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    Hippo signaling pathway reveals a spatio-temporal correlation with the size of primordial follicle pool in mice. Xiang Cheng,Li Jia,Hu Liaoliao,Huang Jian,Luo Tao,Zhong Zhisheng,Zheng Yuehui,Zheng Liping Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology BACKGROUND:The Hippo signaling pathway, a highly conserved cell signaling system, exists in most multicellular organisms and regulates cell proliferation, differentiation, and apoptosis. It has been reported that the members of Hippo signaling are expressed in mammalian ovaries, but the exact functions of this pathway in primordial follicle development remains unclear. METHODS:To analyze the spatio-temporal correlation between the core component of Hippo pathway and the size of primordial follicle pool, Western blot, Real-time PCR and immunohistochemistry were used, and the expression and localization of MST1, LATS2 and YAP1 mRNA and protein were examined in 3 d, 1 m, 5 m, 16 m postnatal mice ovary and the culture model of mice primordial follicle in vitro. RESULTS:Both the protein and mRNA expression of the MST1 and LATS2 were decreased significantly as mouse age increased (p < 0.05), however, the mRNA expression of them increased significantly in 16 m compared with 5 m as well as the protein expression of LATS2.The expression of YAP showed the opposite trend, and the significant protein expression of pYAP was increased before 1 m, after which no significant change was observed. Moreover, the ratio of pYAP/YAP decreased significantly. Culturing ovaries for 8 d in vitro resulted in the activation of primordial follicles in 3 d postnatal mice ovaries, and these developed into primary follicles with the expression of PCNA increasing significantly (p < 0.05). The mRNA and protein expression of MST and LATS decreased significantly (p < 0.05), and the expression of YAP increased significantly (p < 0.05, p < 0.01), whereas the ratio of pYAP/YAP decreased significantly (p < 0.05). CONCLUSION:The above results reveal that the expression of the core components of Hippo pathway changed during mouse follicular development, especially before and after primordial follicle activation in vitro. The primordial follicle activation may be related to the significant decrease of the ratio of pYAP1/YAP1. In conclusion, Hippo signaling pathway expressed in mice ovaries and have spatio-temporal correlation with the size of primordial follicle pool. 10.1159/000369752
    Ovarian Germline Stem Cells (OGSCs) and the Hippo Signaling Pathway Association with Physiological and Pathological Ovarian Aging in Mice. Li Jia,Zhou Fangyue,Zheng Tuochen,Pan Zezheng,Liang Xia,Huang Jian,Zheng Liping,Zheng Yuehui Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology BACKGROUND:The Hippo signaling pathway plays fundamental roles in stem cell maintenance in a variety of tissues and has thus implications for stem cell biology. Key components of this recently discovered pathway have been shown to be associated with primordial follicle activation. However, whether the Hippo signaling pathway plays a role in the development of Ovarian Germline Stem Cells (OGSCs) during physiological and pathological ovarian aging in mice is unknown. METHODS:Mice at the age of 7 days (7D), or of 2, 10, or 20 months (2M, 10M, 20M) and mice at 2M treated with TPT and CY/BUS drugs were selected as physiological and pathological ovarian aging models, respectively. Immunohistochemistry was used to assess the development of follicles, and the co-localization of genes characteristic of OGSCs with MST1, LATS2 and YAP1 was assessed by immunofluorescence, western blotting and real-time PCR methods. RESULTS:The Hippo signal pathway and MVH/OCT4 genes were co-expressed in the mouse ovarian cortex. The level and co-localization of LATS2, MST1, MVH, and OCT4 were significantly decreased with increased age, but YAP1 was more prevalent in the mouse ovarian cortex of 2M mice than 7D mice and was not observed in 20M mice. Furthermore, YAP1, MVH, and OCT4 were gradually decreased after TPT and CY/BUS treatment, and LATS2 mRNA and protein up-regulation persisted in TPT- and CY/BUS-treated mice. However, the expression of MST1 was lower in the TPT and CY/BUS groups compared with the control group. In addition, pYAP1 protein showed the highest expression in the ovarian cortexes of 7D mice compared with 20M mice, and the value of pYAP1/YAP1 decreased from 7D to 20M. Moreover, pYAP1 decreased in the TPT- and CY/BUS-treated groups, but the value of pYAP1/YAP1 increased in these groups. CONCLUSION:Taken together, our results show that the Hippo signaling pathway is associated with the changes that take place in OGSCs during physiological and pathological ovarian aging in mice. Thus, the Hippo signaling pathway may be involved in the development schedule of OGSCs. 10.1159/000430144
