 32.3%), compared with DOR-COC score of I (16, 64%) and NOR-COC score of I groups (20, 76.9%) (p = 0.001), respectively. Hsp70 expression levels were positively correlated with the number of day 3 good-quality embryo and negatively correlated with estradiol levels in the DOR group. CONCLUSION:Our data suggest that COC score of 0 is associated with increased expression levels of apoptotic proteins, decreased expression levels of anti-apoptotic protein, and poor ICSI clinical outcomes in women with and without DOR. 10.1007/s10815-022-02422-2
    Hydrogen-rich Water Exerting a Protective Effect on Ovarian Reserve Function in a Mouse Model of Immune Premature Ovarian Failure Induced by Zona Pellucida 3. He Xin,Wang Shu-Yu,Yin Cheng-Hong,Wang Tong,Jia Chan-Wei,Ma Yan-Min Chinese medical journal BACKGROUND:Premature ovarian failure (POF) is a disease that affects female fertility but has few effective treatments. Ovarian reserve function plays an important role in female fertility. Recent studies have reported that hydrogen can protect male fertility. Therefore, we explored the potential protective effect of hydrogen-rich water on ovarian reserve function through a mouse immune POF model. METHODS:To set up immune POF model, fifty female BALB/c mice were randomly divided into four groups: Control (mice consumed normal water, n = 10), hydrogen (mice consumed hydrogen-rich water, n = 10), model (mice were immunized with zona pellucida glycoprotein 3 [ZP3] and consumed normal water, n = 15), and model-hydrogen (mice were immunized with ZP3 and consumed hydrogen-rich water, n = 15) groups. After 5 weeks, mice were sacrificed. Serum anti-Müllerian hormone (AMH) levels, granulosa cell (GC) apoptotic index (AI), B-cell leukemia/lymphoma 2 (Bcl-2), and BCL2-associated X protein (Bax) expression were examined. Analyses were performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA) software. RESULTS:Immune POF model, model group exhibited markedly reduced serum AMH levels compared with those of the control group (5.41 ± 0.91 ng/ml vs. 16.23 ± 1.97 ng/ml, P = 0.033) and the hydrogen group (19.65 ± 7.82 ng/ml, P = 0.006). The model-hydrogen group displayed significantly higher AMH concentrations compared with that of the model group (15.03 ± 2.75 ng/ml vs. 5.41 ± 0.91 ng/ml, P = 0.021). The GC AI was significantly higher in the model group (21.30 ± 1.74%) than those in the control (7.06 ± 0.27%), hydrogen (5.17 ± 0.41%), and model-hydrogen groups (11.24 ± 0.58%) (all P < 0.001). The GC AI was significantly higher in the model-hydrogen group compared with that of the hydrogen group (11.24 ± 0.58% vs. 5.17 ± 0.41%, P = 0.021). Compared with those of the model group, ovarian tissue Bcl-2 levels increased (2.18 ± 0.30 vs. 3.01 ± 0.33, P = 0.045) and the Bax/Bcl-2 ratio decreased in the model-hydrogen group. CONCLUSIONS:Hydrogen-rich water may improve serum AMH levels and reduce ovarian GC apoptosis in a mouse immune POF model induced by ZP3. 10.4103/0366-6999.190668
    Circular is associated with ovarian function and assisted reproductive technology outcomes through modulating the proliferation and steroidogenesis of granulosa cells. Cai Hongcai,Chang Tianli,Li Yamin,Jia Yinzhao,Li Huiying,Zhang Mengdi,Su Ping,Zhang Ling,Xiang Wenpei Aging circRNAs are present in human ovarian tissue, but how they regulate ovarian function remains unknown. In the current study, we investigated the levels of circRNAs in granulosa cells (GCs) derived from human follicular fluid, explored their correlation with female ovarian reserve function and clinical outcomes of assisted reproduction technique (ART), and investigated their effects on the biological functions of GC cell lines (COV434) . We identified that the levels of in GCs decreased gradually with aging ( < 0.01) and was positively correlated with AMH (r = 0.45, < 0.01) and AFC (r = 0.32, < 0.01), but not with FSH and estradiol ( > 0.05). Additionally, was related to the number of oocytes obtained, and good quality embryo rates. Silencing in GCs could markedly up-regulate the expression of apoptosis-related factors, reduce cell proliferation activity, inhibit the expression of steroid hormone synthesis-related factors, and prohibit the synthesis of estradiol. On the contrary, over-expression of had the opposite effect. is expected to become a novel biomarker for predicting the outcomes of ART, and may participate in the regulation of ovarian function by affecting the proliferation and apoptosis of GCs and steroid hormone synthesis. 10.18632/aging.202699
    Mir-484 contributes to diminished ovarian reserve by regulating granulosa cell function via YAP1-mediated mitochondrial function and apoptosis. International journal of biological sciences Women with diminished ovarian reserve (DOR) have reduced fertility, but the underlying regulation of ovarian function remains unknown. Although differential microRNA (miRNA) expression has been described in several ovarian disorders, little is known about the role of miRNAs in the pathogenesis of DOR. In this study, we investigated the expression levels of miR-484 in granulosa cells (GCs) derived from human follicular fluid, and explored their correlation with female ovarian reserve function as well as clinical outcomes of assisted reproduction technology (ART). Additionally, we investigated the effects of miR-484 on the biological functions of GC cell lines . We found that miR-484 was highly expressed in GCs from DOR patients and was correlated with decreasing AMH levels and AFC, as well as increasing FSH levels, but not with LH, progesterone, or estradiol. Additionally, miR-484 was negatively related to the number of retrieved oocytes and the ratio of high-quality embryos. Moreover, we found that miR-484 repressed the proliferation of GCs and induced apoptosis, which can in part be attributed to mitochondrial dysfunction. Conversely, silencing miR-484 had the opposite effect. Multiple approaches, including bioinformatic analysis, RNA-seq, qPCR, immunofluorescence, western blotting and luciferase reporter assays, identified YAP1 as a direct target of miR-484 in GCs. Additionally, reintroduction of YAP1 rescued the effects of miR-484 in GCs. The present study indicates that miR-484 can directly target the mRNA of YAP1, induce mitochondrial dysfunction, and consequently reduce the viability and promote the apoptosis of granulosa cells, which contributes to the pathogenesis of DOR. 10.7150/ijbs.68028
