Mechanism of Periovulatory Leukocyte Infiltration into the Ovary

项目总结(见说明)： 免疫系统和生殖系统之间的串扰极大地影响生育能力。特别是，研究表明，严格的时空控制白细胞渗入卵巢是成功排卵的关键。一旦被渗透，白细胞在排卵前卵巢中分化为一群促炎或抗炎细胞，并在排卵和黄体形成中发挥关键作用。在这项研究中，我们将检验孕酮(P4)和前列腺素(PG)在将白细胞招募到排卵前卵巢中起关键作用的假设。目的1.确定P4和PG的调节机制(S) 白细胞渗入。我们首先通过用RU486、消炎痛和赋形剂处理循环成年大鼠来确定P4和PG在WBC渗透中的作用，并用流式细胞仪和免疫组织化学方法检测其对WBC的数量、数量和定位的影响，同时用ELISA法和qPCR法对血清、卵巢细胞和WBC中趋化因子、细胞因子及其受体和细胞黏附分子(CAM)的表达进行功能分析。目的2.确定P4和PG是否调节 白细胞在灵长类卵巢中的浸润。为此，我们将研究P4和PG对灵长类动物白细胞渗透的调节作用。与啮齿动物类似，包括人类在内的灵长类动物也需要P4和PG才能正常排卵。 因此，我们推测P4和PG可能调节WBC在灵长类卵巢的渗透。用三环烷(3|3HSD抑制剂，抑制孕酮合成)、塞来昔布(选择性COX-2抑制剂)或在卵巢刺激下使用赋形剂处理灵长类动物，可抑制P4和PG的作用。 将确定对WBC的数量、种群和定位以及炎症介质表达的影响。一旦确定了猴体内P4和PG调节的WBC亚型和炎症介质，将确定其同源物在人卵巢中的定位和表达模式。目的3.探讨排卵前脾WBC释放的调节机制。我们将确定触发(S)和作用机制的脾白细胞从脾释放。我们假设黄体生成素、循环中白细胞浓度降低或血管紧张素-11(唯一已知的脾白细胞释放的触发物)是触发因素。为了确定触发因素(S)，对去卵巢大鼠进行候选触发因素(促黄体生成素和血管紧张素-11)处理，或通过向大鼠灌流血浆来人工降低循环白细胞浓度。通过计数脾和外周血中的WBC数来衡量对脾WBC释放的影响。

PROJECT SUMMARY (See instructions): Crosstalk between the immune and reproductive systems dramatically impacts fertility. In particular, studies have shown that a tight spatiotemporal control of leukocyte infiltration into the ovary is pivotal for successful ovulation. Once infiltrated, the leukocytes differentiate into a cohort of pro- or anti-infiammatory cells in the preovulatory ovary and play critical roles in ovulation and luteal formation. In this study, we will test the hypothesis that progesterone (P4) and prostaglandins (PG) play pivotal roles in recruiting leukocytes into the preovulatory ovary. Aim 1. To determine the mechanism(s) by which P4 and PG regulate WBC infiltration. We will first determine the roles of P4 and PG on WBC infiltration by treating cycling adult rats with RU486, indomethacin and vehicle, and measuring the effect on the numbers, populations and localization of infiltrating WBC by flow cytometry and immunohistochemistry together with a functional analysis of the expression of chemokines, cytokines, their receptors and cell adhesion molecules (CAMs) in serum, on ovarian cells and WBC by ELISA and qPCR. Aim 2. To determine whether P4 and PG regulate WBC infiltration in primate ovary. In this Aim, we will investigate P4 and PG regulated WBC infiltration in primates. Similar to rodents, primates, including humans also require P4 and PG for normal ovulation. Therefore, we hypothesize that P4 and PG will regulate WBC infiltration in the primate ovary. The actions of P4 and PG will be inhibited by treating primates (Cynomolgous monkeys) with trilostane (3|3HSD inhibitor; inhibits progesterone synthesis), celecoxib (selective COX-2 inhibitor) or vehicle under ovarian stimulation. The effects on the numbers, populations and localization of WBC together with the expression of infiammatory mediators will be determined. Upon the identification of the P4- and PG-regulated WBC subtypes and infiammatory mediators in the monkey, the localization and expression patterns of their homologs will be determined in the human ovary. Aim 3. To determine the regulatory mechanism of preovulatory splenic WBC release. We will identify the trigger(s) and the mechanism of action of splenic WBC release from spleen. We hypothesize that either LH, decreased concentration of circulating WBC or angiotensin-ll (the only known trigger of splenic leukocyte release) act as the trigger. To identify the trigger(s), ovariectomized rats will be treated with candidate triggers (LH and angiotensin-ll) or artificially reduce the concentration of circulating WBC by perfusing rats with plasma. The effects on splenic WBC release will be measured by counting WBC numbers in spleen and peripheral blood.