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相互作用会严重影响生育能力。特别是，研究表明，严格的时空控制白细胞浸润到卵巢是成功排卵的关键。一旦浸润，白细胞在排卵前卵巢中分化为一组促炎或抗炎细胞，并在排卵和黄体形成中起关键作用。在这项研究中，我们将测试的假设，孕激素（P4）和三尖杉酯碱（PG）在募集白细胞进入排卵前卵巢发挥关键作用。目标1。确定P4和PG调节的机制 白细胞浸润。我们将首先通过用RU 486、吲哚美辛和赋形剂处理周期性成年大鼠来确定P4和PG对WBC浸润的作用，并通过流式细胞术和免疫组织化学测量对浸润的WBC的数量、群体和定位的影响，以及血清中趋化因子、细胞因子及其受体和细胞粘附分子（CAM）表达的功能分析，通过ELISA和qPCR对卵巢细胞和WBC的影响。目标2.为了确定P4和PG是否调节 灵长类动物卵巢中的白细胞浸润。在这个目的中，我们将研究P4和PG调节灵长类动物的白细胞浸润。与啮齿类动物类似，灵长类动物，包括人类，也需要P4和PG才能正常排卵。 因此，我们假设P4和PG将调节灵长类动物卵巢中的WBC浸润。P4和PG的作用将通过用trilostane（3）处理灵长类动物（食蟹猴）而被抑制|3 HSD抑制剂;抑制孕酮合成）、塞来昔布（选择性考克斯-2抑制剂）或赋形剂。 将确定对WBC数量、群体和定位以及炎症介质表达的影响。在猴中鉴定P4和PG调节的WBC亚型和炎症介质后，将确定其同源物在人卵巢中的定位和表达模式。目标3。探讨排卵前脾脏白细胞释放的调节机制。我们将确定脾脏WBC从脾脏释放的触发因素和作用机制。我们假设LH、循环WBC浓度降低或血管紧张素II（脾白细胞释放的唯一已知触发因子）作为触发因子。为了鉴定触发物，卵巢切除大鼠将用候选触发物（LH和血管紧张素-II）处理或通过用血浆灌注大鼠来人工降低循环WBC的浓度。将通过计数脾脏和外周血中的WBC数量来测量对脾脏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.