    Melatonin protects against excessive autophagy-induced mitochondrial and ovarian reserve function deficiency though ERK signaling pathway in Chinese hamster ovary (CHO) cells. Xie Q E,Wang M Y,Cao Z P,Du X,Ji D M,Liang D,Cao Y X,Liu Y J Mitochondrion Excessive autophagy-induced follicular atresia of ovarian granulosa cells might be one of the pathogenesis of Premature Ovarian Insufficiency (POI), and melatonin (MT) exerted many beneficial effects on mitochondria. However, there was little report regarding the beneficial effects of MT on excessive autophagy-induced mitochondrial and ovarian reserve function deficiency, and the mechanisms have not been clearly identified. Autophagy played a protective role in cells survival, however, high level of autophagy could lead to cell death. In this report, firstly, Chinese hamster ovary cell damage model stably expressing EGFP-LC3 was established. Next, we systematically investigated the protective effects of MT on mitochondrial and ovarian reserve function and molecular mechanisms using this cell damage model. Our results revealed that 10 M MT not only protected against the decline of anti-mullerian hormone (AMH) expression induced by excessive autophagy, but also rescued excessive autophagy-induced impairment of mitochondrial expression and mitochondrial membrane potential. Furthermore, MT protected against excessive autophagy-induced decrease of nucleus-encoded proteins including SDHA and mitofilin, and mitochondrial dynamic-related proteins including OPA1, MFN2, and DRP1. MT also decreased mitochondrial oxidative stress, increased antioxidant enzyme superoxide dismutase 2 (SOD2) expression and ameliorated the G2/M cell cycle arrest induced by excessive autophagy. Finally, MT inhibited excessive autophagy-induced activation of extracellular signal regulated kinase (ERK) signaling pathway. In conclusion, our study showed that MT rescued impairment of mitochondrial and ovarian reserve function, and production of mitochondrial ROS and cell cycle arrest induced by excessive autophagy through down-regulated ERK pathway, implying the potential therapeutic drug target for POI. 10.1016/j.mito.2021.09.009
    Identification of microRNAs in granulosa cells from patients with different levels of ovarian reserve function and the potential regulatory function of miR-23a in granulosa cell apoptosis. Luo Haining,Han Ying,Liu Jiao,Zhang Yunshan Gene This study aimed to determine the microRNA (miRNA) profiles in granulosa cells (GCs) from the follicular fluid (FF) of patients with varying levels of ovarian reserve function. We included 45 women undergoing in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatment. After collecting GCs from each patient, total RNA was extracted from 12 samples. Using Illumina/deep-sequencing technology, we analyzed the small RNAs in each group. Using the R package, we identified the differentially expressed (DE) miRNAs among patients with varying levels of ovarian reserve function. We identified 20 conserved and 3 novel miRNAs that were upregulated in the poor ovarian response (POR) group and 30 conserved miRNAs and 1 novel miRNA that were upregulated in the polycystic ovary syndrome (PCOS) group. Bioinformatics analysis revealed complementary pairing between miR-23a and the 3'-untranslated region (UTR) of the Sirt1 mRNA. miR-23a can regulate SIRT1 protein expression at the posttranscriptional level in GCs. Overexpressing miR-23a can inhibit the expression of SIRT1, decrease the stimulatory effect of SIRT1 on the ERK1/2 pathway, inhibit the expression of p-ERK1/2, and increase apoptosis in GCs. Previous studies confirmed that miR-23a targets SIRT1 and promotes apoptosis in GCs by inhibiting the ERK1/2 signaling pathway. This study provides a novel perspective regarding the role of miRNAs in the regulation of human GC apoptosis in vitro. 10.1016/j.gene.2018.11.025
    Moxibustion alleviates decreased ovarian reserve in rats by restoring the PI3K/AKT signaling pathway. Journal of integrative medicine OBJECTIVE:Moxibustion, a common therapy in traditional Chinese medicine, has potential benefits for treating decreased ovarian reserve (DOR). The present study investigates the protective effect of moxibustion in a rat model of DOR and explores the possible mechanisms. METHODS:Sixty-four female Sprague-Dawley rats were randomly divided into four groups: control, DOR, moxibustion (MOX), and hormone replacement therapy (HRT). The DOR rat model was established by intragastric administration of 50 mg/kg Tripterygium glycoside suspension (TGS), once daily for 14 days. MOX and HRT treatments were given from the day TGS administration was initiated. The ovarian reserve function was evaluated by monitoring the estrus cycle, morphological changes in ovaries, levels of serum estradiol (E), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and anti-Mullerian hormone (AMH), pregnancy rate and embryo numbers. Terminal-deoxynucleotidyl transferase-mediated nick-end-labeling staining was used to identify ovarian granulosa cell apoptosis, while the protein and mRNA expressions of Bax, B-cell lymphoma-2 (Bcl-2), phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) in ovarian tissues were examined by immunohistochemistry, Western