FGF SIGNALING AND ZINC FINGER PROTEINS IN GONAD DEVELOPMENT AND REPRODUCTION

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 磷脂酰肌醇3-激酶（PI 3 K）信号转导在卵泡膜和间质（TI）细胞中的体内功能的表征是我们在过去一年的COBRE支持的研究重点。卵泡膜细胞由卵巢间质细胞分化而来。卵泡膜细胞的主要功能是产生雄激素，用于颗粒细胞中雌激素的生物合成和基因组作用。与卵泡膜细胞相关的主要疾病是多囊卵巢综合征（PCOS），其中经常观察到雄激素过多和其他卵巢缺陷。然而，在卵巢中的膜和间质（TI）细胞中雄激素产生的关键信号通路的体内功能以及PCOS的病因学仍有待表征。磷酸酶和张力蛋白同源物（Pten）是PI 3 K信号传导的负调节剂。最近对Pten和其他PI 3 K信号分子的研究表明，PI 3 K信号（特别是在卵母细胞中）在正常卵巢发育和女性生育力中起着关键作用。然而，这种信号在TI细胞中的体内功能仍有待表征。为了探索PI 3 K信号转导在体内TI细胞中的功能，我们产生了TI细胞特异性Pten突变体（tPtenMT）小鼠。在tPtenMT卵巢中观察到PI 3 K信号下游分子如3-磷酸肌醇依赖性蛋白激酶1（PDK 1）和蛋白激酶B（AKT）的磷酸化增加，表明TI细胞中Pten的缺失导致卵巢中PI 3 K信号过度活化。更重要的是，在tPtenMT小鼠中观察到雌性生育力的过早丧失以及异常的发情周期和自然排卵。tPtenMT小鼠中的这些生理缺陷是由于卵巢发育异常，因为卵巢中存在各种组织学缺陷（例如，卵巢增大）。tPtenMT卵巢中出现的缺失可能是由于TI细胞中Pten的缺失所致，并且主要缺陷应该在TI细胞中。为了支持这一点，在tPtenMT小鼠中观察到雄激素过量，并且在tPtenMT卵巢和TI细胞中以及在促黄体生成激素（LH）处理的Pten-siRNA慢病毒敲低TI细胞中观察到Lhcgr和几种对雄激素生物合成至关重要的类固醇生成酶基因的表达升高。LHCGR位于最近确定的PCOS易感基因座上。TI细胞中通过PI 3 K信号调节LH和Lhcgr在雄激素产生中的作用可能是某些类型PCOS的潜在病因。与这一假设一致，在年轻成年tPtenMT小鼠中显示的异常模拟了人类的PCOS。与PCOS患者一样，tPtenMT小鼠能够对促性腺激素作出反应而排卵。此外，API-2，一种特异性AKT抑制剂，能够减轻年轻成年tPtenMT小鼠中显示的卵巢增大和卵巢重量增加。总之，我们的研究结果表明，TI细胞中的Pten对正常卵巢发育和女性生育力至关重要，TI细胞中PI 3 K信号的过度激活可能导致年轻人出现PCOS样综合征。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Characterization of the in vivo function of phosphatidylinositol 3-kinase (PI3K) signaling in theca and interstitial (TI) cells was our research focus in the past year of COBRE support. Theca cells are differentiated from ovarian interstitial cells. The major function of theca cells is to produce androgen for estrogen biosythesis and genomic action in granulosa cells. The principal disorder associated with theca cells is polycystic ovary syndrome (PCOS) in which an excess of androgen and other ovarian defects are often observed. However, the in vivo function of signaling pathway(s) key for androgen production in theca and interstitial (TI) cells in the ovary remains to be characterized, as well as the etiology of PCOS. Phosphatase and tensin homolog (Pten) is a negative regulator of PI3K signaling. Recent studies of Pten and other PI3K signaling molecules have shown that PI3K signaling (especially in the oocyte) plays a critical role in normal ovarian development and female fertility. However, the in vivo function of this signaling in TI cells remains to be characterized. To explore the function of PI3K signaling in TI cells in vivo, we have generated TI-cell specific Pten mutant (tPtenMT) mice. Increased phosphorylation of PI3K signaling downstream molecules such as 3-phosphoinositide-dependent protein kinase 1 (PDK1) and protein kinase B (AKTs) were observed in tPtenMT ovaries, indicating that deletion of Pten in TI cells causes an overactivation of PI3K signaling in the ovary. More importantly, premature loss of female fertility with aberrant estrous cycle and natural ovulation were observed in tPtenMT mice. These physiological defects in tPtenMT mice were due to abnormal ovarian development, as there existed a variety of histological defects in the ovary (e.g., ovary enlargement). Abnormalities displayed in tPtenMT ovaries should result from deletion of Pten in TI cells, and the primary defect should be in TI cells. To support this, androgen excess was observed in tPtenMT mice and elevated expression of Lhcgr and several steroidogenic enzyme genes key for androgen biosynthesis in tPtenMT ovaries and TI cells, as well as in luteinizing hormone (LH)-treated Pten-siRNA lentiviral knockdown TI cells. LHCGR is located on a recently identified PCOS susceptibility locus. Modulation of the LH and Lhcgr action in androgen production by PI3K signaling in TI cells could be an underlying etiology of certain types of PCOS. Consistent with this hypothesis, the abnormalities displayed in young adult tPtenMT mice mimic PCOS in humans. Like PCOS patients, tPtenMT mice were able to ovulate in response to gonadotropins. Moreover, API-2, a specific AKT inhibitor, was able to attenuate the ovary enlargement and the increase of ovary weight displayed in young adult tPtenMT mice. Together, our results suggest that Pten in TI cells is critical for normal ovarian development and female fertility and that overactivation of PI3K signaling in TI cells can lead to development of PCOS-like syndrome in young adulthood.