Follicular morphogenesis during perinatal development

描述(由申请人提供)：原始卵泡的形成和发育是成功的卵泡发生和生育的必备事件。这些步骤的缺陷会导致卵巢发育不全或卵巢早衰，最终导致不孕。我们的长期目标是阐明控制原始卵泡形成和发育的调节机制，作为发展改进人类不孕不育和避孕治疗的必要前提。尽管有证据表明生长分化因子9(GDF9)或E2刺激腔前卵泡发育，但对它们在原始卵泡发生中的作用及其机制几乎一无所知。此续订申请的重点是填补这一知识缺口。在上一次资助期间，我们已经表明，在胎儿发育过程中FSH的失活会扰乱原始卵泡的发生，这与GDF9和雌激素受体(ESR)的低表达有关。此外，FSH可上调GDF9和ESR的表达。我们的初步结果表明：(1)体外抑制内源性GDF9的表达抑制原始卵泡的发生；(2)E2刺激体外原始卵泡的形成；(3)胎儿暴露于FSH抗血清中，阻断GDF9受体的表达，如骨形态发生蛋白受体II(BMPRII)和转化生长因子B受体I型(TBRI)，并阻断ESR1和ESR2的表达；(4)抑制G蛋白偶联受体30(GPR30)的siRNA，在体外跨膜ESR抑制原始卵泡的形成。基于这些观察，我们推测GDF9和E2通过GDF9受体(BMPRII和TbRI)和ESR(ESR1，ESR2和GPR30)的协同作用促进体细胞分化为颗粒细胞，导致原始卵泡的形成。我们进一步推测，FSH通过调节GDF9和E2受体的表达来调节它们的作用。GDF9的表达在体细胞分化为颗粒前细胞导致原始卵泡形成过程中的生理意义。我们将确定卵巢内GDF9的必要性及其作用机制。2.探讨血沉在体细胞分化为颗粒前细胞形成原始卵泡过程中的生理意义。我们将确定关于GDF9行动的经典ESR和GPR30的必要性。我们将使用胎儿和新生仓鼠卵巢在体内和体外(原始卵泡直到生命第8天才出现)，RNAi，反义寡核苷酸，药理学，形态学和分子方法来解决特定的目的。该项目的成功完成将促进我们对原始卵泡形态发生机制的理解。综述：原始卵泡存量代表了哺乳动物物种(包括人类)整个生殖生命的不可再生的卵泡储备，并决定了物种的繁殖力和繁殖力。本研究的目的是阐明控制原始卵泡形成和发育的调控机制。这一结果将对人类不孕不育和避孕的改进或新的治疗方法的发展产生重大影响。

DESCRIPTION (provided by applicant): Formation and development of primordial follicles are obligatory events for successful folliculogenesis and fertility. Defects in these steps lead to ovarian dysgenesis or premature ovarian failure and ultimately infertility. Our long-term goal is to elucidate the regulatory mechanisms controlling the formation and development of the primordial follicles as a necessary prerequisite to the development of improved therapeutic management of human infertility and contraception. Although evidence suggests that growth differentiation factor 9 (GDF9) or E2 stimulates preantral follicle development, virtually nothing is known about their role in primordial folliculogenesis or the mechanisms therein. This renewal application is focused on filling this knowledge gap. During the last funded period, we have shown that inactivation of FSH during fetal development disrupts primordial folliculogenesis, which correlates with low expression of GDF9 and estrogen receptors (ESR). Further, FSH treatment upregulates GDF9 and ESR expression. Our preliminary results indicate that (1) suppression of endogenous GDF9 expression in vitro retards primordial folliculogenesis, (2) E2 stimulates primordial follicle formation in vitro, (3) fetal exposure to an FSHantiserum blocks the expression of the GDF9 receptors, such as bone morphogenetic protein receptor II [BMPRII] and transforming growth factor B receptor type I [TBRI], and blocks the expression of ESR1 and ESR2, and (4) siRNA knockdown of a G-protein coupled receptor 30 (GPR30), a transmembrane ESR in vitro attenuates the formation of primordial follicles. Based on these observations we hypothesize that GDF9 and E2 promote the differentiation of somatic cells into granulosa cells leading to the formation of primordial follicles by mechanisms that involve a concerted action of the GDF9 receptors (BMPRII and TBRI), and the ESR (ESR1, ESR2 and GPR30). We further postulate that FSH regulates the action of GDF9 and E2 by modulating the expression of their receptors. The hypothesis will be tested with the following specific aims: 1. To reveal the physiological importance of GDF9 expression in the differentiation of somatic cells into pregranulosa cells leading to primordial follicle formation. We will determine the need for intraovarian GDF9, and its mechanisms of action. 2. To examine the physiological importance of the ESR in the differentiation of somatic cells into pregranulosa cells leading to primordial follicle formation. We will determine the need for the classic ESR and GPR30 with respect to GDF9 action. We will use fetal and neonatal hamster ovaries in vivo and in vitro (primordial follicles do not appear until 8th day of life), RNAi, antisense oligodeoxynucleotides, pharmacological, morphological and molecular approaches to address the specific aims. The successful completion of this project should advance our understanding of the mechanisms involved in the morphogenesis of primordial follicles. Lay summary: The primordial follicle stock represents a nonrenewable follicular reserve for the entire reproductive life of the mammalian species, including the human, and determines the fertility and fecundity of the species. The purpose of the proposed research is to elucidate the regulatory mechanisms controlling the formation and development of the primordial follicles. The outcomes will have a significant impact on the development of improved or novel therapeutic management of human infertility and contraception.