blot and quantitative reverse transcription-polymerase chain reaction. RESULTS:Compared with the DOR group, MOX improved the disordered estrous cycle, promoted follicular growth, reduced the number of atresia follicles, increased the concentrations of serum E and AMH, and decreased serum FSH and LH concentrations. More importantly, the pregnancy rate and embryo numbers in DOR rats were both upregulated in the MOX treatment group, compared to the untreated DOR model. Further, we found that the MOX group had reduced apoptosis of ovarian granulosa cells, increased Bcl-2 expression and reduced expression of Bax. Furthermore, the PI3K/AKT signaling pathway was triggered by the moxibustion treatment. CONCLUSION:Moxibustion improved ovarian function and suppressed apoptosis of ovarian granulosa cells in a rat model of DOR induced by TGS, and the mechanism may involve the PI3K/AKT signaling pathway. 10.1016/j.joim.2022.01.007
    Gene expression patterns in granulosa cells and oocytes at various stages of follicle development as well as in in vitro grown oocyte-and-granulosa cell complexes. Munakata Yasuhisa,Kawahara-Miki Ryoka,Shiratsuki Shogo,Tasaki Hidetaka,Itami Nobuhiko,Shirasuna Koumei,Kuwayama Takehito,Iwata Hisataka The Journal of reproduction and development Follicle development is accompanied by proliferation of granulosa cells and increasing oocyte size. To obtain high-quality oocytes in vitro, it is important to understand the processes that occur in oocytes and granulosa cells during follicle development and the differences between in vivo and in vitro follicle development. In the present study, oocytes and granulosa cells were collected from early antral follicles (EAFs, 0.5-0.7 mm in diameter), small antral follicles (SAFs, 1-3 mm in diameter), large antral follicles (LAFs, 3-7 mm in diameter), and in vitro grown oocyte-and-granulosa cell complexes (OGCs), which were cultured for 14 days after collection from EAFs. Gene expression was analyzed comprehensively using the next-generation sequencing technology. We found top upstream regulators during the in vivo follicle development and compared them with those in in vitro developed OGCs. The comparison revealed that HIF1 is among the top regulators during both in vivo and in vitro development of OGCs. In addition, we found that HIF1-mediated upregulation of glycolysis in granulosa cells is important for the growth of OGCs, but the cellular metabolism differs between in vitro and in vivo grown OGCs. Furthermore, on the basis of comparison of upstream regulators between in vivo and in vitro development of OGCs, we believe that low expression levels of FLT1 (VEGFA receptor), SPP1, and PCSK6 can be considered causal factors of the suboptimal development under in vitro culture conditions. 10.1262/jrd.2016-022
    Relationship between the number of cells surrounding oocytes and energy states of oocytes. Munakata Yasuhisa,Ichinose Tomoya,Ogawa Kaori,Itami Nobuhiko,Tasaki Hidetaka,Shirasuna Koumei,Kuwayama Takehito,Iwata Hisataka Theriogenology Lipid content, ATP content, and histone acetylation are thought to reflect the energy state of cells. In addition, the energy state closely associates with growth and developmental ability of oocytes. Oocyte growth is accompanied by active proliferation of the surrounding granulosa cells (GCs), and GCs play a key role in the provision of energy substrates to the oocytes. In the present study, we first examined the relationship among the average number of GCs per follicle, the average number of cumulus cells (CCs) per oocyte, and the average lipid content in oocytes that developed in vivo within individual donor gilts. Second, we validated the relationship between the number of cells surrounding oocytes and the energy states of oocytes by using an IVC system of oocyte granulosa cell complexes (OGCs) derived from early antral follicles. We collected cumulus cells and oocyte complexes (COCs) from antral follicles (3-5 mm in diameter) and found that average lipid content in oocytes significantly correlated with the average number of both GCs/follicle and CCs/oocyte (P < 0.05). In the next series of experiments, we collected OGCs from early antral follicles (0.5-0.7 mm in diameter), and cultured them for 14 days, and then determined the cell number of OGCs, as well as the lipid content, ATP content, and acetylation level of H4K12 in oocytes grown in vitro. In addition, glucose consumption by OGCs was calculated from the sample media collected at Days 13 and 14. The lipid content of oocytes grown in vitro, significantly correlated with the number of cells surrounding the oocytes (P < 0.01) and with the level of glucose consumption by OGCs (P < 0.01). In addition, both ATP content and H4K12 acetylation levels of oocytes grown in vitro significantly correlated with the number of cells surrounding the oocytes (P < 0.05) and glucose consumption by OGCs (P < 0.05). In conclusion, the lipid content of oocytes depends on the number of cells surrounding the oocytes, and glucose uptake by OGCs is crucial for lipid content and ATP content, and H4K12 acetylation in oocytes. 10.1016/j.theriogenology.2016.05.036