    The Expression of Markers Related to Ovarian Germline Stem Cells in the Mouse Ovarian Surface Epithelium and the Correlation with Notch Signaling Pathway. Pan Zezheng,Sun Mengli,Li Jia,Zhou Fangyue,Liang Xia,Huang Jian,Zheng Tuochen,Zheng Liping,Zheng Yuehui Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology BACKGROUND/AIMS:Ovarian germline stem cells (OGSCs) have been shown to mainly exist in the ovarian surface epithelium (OSE), but the activity changes of germline stem cells during different reproductive stages and the potential regulatory signaling pathway are still unknown. The Notch signaling pathway plays a key role in cell development, primordial follicles and stem cell proliferation. However, whether it plays a role in the proliferation of OGSCs is unknown. Here, we analyzed the activity changes of germline stem cells and the correlation between germline stem cells and the Notch signaling pathway. METHODS:The expression of germline stem cell markers Mvh, Ooc4 and the Notch molecules Notch1, Hes1, and Hes5 were detected during 3 days (3d), and 2, 12, 20 months (2m, 12m, 20m) mouse ovarian surface epithelium samples. DAPT, a specific inhibitor of the Notch pathway, was used to observe the influence of Notch signaling in the germline stem cells. RESULTS:The results showed that the levels of MVH and OCT4 decreased substantially with reproductive age in ovarian surface epithelium, and the same tendency was detected in the Notch signaling molecules Notch1, Hes1 and Hes5. Dual-IF results showed that the germline stem cell markers were co-expressed with Notch molecules in the ovarian surface epithelium. While, the expression of MVH and OCT4 were reduced when the ovaries were treated with DAPT and the levels were attenuated with increasing dose of DAPT. CONCLUSION:Taken together, our results indicate that the viability of OGSCs decreased with the age of the mouse ovaries, and the activity of OGSCs in the ovarian surface epithelium may be related to the Notch signaling pathway. 10.1159/000438586
    Ovarian Stem Cell Nests in Reproduction and Ovarian Aging. Ye Haifeng,Zheng Tuochen,Li Wei,Li Xiaoyan,Fu Xinxin,Huang Yaoqi,Hu Chuan,Li Jia,Huang Jian,Liu Zhengyv,Zheng Liping,Zheng Yuehui Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology The fixed primordial follicles pool theory, which monopolized reproductive medicine for more than one hundred years, has been broken by the discovery, successful isolation and establishment of ovarian stem cells. It has brought more hope than ever of increasing the size of primordial follicle pool, improving ovarian function and delaying ovarian consenescence. Traditional view holds that stem cell aging contributes to the senility of body and organs. However, in the process of ovarian aging, the main factor leading to the decline of the reproductive function is the aging and degradation of ovarian stem cell nests, rather than the senescence of ovarian germ cells themselves. Recent studies have found that the immune system and circulatory system are involved in the formation of ovarian germline stem cell niches, as well as regulating the proliferation and differentiation of ovarian germline stem cells through cellular and hormonal signals. Therefore, we can improve ovarian function and delay ovarian aging by improving the immune system and circulatory system, which will provide an updated program for the treatment of premature ovarian failure (POF) and infertility. 10.1159/000484114
    Tri-ortho-cresyl phosphate (TOCP) induced ovarian failure in mice is related to the Hippo signaling pathway disruption. Hu Liaoliao,Peng Tingting,Huang Jian,Su Tie,Luo Ruichen,Zheng Yuehui,Zhong Zhisheng,Yu Peiling,Nie Kun,Zheng Liping Reproductive toxicology (Elmsford, N.Y.) As a plasticizer widely used in society, tri-ortho-cresyl phosphate (TOCP) is reported to inhibit spermatogenesis and growth of spermatogonial stem cells. However, its effects on female reproductive system are virtually unknown. The present study investigated the effects of TOCP on ovarian follicle development by using mouse model of chronic TOCP exposure, and examined the expression of the core components of the Hippo pathway, which had been proven to be crucial for ovarian follicle development. Furthermore, through up-regulation of Hippo-yes-associated protein 1 (Yap1) in ovaries, the potential protective effects of Yap1 over-expression on TOCP-induced ovarian dysfunction were observed. The results showed that TOCP impaired ovarian function in a dose-dependent manner, and the expression of the Hippo pathway changed significantly in TOCP-exposed ovaries. Further, YAP1 over-expression partially reversed the TOCP-induced ovarian impairment. Collectively, these data indicate that the Hippo pathway is involved in the mechanism by which TOCP elicits ovarian function impairment. 10.1016/j.reprotox.2018.10.007
