BOVINE AND BABOON OOCYTE DEVELOPMENT IN VITRO

在雌性哺乳动物中，大多数卵母细胞存在于原始卵泡中 休息阶段。实际上，人们对这种机制一无所知 调节这些毛囊进入生长池的运动 毛囊。目前，静止的原始卵泡池是一种资源。 未开发的，可以作为体外培养的材料来源 成熟(IVM)和体外受精(IVF)加速发育 改良家养物种，保护濒危物种，并提供 缓解妇女不孕不育的替代方法。我们有 开发了一种支持原始细胞激活的培养系统 牛和狒狒卵巢皮质器官培养中的卵泡。大脑皮层 外植体在无血清培养条件下存活至少7-10年。 天数和高比例的原始毛囊离开休息池 并进展到初级卵泡阶段，但只有几个卵泡 向多层次次要阶段发展。我们现在建议在此基础上 这些惊人的发现并启动了一个新的项目来确定文化 支持卵泡在体外进一步发育的条件。 虽然牛将是这些研究的主要动物模型，但我们 将与灵长类动物模型--狒狒--进行平行研究，因为 我们有独一无二的途径，以很少的成本获得胚胎狒狒的卵巢。 总体假设是启动原始卵泡的生长 至卵巢皮质器官培养的次级卵泡期， 然后用酶法或机械方法分离次级毛囊和 他们进一步培养成单独的卵泡，将允许卵泡 在体外生长到卵母细胞可以成熟的阶段 体外培养。第一个具体目标是检验牛和狒狒 卵泡在体外可以从原始阶段发育到第二阶段。 前提是可以改善培养条件以延长存活时间。 具体目标#2是开发养牛次级牛的方法 卵泡发育到体外成熟的能力阶段。这些实验是 朝着最终目标迈出关键一步，使用大量的 哺乳动物卵巢中原始卵泡作为遗传资源的研究 改良家畜，保护濒危物种，以及 缓解人类不孕不育。

In female mammals most oocytes reside in primordial follicles in a resting stage. Virtually nothing is known about the mechanisms that regulate the movement of these follicles into the pool of growing follicles. The pool of resting primordial follicles is a resource, as yet untapped, that could be exploited as source of material for in vitro maturation (IVM) and in vitro fertilization (IVF) to hasten genetic improvement of domestic species, preserve endangered species, and provide alternative methods for alleviating infertility in women. We have developed a culture system that supports activation of primordial follicles in organ cultures of bovine and baboon ovarian cortex. Cortical explants survive in culture in serum-free conditions for at least 7-10 days and a high percentage of primordial follicles leave the resting pool and progress to the primary follicle stage, but only a few follicles develop to the multilayered secondary stage. We now propose to build on these striking findings and initiate a new project to determine culture conditions that will support further development of follicles in vitro. Although cattle will be the primary animal model for these studies, we will pursue parallel studies with a primate model, the baboon, because we have unique access to fetal baboon ovaries at little cost. The overall hypothesis is that initiating growth of primordial follicles to the secondary follicle stage in organ cultures of ovarian cortex, followed by enzymatic or mechanical isolation of secondary follicles and their further culture as individual follicles, will allow follicular growth to progress in vitro to a stage where oocytes can be matured in vitro. Specific Aim #1 is to test the hypothesis that bovine and baboon follicles can develop in vitro from the primordial to the secondary stage provided that culture conditions can be improved to extend survival. Specific Aim #2 is to develop methods for growing bovine secondary follicles to the stage of competence for IVM. These experiments are crucial steps towards the ultimate goal of using the large numbers of primordial follicles in mammalian ovaries as a resource for genetic improvement of domestic animals, preservation of endangered species, and alleviation of human infertility.