    [Triptolide induces autophagy of ovarian granulosa cells via PI3K/AKT/m TOR pathway]. Bai Jun,Wu Ye-Ke,Wu Ke-Ming,Zhu Hong-Li,Li Nan,Chen Mei,Liu Li-Xiu Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China journal of Chinese materia medica The aim of this paper was to observe the concentration,time and mechanism of autophagy induced by triptolide( TP) in ovarian granulosa cells( OGCs). CCK-8 method was used to compare the inhibitory effects of TP at different concentrations on primary cultured rat OGCs and IC50 was calculated. The effects of TP at different concentrations and time points on the expression of OGCs autophagy factor protein and the cascade of PI3 K/AKT/m TOR pathway were detected by Western blot. The effects of TP,autophagy inducer( brefeldin A) and PI3 K/m TOR inhibitor( NVP-BEZ235) on the expression of PI3 K/AKT/m TOR cascade and autophagy related factor protein were detected by Western blot. The results show that the IC50 of different concentrations of TP on OGCs of rat ovary was14. 65 μmol·L-1,and the minimum inhibitory concentration of TP was 0. 1 μmol·L-1( 100 nmol·L-1). Compared with the control group,the expression levels of beclin1 and LC3Ⅱ in each group were significantly higher than those in the control group( P<0. 05 or P<0. 01). After 12 hours of treatment with TP,brefeldin A and NVP-BEZ235,respectively,compared with the control group,TP could significantly promote the expression level of downstream autophagy effect or molecule beclin1,LC3Ⅱ and inhibit the expression level of LC3Ⅰ,p62 protein( P<0. 05 or P< 0. 01). Moreover,the expression of beclin1 and LC3Ⅱ/LC3Ⅰ in TP group was higher than that in brefeldin A group( P<0. 05 or P<0. 01),and the expression of p62 in TP group was lower than that in brefeldin A group( P<0. 05 or P<0. 01). At the same time,TP could significantly inhibit the expression of p-PI3 K,p-AKT,p-mTOR protein,and the inhibitory effect of TP was better than that of NVP-BEZ235 group. This study suggests that 100 nmol·L-1 TP could induce OGCs autophagy successfully in cultured rat ovary for 12 h; TP may induce OGCs autophagy by inhibiting PI3 k/Akt/m TOR signaling pathway. 10.19540/j.cnki.cjcmm.20190320.401
    Cyclophosphamide promotes the proliferation inhibition of mouse ovarian granulosa cells and premature ovarian failure by activating the lncRNA-Meg3-p53-p66Shc pathway. Xiong Ying,Liu Te,Wang Suwei,Chi Huiying,Chen Chuan,Zheng Jin Gene The dysfunction of ovarian granulosa cells (OGCs) directly affects the premature ovarian failure (POF). In vivo experiments showed that cyclophosphamide significantly induced mouse ovarian atrophy and proliferation inhibition of OGCs. The expressions of p53, p66Shc and p16 were significantly higher in OGCs of the cyclophosphamide treatment group. MTT assay showed that cyclophosphamide effectively inhibited the proliferation of OGCs in vitro. SA-β-Gal staining showed that the OGCs in the cyclophosphamide treatment group had many senescent cells. And, the expression of p53, p66Shc, p16 and cleaved caspase-3 in the OGCs of the cyclophosphamide treatment group significant increases. The Northern blot showed that the intensity of the lncRNA-Meg3 hybridization signal of the OGCs in the cyclophosphamide treatment group was significantly higher than that in the control group. ChIP results confirmed the significant increase in the obtained p66Shc promoter DNA fragment, which was enriched on p53 protein, in the OGCs treated with cyclophosphamide. When cyclophosphamide treatment was conducted after siRNA-Meg3 was used, the expression of endogenous lncRNA-Meg3, p53, p66Shc, p16 and cleaved caspase-3 was significantly lower than that in the siRNA-Mock control group. In summary, cyclophosphamide promotes the proliferation inhibition of mouse OGCs and premature ovarian failure by activating the lncRNA-Meg3-p53-p66Shc pathway. 10.1016/j.gene.2016.10.011
    Dehydroepiandrosterone as a potential agent to slow down ovarian aging. Lin Li-Te,Cheng Jiin-Tsuey,Wang Peng-Hui,Li Chia-Jung,Tsui Kuan-Hao The journal of obstetrics and gynaecology research AIM:Ovarian aging, which leads to diminished ovarian reserve and decreased oocyte quality, is highly associated with poor reproductive outcomes. It has been suggested that dehydroepiandrosterone (DHEA) might be able to temporarily slow down the aging process. This study attempted to investigate the clinical benefits of DHEA in older patients and the anti-senescence effect of DHEA on cumulus cells (CC) and human ovarian granulosa cells (HO23 cell line). METHODS:This prospective study enrolled 88 patients who underwent in vitro fertilization (IVF), including 30 younger patients (aged ≤ 37 years) and 58 older patients (aged > 37 years). Older patients were assigned to receive DHEA treatment or not prior to the IVF cycle. CC were obtained from all patients after oocyte retrieval and the HO23 granulosa cell line was used for in vitro studies. Senescence-associated β-galactosidase (SA-β-gal) was used as a biomarker of senescence. RESULTS:In older patients, following DHEA supplementation, a greater number of transferred embryos and a higher fertilization rate were observed compared with those in patients without DHEA supplementation. However, the clinical pregnancy rate was not significantly increased following DHEA supplementation. Additionally, treatment with DHEA resulted in significantly reduced SA-β-gal staining in both CC and HO23 cells. CONCLUSION:DHEA supplementation ameliorated IVF outcomes but without a consequence on pregnancy rate in older patients and decreased SA-β-gal activity in CC and HO23 cells, suggesting that DHEA might be used as a possible intervention to slow down ovarian aging. 10.1111/jog.13456
