The Primate Corpus Luteum: Functional Regression and Cardiovascular Impacts

简介（由申请人提供）：候选人Randy Bogan博士，俄勒冈国家灵长类动物研究中心（ONPRC）博士后。Bogan博士的研究生研究利用羊作为实验模型来研究黄体（CL）控制黄体酮（P4）合成的机制。在此期间，Bogan博士对将他的研究应用于人类健康产生了兴趣。因此，他来到ONPRC进行博士后培训，这将进一步提高他在哺乳动物CL调节方面的专业知识，使用与翻译相关的非人灵长类动物模型。在妊娠的前6-7周，需要CL产生P4来维持妊娠，而在此期间，妊娠丢失的概率与P4水平呈负相关。因此，了解灵长类动物在黄体期结束时停止P4产生（称为功能回归）的机制，对预防早期妊娠流产和提高辅助生殖技术的效率具有重要意义。然而，导致灵长类动物CL功能退化的机制尚不清楚。该候选人先前确定了逆向胆固醇转运（RCT）系统作为灵长类动物CL功能退化和最终黄体溶解（完全结构降解）的潜在介质。由于RCT系统的研究也对心血管疾病有影响，他还发现恒河猴在月经周期的不同阶段脂蛋白水平的变化与患心血管疾病的风险降低是一致的。这些变化可能是由于卵巢对脂蛋白代谢的直接影响，和/或卵巢分泌的类固醇对肝脏或其他器官有全身性影响。候选人的长期职业目标是成为一名生殖生理学独立调查员，以帮助改善人类的生殖健康，为了实现这一目标，候选人确定了三个需要额外培训的直接领域，以确保作为一名独立调查员取得成功：1)进一步发展心血管疾病及其危险因素研究方面的专业知识，2)获得进行临床研究所需的知识和技能，3)获得足够的经验以进行有效的实验室管理。该提案将利用OHSU的科学环境，OHSU拥有仅有的八个国家灵长类动物研究中心之一，以及可以进行临床研究和培训的医学校园。生殖和心血管生理学领域的专家，具有非人灵长类动物和临床研究经验，已被招募来监督候选人的培训和研究。正式的课程和培训目标是为了满足上述的直接目标而制定的。该研究计划将解决一个新的假设，即由肝x受体（LXR） 1和/或2调节的胆固醇摄取和外排活动，决定黄体甾体原性寿命，并降低患心血管疾病的风险。为了验证这一假设，在K99指导阶段进行的第一个目标将使用分子研究来确定LXR1和LXR2在灵长类动物CL回归期间诱导RCT系统中的个体重要性，并且在体外研究中将确定激活LXR或单个同种异构体诱导的灵长类动物CL基因表达的全基因组变化。每个LXR异构体与已知LXR靶基因的启动子区域的直接结合将在体内自发的黄体溶解过程中确定，DNA微阵列分析将在LXR激动剂存在的情况下对含有两个或单个LXR异构体的黄体细胞进行。独立阶段的研究包括目标2和目标3。第二个目标将利用直接输注合成LXR激动剂在CL内，以确定是否过早激活RCT系统可以缩短CL的寿命。此外，我们将确定模拟早孕是否会阻止LXR激活和非受孕周期结束时通常观察到的RCT，以及LXR激动剂替代是否可以逆转绒毛膜促性腺激素（CG）的作用。血清雌二醇（E2）和P4水平，以及月经的第一天，将被用作黄体寿命的标志。确定已知LXR靶基因和脂蛋白受体的表达、内源性LXR配体水平和脂质定位将提供RCT活性评估，凋亡标志物将评估CL结构完整性。第三个具体目标是利用恒河猴来确定E2和P4周期性水平诱导的心血管危险因素的变化，与直接卵巢内效应诱导的心血管危险因素的变化相比。切除卵巢的猴子将接受E2和P4植入物来模拟人工月经周期，并测量血清中高、低和极低密度脂蛋白（分别为HDL、LDL和VLDL）和甘油三酯的水平，作为心血管风险的指标。此外，将对女性进行实验，以确定月经周期引起的心血管危险因素的变化，以及通过口服激素类避孕药抑制卵巢活动如何影响这些因素。这项工作是独一无二的，因为RCT系统尚未在任何物种的CL中进行过研究，它可能是迄今尚未确定的导致灵长类CL功能退化的信号。此外，将探讨这些发现的跨学科意义，可能产生与减少妇女心血管疾病和更安全地使用激素口服避孕药和激素替代疗法相关的重要发现。

DESCRIPTION (provided by applicant): The candidate, Dr. Randy Bogan, is a postdoctoral fellow at the Oregon National Primate Research Center (ONPRC). Dr. Bogan's graduate research utilized sheep as an experimental model to study mechanisms controlling the synthesis of progesterone (P4) by the corpus luteum (CL). During this experience, Dr. Bogan developed an interest in the applications of his research to human health. Therefore, he came to the ONPRC to conduct postdoctoral training that would further his expertise in the regulation of the mammalian CL using a translationally-relevant nonhuman primate model. P4 production from the CL is required to maintain pregnancy in women through the first 6-7 weeks of gestation with the probability of pregnancy loss during this period being inversely related to P4 levels. Therefore, understanding the mechanisms that halt P4 production (termed functional regression) in the primate CL at the end of the luteal phase has implications for preventing early pregnancy loss, and improving the efficiency of assisted reproduction technologies. However, the mechanisms causing functional regression of the primate CL are not known. The candidate previously identified the reverse cholesterol transport (RCT) system as a potential mediator of functional regression and ultimately luteolysis (complete structural degradation) in the primate CL. As study of the RCT system also has implications for cardiovascular disease, he additionally discovered changes in lipoprotein levels in rhesus macaques at distinct phases of the menstrual cycle that are consistent with a reduced risk of developing cardiovascular disease. These changes may be due to direct ovarian effects on lipoprotein metabolism, and/or steroids secreted from the ovary having systemic effects on the liver or other organs. In keeping with the candidate's long term career goal of becoming an independent investigator in reproductive physiology to help improve the reproductive health of humans, the candidate has identified