ROLE OF THE OOCYTE IN FOLICLE DEVELOPMENT AND ATRESIA

卵母细胞在卵泡发育和闭锁中的作用

• 批准号: 6323358
• 负责人: GREGORY F ERICKSON
• 金额: $ 19.3万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6323358
• 关键词: RNase protection assay; biological signal transduction; clinical research; egg /ovum; estrogens; female; gene expression; graafian follicles; growth factor receptors; hormone regulation /control mechanism; human subject; immunocytochemistry; in situ hybridization; laboratory rat; menstrual cycle; oogenesis; ovary disorder; ovulation; polycystic ovary syndrome; prolactin; protein localization; testosterone; transforming growth factors

Questions concerning the regulation of folliculogenesis are clinically important because alterations in the process can lead to pathophysiology and infertility. Recently, a novel TGF-Beta family member, GDF-9 is ovary specific, being localized exclusively to oocytes. The importance of GDF-9 deficient mice is arrested and the females are infertile. Accordingly, GDF-9 secreted by the egg is obligatory for folliculogenesis and fertility. The clinical relevance of this new concept is demonstrated by the presence of GDF- 9 mRNA in the human oocyte. At present, virtually nothing is known about the mechanisms controlling GDF-9 expression, and nothing is known about the target cells for GDF-9 and the biological process that GDF-9 regulates. The purpose of this grant is to fill these fundamental gaps in our knowledge of GDF-9 in rat and human ovaries. Three specific aims are proposed: AIM1. Determine the cellular localization and changing pattern of expression of GDF-9 mRNA and protein by in situ hybridization and immunocytochemiestry, respectively, in human (normal and abnormal) and rat ovaries at various reproductive states and during aging. The latter could have implications for aged-related infertility. Novel preliminary data show a dramatic decrease in GDF-9 mRNA during atresia. AIM 2. Test the hypothesis that atretogenic endocrine and autocrine/paracrine ligands negatively regulate GDF-9 expression and promote atresia. Using the immature rat, we will investigate the effects of known atretogenic hormones (testosterone, GnRH, low dose hCG, estrogen withdrawal, prolactin, growth hormone) on oocyte GDF-9 levels, and correlate the effects with changes in granulosa cell mitosis and apoptosis. Novel preliminary data indicate that prolactin and growth hormone cause dramatic decreases in GDF-9 mRNA which are accompanied by the cessation of granulosa mitosis. Aim 3 Using recombinant human GDF-9, we will 1) determine the target cells for GDF-9 in rat and human ovaries by in situ autoradiography; 2) characterize the binding constants of the GDF-9 receptor by Scatchard analysis; and 3) elucidate the biological consequences of GDF-9 action in granulosa cells cultured in serum free medium. Novel preliminary data indicate theat rhGDF-9 acts on granulosa cells to stimulate mitosis. We anticipate that the results from our experiments will also suggest new targets and avenues for managing fertility and infertility.

有关调节卵泡发生的问题在临床上很重要，因为该过程的改变会导致病理生理和不育。 最近，一种新型的TGF-beta家族成员GDF-9是卵巢特异性的，仅本地化为卵母细胞。 GDF-9缺乏小鼠的重要性被捕，女性是不育的。 因此，卵子分泌的GDF-9对于卵泡发生和生育力是必不可少的。 这种新概念的临床相关性证明了人类卵母细胞中GDF-9 mRNA的存在。 目前，关于控制GDF-9表达的机制几乎一无所知，GDF-9的靶细胞和GDF-9调节的生物学过程一无所知。 这笔赠款的目的是在我们对大鼠和人类卵巢中GDF-9的了解中填补这些基本空白。提出了三个具体目标：AIM1。 分别通过原位杂交和免疫细胞化学化学在人（正常和异常）和衰老期间，分别确定了原位杂交和免疫细胞化学化学的细胞定位和变化模式。 后者可能对老年人的不育有影响。 新的初步数据表明，闭锁过程中GDF-9 mRNA急剧下降。 AIM 2。检验以下假设：催化性内分泌和自分泌/旁分泌配体负调节GDF-9表达并促进闭锁。使用未成熟的大鼠，我们将研究已知的催化激素（睾丸激素，GNRH，低剂量HCG，雌激素戒断，催乳素，生长激素）对卵母细胞GDF-9水平的影响，并将其与粒状细胞细胞丝虫病和凋亡的变化相关联。 新的初步数据表明，催乳素和生长激素在GDF-9 mRNA中引起急剧降低，伴随着颗粒丝分裂的停止。 AIM 3使用重组人GDF-9，我们将通过原位放射自显影来确定大鼠和人卵巢中GDF-9的靶细胞； 2）通过SCATCHARD分析表征GDF-9受体的结合常数； 3）阐明了在无血清培养基中培养的颗粒细胞中GDF-9作用的生物学后果。 新的初步数据表明，胰rhgdf-9作用于颗粒细胞以刺激有丝分裂。 我们预计我们的实验结果还将提出管理生育和不育的新目标和途径。