    Long-Term Moderate Oxidative Stress Decreased Ovarian Reproductive Function by Reducing Follicle Quality and Progesterone Production. Shi Liangyan,Zhang Jinjin,Lai Zhiwen,Tian Yong,Fang Li,Wu Meng,Xiong Jiaqiang,Qin Xian,Luo Aiyue,Wang Shixuan PloS one Ovarian aging is a long-term and complex process associated with a decrease in follicular quantity and quality. The damaging effects of reactive oxygen species (ROS) in ovarian aging and ovarian aging-associated disorders have received relatively little attention. Thus, we assessed if the oxidative stress induced by long-term (defined by the Environmental Protection Agency as at least 30 days in duration) moderate ozone inhalation reduced ovarian reserves, decreased ovarian function and induced ovarian aging-associated disorders. The expression of oxidative stress markers and antioxidant enzymes was used to determine the degree of oxidative stress. Ultrastructural changes in ovarian cells were examined via electron microscopy. The ovarian reserve was assessed by measuring multiple parameters, such as the size of the primordial follicle pool and anti-Müllerian hormone (AMH) expression. The estrous cycle, hormone levels and fertility status were investigated to assess ovarian function. To investigate ovarian aging-associated disorders, we utilized bone density and cardiovascular ultrasonography in mice. The levels of oxidized metabolites, such as 8-hydroxy-2´-deoxyguanosine (8-OHdG), 4-hydroxynonenal (4-HNE) and nitrotyrosine (NTY), significantly increased in ovarian cells in response to increased oxidative stress. The ultrastructural analysis indicated that lipid droplet formation and the proportion of mitochondria with damaged membranes in granulosa cells were markedly increased in ozone-exposed mice when compared with the control group. Ozone exposure did not change the size of the primordial follicle pool or anti-Müllerian hormone (AMH) expression. The estrogen concentration remained normal; however, progesterone and testosterone levels decreased. The mice exposed to ozone inhalation exhibited a substantial decrease in fertility and fecundity. No differences were revealed by the bone density or cardiovascular ultrasounds. These findings suggest that the decreased female reproductive function caused by long-term moderate oxidative damage may be due to a decrease in follicle quality and progesterone production. 10.1371/journal.pone.0162194
    Effect of resveratrol and metformin on ovarian reserve and ultrastructure in PCOS: an experimental study. Furat Rencber Selenay,Kurnaz Ozbek Sema,Eraldemır Ceyla,Sezer Zehra,Kum Tugba,Ceylan Sureyya,Guzel Elif Journal of ovarian research BACKGROUND:PCOS is a reproductive hormonal abnormality and a metabolic disorder. It is frequently associated with insulin resistance, hyperandrogenism, chronic inflammation, and oxidative stress. We aim to investigate the potential therapeutic effects of combined therapy of resveratrol and metformin on polycystic ovaries via SIRT1 and AMPK activation. METHODS:Wistar albino rats were divided into control and experimental (PCOS) groups. DHEA-induced PCOS rats were given resveratrol (20 mg/kg/day), metformin (300 mg/kg/day) and combined therapy. At the end of the experiment, the body and ovarian weight of rats were measured and blood samples were analyzed for FSH, LH, testosterone, AMH, TNF-α and MDA levels. Histopathological evaluation of ovaries were carried out by light and electron microscopy. SIRT1 and AMPK immunreactivity and TUNEL assay were scored. Data were statistically analyzed by SPSS programme. RESULTS:Metformin and combined treatment groups reduced the body and ovary weights compared to the PCOS group. Serum testosterone levels were significantly higher in the PCOS group than in the control group and this was reduced when PCOS was treated with all but especially resveratrol. All the treatment groups decreased LH, LH/FSH, TNF-α and tissue AMH levels which were induced in the PCOS group, whereas metformin was unable to improve the increased MDA and plasma AMH levels. Treatment with resveratrol and/or metformin ameliorated the elevated number of secondary and atretic follicles and the decreased number of Graafian follicles in the PCOS group, which indicates the effect of the treatments on the maintenance of folliculogenesis. Light and electron microscopic findings supported the analysis of follicular count. Increased number of TUNEL (+) granulosa cells in the PCOS group were reduced significantly in the treatment groups. Resveratrol and metformin increased SIRT1 and AMPK immunreactivity, respectively, compared to the PCOS group. CONCLUSIONS:The results suggest that combined therapy of metformin and resveratrol may improve the weight gain, hormone profile and ovarian follicular cell architecture by inducing antioxidant and antiinflammatory systems via SIRT1 and AMPK activation in PCOS. 10.1186/s13048-018-0427-7
    FMR1 and AKT/mTOR signalling pathways: potential functional interactions controlling folliculogenesis in human granulosa cells. Rehnitz Julia,Alcoba Diego D,Brum Ilma S,Hinderhofer Katrin,Youness Berthe,Strowitzki Thomas,Vogt Peter H Reproductive biomedicine online Granulosa cells (GCs) play a major role in folliculogenesis and are crucial for oocyte maturation and growth. In these cells, the mTOR/AKT signalling pathway regulates early folliculogenesis by maintaining the dormancy of primordial follicles, while FSH induces their further differentiation and maturation. Because changes in number of CGG triplets in FMR1 exon 1 (below or beyond normal values of 26-34 triplets) affect ovarian reserve and pre-mutations containing >54 CGG triplets represent a known risk factor for premature ovarian insufficiency/failure, we investigated in the human GC model (COV434) how FMR1/FMRP and mTOR/AKT are expressed and potentially interact during GC proliferation. As FMR protein (FMRP) is expressed mainly in human ovarian GCs, we used these after inducing their proliferation using recombinant FSH (rFSH) and the repression of the mTOR/AKT signalling pathway. We showed that AKT and mTOR expression levels significantly increase after stimulation with rFSH, while S6K and FMR1 expression decrease. After inhibiting mTOR and AKT, FMR1 and S6K expression significantly increased. Only AKT inhibition led to decreased FMRP levels, as expected due to the known FMR1/FMRP negative feedback loop. But rFSH and the mTOR inhibition increased them, indicating a decoupling of this FMR1/FMRP negative feedback loop in our model system. 10.1016/j.rbmo.2017.07.016
