The Hippo Signaling Pathway Regulates Ovarian Function via the Proliferation of Ovarian Germline Stem Cells
The Hippo Signaling Pathway Regulates Ovarian Function via the Proliferation of Ovarian Germline Stem Cells
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Hippo 信号通路通过卵巢生殖干细胞的增殖调节卵巢功能
DOI：
10.1159/000464113
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发表时间：
2017
影响因子：
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通讯作者：
Zheng Yuehui
中科院分区：
文献类型：
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作者：
Ye Haifeng;Li Xiaoyan;Zheng Tuochen;Hu Chuan;Pan Zezheng;Huang Jian;Li Wei;Li Jia;Zheng Yuehui
目的：提高卵巢种系干细胞（OGSCs）的分离、鉴定和培养水平，明确Hippo信号通路效应因子YAP1与OGSCs体外增殖分化的关系，明确Hippo信号通路对卵巢功能的主要作用。方法：采用两步酶法分离和磁分离法，通过检测mvh、Oct4、Nanog、FragilisandStellamarkers的表达，分离鉴定ogsc。然后选择Hippo信号通路中的主要效应分子YAP1作为本研究的靶基因。含有过表达的YAP1或YAP1靶向shRNA的慢病毒被转导到ogsc中。通过将过表达YAP1或YAP1 shRNA的慢病毒载体微注射到不育小鼠模型或育龄自然小鼠体内，观察Hippo信号通路调控对卵巢增殖、分化、生殖及内分泌功能的影响。结果：(1)在离体干细胞中观察到mvh、Oct4、Nanog、FragilisandStellamarkers的特异性表达。因此，分离的细胞被初步鉴定为OGSCs。(2)在离体OGSCs中观察到LATS2、MST1、YAP1和MVH的共表达。与GFP对照相比，过表达YAP1的OGSCs中mvhandoct4的表达水平显著升高。与此一致，mvhandoct4水平在表达yap1靶向shRNA的细胞中显著降低。(3) YAP1在不育小鼠模型中过表达14-75天后，我们检测到卵巢卵泡再生，原始卵泡激活，出生率增加，同时E2和FSH水平升高。(4)然而，我们检测到健康育龄小鼠在表达YAP1 shRNA后，卵巢卵泡减少，出生率降低，血清E2和FSH下降。结论：成功建立了OGSCs的分离、鉴定和培养方法。进一步的研究结果表明，分离的OGSCs可以特异性识别Hippo信号分子，并且YAP1的表达可用于调节OGSCs的增殖和分化，以及小鼠卵巢功能。本研究提示Hippo信号通路可能是调控小鼠卵巢功能重塑的一个新的分子靶点。
Objective:To improve the separation, identification and cultivation of ovarian germline stem cells (OGSCs), to clarify the relationship between the Hippo signaling pathway effector YAP1 and the proliferation and differentiation of OGSCsin vitroand to identify the major contribution of Hippo signaling to ovarian function.Methods:Two-step enzymatic separation processes and magnetic separation were used to isolate and identify OGSCs by determining the expression ofMvh, Oct4, Nanog, FragilisandStellamarkers. Then, YAP1, as the main effector molecule in the Hippo signaling pathway, was chosen as the target gene of the study. Lentivirus containing overexpressed YAP1 or a YAP1-targeted shRNA was transduced into OGSCs. The effects of modulating the Hippo signaling pathway on the proliferation, differentiation, reproduction and endocrine function of ovaries were observed by microinjecting the lentiviral vectors with overexpressed YAP1 or YAP1 shRNA into infertile mouse models or natural mice of reproductive age.Results:(1) The specific expression ofMvh, Oct4, Nanog, FragilisandStellamarkers was observed in isolated stem cells. Thus, the isolated cells were preliminarily identified as OGSCs. (2) The co-expression of LATS2, MST1, YAP1 and MVH was observed in isolated OGSCs.MvhandOct4expression levels were significantly increased in OGSCs overexpressing YAP1 compared to GFP controls. Consistently,MvhandOct4levels were significantly decreased in cells expressing YAP1-targeted shRNA. (3) After 14-75 days of YAP1 overexpression in infertile mouse models, we detected follicle regeneration in ovaries, the activation of primordial follicles and increased birth rate, accompanied by increasing levels of E2 and FSH. (4) However, we detected decreasing follicles in ovaries, lower birth rate, and decreasing E2 and FSH in serum from healthy mice of reproductive age following YAP1 shRNA expression.Conclusion:Methods for the isolation, identification and culture of OGSCs were successfully established. Further results indicate that isolated OGSCs can specifically recognize Hippo signaling molecules and that manipulation of YAP1 expression can be used to regulate the proliferation and differentiation of OGSCs, as well as ovarian function in mice. This study suggests that the Hippo signaling pathway may represent a new molecular target for the regulation of mouse ovarian functional remodeling.