three immediate areas where additional training is needed to ensure success as an independent investigator: 1) further develop research expertise regarding the study of cardiovascular disease and its risk factors, 2) obtain the necessary knowledge and skills to conduct clinical research, and 3) gain sufficient experience to allow for effective laboratory management. This proposal will take advantage of the scientific environment at OHSU, which has one of only eight National Primate Research Centers, as well as a medical campus where clinical research and training can be performed. Experts in the fields of reproduction and cardiovascular physiology, with both nonhuman primate and clinical research experience, have been recruited to oversee the candidate's training and research. Formal classes and training objectives are laid out to meet the aforementioned immediate goals. The research plan will address the novel hypothesis that cholesterol uptake and efflux activities, regulated by liver x receptor (LXR) 1 and/or 2, determine luteal steroidogenic lifespan and reduce the risk of developing cardiovascular disease. To test this hypothesis, the first aim conducted during the K99 mentored phase will use molecular studies to determine the individual importance of LXR1 and LXR2 in inducing the RCT system during regression of the primate CL, and in vitro studies will identify genome-wide changes in gene expression of the primate CL induced by activation of both or individual LXR isoforms. Direct binding of each LXR isoform to promoter regions of known LXR target genes will be determined during spontaneous luteolysis in vivo, and DNA microarray assays will be performed on luteal cells containing both or individual LXR isoforms in the presence of an LXR agonist. The independent phase research includes aims 2 and 3. The second aim will utilize direct infusion of a synthetic LXR agonist within the CL to determine if premature activation of the RCT system can shorten CL lifespan. Also, we will determine if simulating early pregnancy prevents LXR activation and RCT normally observed at the end of non-conception cycles, and whether LXR agonist replacement can reverse the effects of chorionic gonadotropin (CG). Serum levels of estradiol (E2) and P4, as well as the first day of menstruation, will be used as markers of luteal lifespan. Determining the expression of known LXR target genes and lipoprotein receptors, endogenous LXR ligand levels, and lipid localization will provide an assessment of RCT activity, and markers of apoptosis will evaluate CL structural integrity. The third specific aim will utilize rhesus macaques to determine changes in cardiovascular risk factors induced by cyclical levels of E2 and P4, versus those induced by direct intra-ovarian effects. Ovariectomized monkeys will receive E2 and P4 implants to simulate an artificial menstrual cycle and levels of high, low, and very-low density lipoproteins (HDL, LDL, VLDL, respectively) and triglycerides in serum will be measured as indicators of cardiovascular risk. Also, experiments will be performed using women to determine menstrual cycle-induced changes in cardiovascular risk factors and how inhibiting ovarian activity via hormonal oral contraceptives affects these factors. This work is unique as the RCT system has not been investigated in the CL of any species, and it may be the as-yet unidentified signal causing functional regression of the primate CL. Additionally, cross-disciplinary implications of these findings will be explored that may yield important findings with relevance to reducing cardiovascular disease in women and safer administration of hormonal oral contraceptives and hormone replacement therapies.