    Altered microRNA and Piwi-interacting RNA profiles in cumulus cells from patients with diminished ovarian reserve. Chen Dawei,Zhang Zhiguo,Chen Beili,Ji Dongmei,Hao Yan,Zhou Ping,Wei Zhaolian,Cao Yunxia Biology of reproduction Diminished ovarian reserve (DOR) is defined as decreased number or quality of follicles and oocytes in a woman at childbearing age. It is estimated that up to 10% of women in the general population may suffer from DOR. This study aims to comprehensively characterize microRNA (miRNA) and Piwi-interacting RNA (piRNA) expression profiles in cumulus cells of DOR patients. Cumulus cells were collected from 20 women of similar chronological age who received assisted reproductive technology treatment: 10 with DOR and 10 with normal ovarian reserve (NOR). The small RNAs were extracted from each sample and reverse transcribed. Deep sequencing and bioinformatic analysis were performed to identify the small noncoding RNA profiles. The rRNAs were the most abundant small RNA class in cumulus cells derived from human MII oocytes, following were miRNAs and tRNAs. Twenty-six piRNAs, 79 annotated miRNAs, and 5 novel miRNAs were identified differentially expressed. In DOR group, the chromosomal strand bias patterns of piRNAs on chromosome 1, 3, 5, and X were distinct from its counterpart in NOR group. The involved biological pathways from the putative target genes of differentially expressed miRNAs were enriched by using GO analysis and KEGG pathway annotations, and mTOR pathway and meiosis-associated biological processes were enriched. This study provided additional information on the dysfunctions of cumulus cells in patients with diminished ovarian reserve. Future investigations will involve the characterization of specific functional roles of noncoding small RNA in ovarian reserve regulation. 10.1093/biolre/iox062
    Low MFN2 expression related to ageing in granulosa cells is associated with assisted reproductive technology outcome. Wang Lingjuan,Song Su,Liu Xuemei,Zhang Mengdi,Xiang Wenpei Reproductive biomedicine online RESEARCH QUESTION:Is low MFN2 expression associated with ageing in granulosa cells as well as assisted reproductive technology (ART) outcome, and what is the underlying mechanism of action of MFN2? DESIGN:In a prospective study, fresh granulosa cells were obtained from 161 women aged 20-40 years who underwent IVF with embryo transfer and who were divided into two groups: the diminished ovarian reserve (DOR) group (n = 51) and the control group (n = 110). Patient characteristics including age, infertility duration, body mass index, FSH, anti-Müllerian hormone (AMH), antral follicle count (AFC) and husband's semen parameters and granulosa cell MFN2 expression levels, cell apoptosis, mitochondrial membrane potential (ΔΨm) and ATP levels were analysed. RESULTS:There were no significant differences between the DOR and control groups in terms of age, infertility duration and husband'' semen parameters; however, significant (P< 0.05) changes were found between the two groups in FSH, AMH and AFC levels. MFN2 expression was remarkably lower in granulosa cells from the DOR group and decreased in both groups as age increased. Furthermore, among young patients, MFN2 levels significantly increased in patients with pregnancy. MFN2 protein levels and cell apoptosis were lower in the MFN2 knockdown (MFN2-siRNA) group than in the control (Cy3-siRNA) group. ΔΨm and ATP levels were reduced in the MFN2-siRNA group compared with the Cy3-siRNA group. CONCLUSIONS:Low MFN2 expression levels in granulosa cells were related to ageing, which may be involved in the clinical outcome of ART by promoting cell apoptosis and affecting mitochondrial function. 10.1016/j.rbmo.2018.10.011
    Effects of hPMSCs on granulosa cell apoptosis and AMH expression and their role in the restoration of ovary function in premature ovarian failure mice. Zhang Hongqin,Luo Qianqian,Lu Xueyan,Yin Na,Zhou Dongli,Zhang Lianshuang,Zhao Wei,Wang Dong,Du Pengchao,Hou Yun,Zhang Yan,Yuan Wendan Stem cell research & therapy BACKGROUND:This study was performed to determine the effects of human placenta mesenchymal stem cell (hPMSC) transplantation on granulosa cell apoptosis and anti-Müllerian hormone (AMH) and follicle-stimulating hormone receptor (FSHR) expression in autoimmune drug-induced premature ovarian failure (POF) mice. The aim of this research is to investigate the mechanisms of hPMSCs on ovarian reserve capacity. METHODS:The POF mice model was established by injection of zona pellucida 3 peptide (pZP3). hPMSC transplantation was conducted by intravenous injection into mice following pZP3 treatment. The follicle number was examined by histopathology. The serum levels of FSH, LH, E, AMH and anti-zona pellucida antibody (AzpAb) were measured by enzyme-linked immunosorbent assay. AMH and FSHR expression in the ovary was analyzed by immunohistochemistry and western blot analysis. Granulosa cell apoptosis of the ovaries was examined by In Situ Cell Death Detection Kit. Granulosa cells were isolated and treated with SiAmh interference and hPMSC supernatant to observe the effects of AMH expression on granulosa cell apoptosis in vitro. RESULTS:The results showed that hPMSC transplantation can significantly recover the estrus cycle in the POF group. Morphological staining showed that the basal follicles and sinus follicles after hPMSC transplantation were higher in POF mice than in those without treatment, and the follicle number was significantly decreased with atresia. The serum levels of FSH, LH and AzpAb in the hPMSC transplantation group were reduced considerably, but the E and AMH levels were significantly increased. After hPMSC transplantation, the AMH and FSHR expression in ovarian tissue was significantly higher than in the POF group as determined by immunochemistry and western blot analysis. The FSHR expression was shown in granulosa cells only, and FSHR expression increases with AMH expressed in the ovary; granulosa cell apoptosis was decreased following hPMSC transplantation. The same results were observed from the in-vitro study. CONCLUSIONS:hPMSC transplantation can significantly improve the serum levels of high gonadotropin and low estrogen of POF mice, promote follicular development, inhibit excessive follicular atresia and granulosa cell apoptosis, and improve the ovarian reserve capacity. The mechanism may be achieved by increasing the expression of AMH and FSHR in ovaries. 10.1186/s13287-017-0745-5
