Molecular Mechanisms of Ovarian Follicular Activation

描述（由申请人提供）：在雌性哺乳动物中，大多数卵母细胞在静止期驻留在原始卵泡中。关于调节这些卵泡进入生长池（卵泡激活）的机制知之甚少。静止的原始卵泡池是一种尚未开发的资源，可以作为材料来源加以利用，为减轻妇女不孕症和繁殖珍贵的家畜和濒危物种提供替代方法。体内卵泡的激活和生长可能受到各种因素（刺激因子和抑制因子）之间微妙平衡的调节。迄今为止，确定启动卵泡生长的信号的努力包括测试个体"候选者"以确定它们是否促进或抑制卵泡激活。虽然使用这一办法取得了进展，但进展缓慢。我们寻求支持，以开发一种互补的方法，基于分子技术的最新进展，以确定在卵泡激活过程中打开或关闭的特定基因。在特异性目的1中，将使用抑制性消减杂交（SSH）来检验特定基因在卵泡激活期间打开（或关闭）的假设。将通过比较新鲜分离的牛卵巢皮质片（原始卵泡高度富集）与培养两天的皮质片（其中90%的卵泡已激活并成为初级卵泡）来确定候选基因。牛为人类卵泡发育提供了一个很好的模型，SSH非常适合检测两种密切相关的细胞类型/组织中的罕见和新序列。特异性目标2旨在通过使用原位杂交定位新鲜分离与培养的牛皮质块中的差异表达序列，检测特异性目标1中鉴定的候选基因在卵泡激活中的潜在作用。在两年后，我们希望已经确定了一些候选基因，这将为卵泡激活的遗传调控提供重要线索。所提出的实验产生的结果将"打开大门"，许多应用，因为它将是第一个（据我们所知）的分子和遗传方法来调节卵泡激活。阐明这些基本机制对开发新的避孕技术和减轻不孕症具有实际意义。

DESCRIPTION (provided by applicant): In female mammals most oocytes reside in primordial follicles in a resting stage. Little is known about the mechanisms that regulate the movement of these follicles into the growing pool (follicle activation). The pool of resting primordial follicles is a resource, yet untapped, that could be exploited as a source of material to provide alternative methods for alleviating infertility in women and propagating valuable domestic animals and endangered species. It is likely that follicular activation and growth in vivo are regulated by a delicate balance among various factors, both stimulators and inhibitors. To date, efforts to determine the signals that initiate follicle growth have consisted of testing individual "candidates" to determine if they promote or inhibit follicle activation. Although progress has been made using this approach, that progress has been slow. We seek support to develop a complementary approach, based on recent advances in molecular techniques, to determine specific genes that are turned on or off during the activation of follicles. In Specific Aim 1, suppressive subtractive hybridization (SSH) will be used to test the hypothesis that specific genes are turned on (or off) during follicle activation. Candidate genes will be determined by comparing freshly isolated pieces of bovine ovarian cortex (highly enriched for primordial follicles) with cortical pieces cultured for two days (in which 90% of follicles have activated and become primary). Cattle provide an excellent model for human follicular development and SSH are ideally suited for the detection of rare and novel sequences in two closely related cell types/tissues. Specific Aim 2 is designed to test candidate genes identified in Specific Aim 1 for a potential role in follicle activation by using in situ hybridization to localize differentially expressed sequences in freshly isolated vs. cultured bovine cortical pieces. At the end of two years, we expect to have identified a number of candidate genes that will provide important clues to the genetic regulation of follicle activation. The results generated by the proposed experiments will "open the door" to many applications, since it will be the first (to our knowledge) molecular and genetic approach to the regulation of follicle activation. Elucidation of these fundamental mechanisms has practical implications for the development of new contraceptive technologies and alleviation of infertility.