    Differentially Expressed lncRNAs After the Activation of Primordial Follicles in Mouse. Zheng Liping,Luo Ruichen,Su Tie,Hu Liaoliao,Gao Fengxin,Zhang Xiaoning Reproductive sciences (Thousand Oaks, Calif.) The activation of primordial follicles is critical to ovarian follicle development, which directly influences female fertility and reproductive life span. Several studies have suggested a role for long noncoding RNAs (lncRNAs) in ovarian function. However, the precise involvement of lncRNAs in the initiation of primordial follicles is still unknown. Here, an in vitro culture model was used to investigate the roles of lncRNAs in primordial follicle activation. We found that primordial follicles in day 3 mouse ovaries were activated after culturing for 8 days in vitro, as indicated by ovarian morphology changes, increases in primary follicle number, and downregulation of mammalian Sterile 20-like kinase messenger RNA (mRNA) and upregulation of growth differentiation factor 9 mRNA. We next examined lncRNA expression profiles by RNA sequencing at the transcriptome level and found that among 60 078 lncRNAs, 6541 lncRNA were upregulated and 2135 lncRNA were downregulated in 3-day ovaries cultured for 8 days in vitro compared with ovaries from day 3 mice. We also found that 4171 mRNAs were upregulated and 1795 were downregulated in the cultured ovaries. Gene ontology and pathway analyses showed that the functions of differentially expressed lncRNA targets and mRNAs were closely linked with many processes and pathways related to ovary development, including cell proliferation and differentiation, developmental processes, and other signaling transduction pathways. Additionally, many novel identified lncRNAs showed inducible expression, suggesting that these lncRNAs may be good candidates for investigating mouse primordial follicle activation. This study provides a foundation for further exploring lncRNA-related mechanisms in the initiation of mouse primordial follicles. 10.1177/1933719118805869
    Protective Effects of Puerarin on Premature Ovarian Failure via Regulation of Wnt/β-catenin Signaling Pathway and Oxidative Stress. Chen Cheng,Li Song,Hu Cong,Cao Weiwei,Fu Qingfeng,Li Jia,Zheng Liping,Huang Jian Reproductive sciences (Thousand Oaks, Calif.) This study was designed to investigate the protective effects of puerarin (PUE), which work via the Wnt/β-catenin signaling pathway, and oxidative stress in the premature ovarian failure (POF) model. Two-month-old female mice were randomly divided into four groups. One group was used as the control, and the other three groups were injected with cyclophosphamide and busulfan to create POF models. Two POF treatment groups were gavaged with 100 or 200 mg/kg PUE for 28 days. Next, the ovaries were fixed, and the numbers of different stage follicles were measured, and the ovarian surface epithelium (OSE) was collected. Oct4 and Mvh expression, Wnt/β-catenin signaling pathway activity, the oxidative stress factors SOD2 and Nrf2, and the apoptosis-related proteins Bcl-2 and Bax were detected by IHC, RT-QPCR, and western blotting. We found that the number of follicles, Oct4 and Mvh expression, and Wnt/β-catenin-signaling activity were reduced in the POF groups (p < 0.05 or p < 0.001). After PUE treatment, the follicle number and the primordial follicle ratio increased (p < 0.01), while the atresia ratio decreased (p < 0.01). In addition, the expression levels of Oct4, Mvh, Wnt1, β-catenin, cyclin D1, SOD2, and Nrf2 showed obvious recovery compared with levels in the POF group (p < 0.01, p < 0.05, or p < 0.001). The Bcl-2/Bax ratio in the POF model had reduced by about 60% compared with the control group (p < 0.001) and improved by about 50% after PUE treatment (p < 0.001). In conclusion, PUE may improve the survival of female reproductive stem cells (FGSCs) and play a protective role against POF via a mechanism involving the Wnt/β-catenin signaling pathway, as well as relieving oxidative stress. Further investigations should focus on the culture of oocytes and FGSCs in vitro in a PUE environment with inhibitors or agonists of the Wnt signaling pathway. 10.1007/s43032-020-00325-0