    miR-221-3p regulates apoptosis of ovarian granulosa cells via targeting FOXO1 in older women with diminished ovarian reserve (DOR). Wei Chaofeng,Xiang Shan,Yu Yi,Song Jingyan,Zheng Mingming,Lian Fang Molecular reproduction and development In our earlier study, we showed that the expression of microRNA-221-3p (miR-221-3p) was significantly lower in women of advanced age with diminished ovarian reserve (DOR) compared with young women with normal ovarian reserve (NOR). Therefore, in this study, we aimed to explore how miR-221-3p regulates apoptosis of granulosa cells and the pathogenesis of DOR. Bioinformatics prediction and dual-luciferase reporter assay were conducted to identify the target gene of miR-221-3p. miR-221-3p expression was manipulated by transfecting KGN cells with miR-221-3p mimics, inhibitor, and negative control. Following transfection, apoptosis of granulosa cells was determined by flow cytometry, and the expression of the target gene was measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis (WB). In addition, the expression of the target gene in granulosa cells of DOR patients and NOR patients was measured. miR-221-3p were found to directly bind the 3' untranslated region of Forkhead box O1 (FOXO1). Transfection with miR-221-3p mimics significantly decreased the apoptosis rate of KGN cells compared with transfection with miR-221-3p inhibitors. The expression level of miR-221-3p was negatively correlated with the messenger RNA and protein levels of the FOXO1 gene. Besides, FOXO1 expression was upregulated in DOR patients. In conclusion, these results provide evidence that downregulation of miR-221-3p expression promotes apoptosis of granulosa cells by upregulating FOXO1 expression, thus serving an important role in DOR pathogenesis. 10.1002/mrd.23457
    Activation of TGF-β1/Smad3 signaling pathway inhibits the development of ovarian follicle in polycystic ovary syndrome by promoting apoptosis of granulosa cells. Shen Haoran,Wang Yao Journal of cellular physiology OBJECTIVE:To investigate the role of the transforming growth factor-β1 (TGF-β1)/Smad3 signaling pathway in development of ovarian follicle by promoting apoptosis of granulosa cells in polycystic ovary syndrome (PCOS). METHODS:A total of 54 female rats were obtained and randomly assigned into the PCOS (n = 27) and control groups ( n = 27). PCOS rat models were constructed using the dehydroepi-androsterone method to observe ovarian morphology and ultrastructure. Chemiluminescence immunoassay was performed to detect the levels of luteinizing hormone, follicle stimulating hormone, estradiol, progesterone, and testosterone. The release of TGF-β1 in follicular fluid and serum was tested by the enzyme-linked immunosorbent assay. RESULTS:Expression of p-Smad3 significantly increased in granulosa cells of PCOS group. In terms of the Smad2 protein, there was no significant difference between the PCOS and control group. In comparison to the control group, the number of normal secondary follicles and normal antral follicles were significantly decreased, whereas the number of hypogenetic secondary follicles undergoing atresia and antral follicles were significantly elevated in the PCOS group. Furthermore, the thickness of granulosa cells decreased and the apoptosis of granulosa cells increased in the PCOS group compared with the control one. CONCLUSION:These results indicate that p-Smad3 may have a close relationship with apoptosis of granulosa cells, the mechanism is that the TGF-β1/Smad3 signaling pathway may inhibit development of ovarian follicle of PCOS by regulating the apoptosis of granulosa cells. 10.1002/jcp.27854
    UFL1 Alleviates LPS-Induced Apoptosis by Regulating the NF-κB Signaling Pathway in Bovine Ovarian Granulosa Cells. Wang Xinling,Li Chengmin,Wang Yiru,Li Lian,Han Zhaoyu,Wang Genlin Biomolecules Ubiquitin-like modifier 1 ligating enzyme 1 (UFL1) is an E3 ligase of ubiquitin fold modifier 1 (UFM1), which can act together with its target protein to inhibit the apoptosis of cells. Lipopolysaccharides (LPS) can affect the ovarian health of female animals by affecting the apoptosis of ovarian granulosa cells. The physiological function of UFL1 on the apoptosis of bovine (ovarian) granulosa cells (bGCs) remains unclear; therefore, we focused on the modulating effect of UFL1 on the regulation of LPS-induced apoptosis in ovarian granulosa cells. Our study found that UFL1 was expressed in both the nucleus and cytoplasm of bGCs. The results here demonstrated that LPS caused a significant increase in the apoptosis level of bGCs in cows, and also dramatically increased the expression of UFL1. Furthermore, we found that UFL1 depletion caused a significant increase in apoptosis (increased the expression of BAX/BCL-2 and the activity of caspase-3). Conversely, the overexpression of UFL1 relieved the LPS-induced apoptosis. In order to assess whether the inhibition of bGCs apoptosis involved in the nuclear factor-κB (NF-κB) signaling pathway resulted from UFL1, we detected the expression of NF-κB p-p65. LPS treatment resulted in a significant upregulation in the protein concentration of NF-κB p-p65, and knockdown of UFL1 further increased the phosphorylation of NF-κB p65, while UFL1 overexpression significantly inhibited the expression of NF-κB p-p65. Collectively, UFL1 could suppress LPS-induced apoptosis in cow ovarian granulosa cells, likely via the NF-κB pathway. These results identify a novel role of UFL1 in the modulation of bGC apoptosis, which may be a potential signaling target to improve the reproductive health of dairy cows. 10.3390/biom10020260
