REGULATION OF THE CORPUS LUTEUM

本提案的总体目标是阐明灵长类动物黄体退化的生理和细胞机制，以及了解妊娠早期黄体寿命延长的机制。我们以前的工作结果已经确定了排卵后黄体细胞中发生的两个显著变化：i）cAMP和PKA依赖性核转录因子CREB的表达停止，和ii）黄体对LH的反应性显著降低，这发生在月经周期的黄体中期至晚期，最终导致黄体退化。本研究的目的是直接验证以下假设：周期中期促性腺激素激增导致细胞内cAMP水平爆发，这是一个细胞内效应物程序，导致LH的营养作用减弱，这是由cAMP依赖性核转录因子CREB的下调调节的。这一假设将使用最先进的生理学和分子方法进行测试。目的1将确定负责在黄体化过程中CREB表达的损失的细胞机制。具体的假设是，排卵cAMP水平的大幅增加指导CREB转录抑制因子ICER的表达，这与CREB和PCNA表达的停止在时间上相关。在目标2中，表达CREB显性负突变体的重组腺病毒载体将用于阻断颗粒细胞中的CREB信号传导。具体的假设是CREB的缺失-CREB和PCNA的表达。在目标2中，表达CREB显性负突变体的重组腺病毒载体将用于阻断颗粒细胞中的CREB信号传导。具体的假设是，CREB介导的转录的损失将直接抑制PCNA表达和细胞增殖，但不会抑制LH对孕酮产生的急性作用。目的3：利用腺病毒载体在猴体内组成性激活cAMP/PKA/CREB信号通路。具体的假设是，这种信号系统的激活将绕过LH受体的下降，并延长灵长类动物黄体的功能寿命。除了提供有关灵长类动物黄体生理学的新信息外，这些研究的结果将进一步加深我们对人类黄体期缺陷原因的理解，黄体期缺陷被认为是不孕症和早孕丢失的重要原因。此外，我们使用腺病毒直接改变体内卵巢蛋白表达的新方法将为了解影响女性健康的其他卵巢疾病（如多囊卵巢疾病）提供新的途径。

The overall goal of this proposal is to elucidate the physiological and cellular mechanisms responsible for the regression of the primate corpus luteum as well as to understand the mechanisms by which the lifespan of the corpus luteum is prolonged during early pregnancy. Results of our previous work have identified two dramatic alterations that occur in luteal cells following ovulation: i) a cessation of the expression of the cAMP and PKA- dependent nuclear transcription factor CREB and ii) a dramatic decrease in the responsiveness of the corpus luteum to LH that occurs during the mid through late luteal phase of the menstrual cycle that is ultimately responsible for luteal regression. The goal of the research presented in the current proposal is to directly test the hypothesis that the eruption in intracellular cAMP levels that occurs in response to the mid-cycle gonadotropin surge sets into play a program of intracellular effectors that result in diminishing trophic effects of LH and this is regulated by the down-regulation of the cAMP-dependent nuclear transcription factor CREB. This hypothesis will be tested using state of the art physiological and molecular approaches. Aim 1 will identify the cellular mechanisms responsible for the loss of CREB expression during luteinization. The specific hypothesis is that the large increase in ovulatory cAMP levels directs the expression of the CREB transcription repressor ICER and this is temporally associated with the cessation of CREB and PCNA expression. In Aim 2, a recombinant adenovirus vector that expresses a dominant-negative mutant of CREB will be used to block CREB signaling in granulosa cells. The specific hypothesis is that the loss of CREB- of CREB and PCNA expression. In Aim 2, a recombinant adenovirus vector that expresses a dominant-negative mutant of CREB will be used to block CREB signaling in granulosa cells. The specific hypothesis is that the loss of CREB-mediated transcription will directly inhibit PCNA expression and cell proliferation but will not inhibit the acute effects of LH on progesterone production. Aim 3 will use adenoviral vectors in vivo in monkeys to constitutively activate the cAMP/PKA/CREB signaling pathway. The specific hypothesis is that activation of this signaling system will bypass the decline in LH receptors and prolong the functional lifespan of the primate corpus luteum. In addition to providing novel information regarding the physiology of the primate corpus luteum, results of these studies will further our understanding of the causes of luteal phase defects in humans which are thought to be a significant cause of infertility and early pregnancy loss. Further, our novel use of adenoviruses to directly alter ovarian protein expression in vivo will provide new ways to approach the understanding of other ovarian disorders that impact upon women's health such as polycystic ovarian disease.