    Morroniside suppresses hydrogen peroxide-stimulated autophagy and apoptosis in rat ovarian granulosa cells through the PI3K/AKT/mTOR pathway. Deng D,Yan J,Wu Y,Wu K,Li W Human & experimental toxicology Previous evidences have indicated that granulosa cells play a critical role in follicular growth. Hydrogen peroxide (HO)-induced oxidative stress has been associated with ovarian granulosa cell apoptosis and ovarian function. Recently, a study highlighted the protective role of morroniside against HO-induced damage. In this study, we aimed to investigate the effects of morroniside on HO-stimulated rat ovarian granulosa cells and its underlying molecular mechanisms. Our results showed that HO treatment suppressed cell survival and increased apoptosis in rat granulosa cells, while treatment with morroniside markedly increased HO-induced granulosa cell survival in a dose-dependent manner (0, 10, 50 and 100 µM). Moreover, treatment with 50 µM morroniside impeded HO-induced cell apoptosis. An elevation in intracellular ROS, MDA, SOD, GSH-Px, and CAT level was observed in HO-induced granulosa cells; however, this effect was abrogated by morroniside treatment. Further studies suggested that administration of morroniside inhibited HO-induced granulosa cell apoptosis and caspase-3 activity. In addition, after morroniside treatment of HO-stimulated granulosa cells, autophagy-related protein (LC3-II/LC3-I ratio) and beclin-1 expression was decreased and p62 level was increased. Interestingly, we found that morroniside treatment activated the PI3K/AKT/mTOR pathway in HO-stimulated granulosa cells. Finally, we showed that treatment with PI3K and mTOR inhibitors reversed the protective effects of morroniside on HO-induced granulosa cells. Taken together, our data suggest that treatment with morroniside decreased apoptosis, autophagy, and oxidative stress in rat granulosa cells through the PI3K/AKT/mTOR pathway. 10.1177/0960327120960768
    Apoptosis of mural granulosa cells is increased in women with diminished ovarian reserve. Fan Yuting,Chang Yajie,Wei Lina,Chen Jianhui,Li Jingjie,Goldsmith Sierra,Silber Sherman,Liang Xiaoyan Journal of assisted reproduction and genetics PURPOSE:To evaluate the relationship between apoptosis of granulosa cells in women with normal ovarian reserve versus diminished ovarian reserve, and relate that to follicular fluid hormones, and to clinical outcomes. METHODS:A prospective cohort study was initiated between October 2015 and June 2016 involving a total of 164 women undergoing IVF/ICSI cycles at a single IVF center. Mural and cumulus granulosa cells, and follicularfluid were collected during oocyte retrieval. Annexin V-FITC/PI apoptosis staining and flow cytometryanalysis were performed to evaluate apoptosis rate of mural granulosa cells and cumulus cells. Follicularfluid hormones were measured by ECLIA. Laboratory and clinical outcomes were analyzed. RESULTS:In mural granulosa cells, early, late and total apoptosis rates were significantly increased in women with diminished ovarian reserve when compare to women with normal ovarian reserve, along with lower AMHand progesterone levels (but higher estradiol levels) in follicular fluid. Early apoptosis rate of cumulus cellswas significantly higher in the non-pregnant group. The apoptosis rate of mural cells was negativelycorrelated with parameters related to ovarian response, oocyte yield, MII egg number, 2pn cleavagenumber, D3 good embryos number, blastocyst formation rate and frozen embryos number. A positivecorrelation was found between mural granulosa cell apoptosis and age. CONCLUSION:A significantly higher apoptosis rate of mural granulosa cells was correlated with worse ovarian response, with fewer egg and embryo numbers in IVF/ICSI, as well as with age. Early apoptosis rate of cumulus cellsmight also have influence on clinical pregnancy. 10.1007/s10815-019-01446-5
    Role of Autophagy in Lysophosphatidylcholine-Induced Apoptosis of Mouse Ovarian Granulosa Cells. Yang Si,Chen Jie,Ma Bingchun,Wang Jinglei,Chen Jiaxiang International journal of molecular sciences Lysophosphatidylcholine (LPC), also known as lysolecithin, is one of the major components of oxidized low-density lipoproteins (ox-LDL). In the pathogenetic process of diverse diseases, LPC acts as a significant lipid mediator. However, no evidence shows that LPC can affect the female reproductive system. In our study, we found that LPC inhibited the cell viability of primary mouse ovarian granulosa cells. Meanwhile, LPC was shown to induce apoptosis, which is accompanied by an increase in apoptosis-related protein levels, such as cleaved caspase-3, cleaved caspase-8 and Bax, as well as a decrease in Bcl-2. The total numbers of early and late apoptotic cells also increased in the LPC-treated cells. These results indicated that LPC could induce apoptosis of mouse ovarian granulosa cells. Furthermore, the increase in autophagy-related protein levels and the number of autophagic vesicles suggested that LPC could induce autophagy. The inhibition of oxidative stress by N-acetyl-L-cysteine (NAC) could rescue the induction of apoptosis and autophagy by LPC, which indicated that oxidative stress was involved in LPC-induced apoptosis and autophagy. Interestingly, the inhibition of autophagy by 3-MA could reserve the inhibition of cell viability and the induction of apoptosis by LPC. In conclusion, oxidative stress was involved in LPC-induced apoptosis, whileautophagy of mouse ovarian granulosa cells and the inhibition of autophagy could alleviate LPC-induced apoptosis. 10.3390/ijms23031479