The Impact of Leukocyte-Derived OSM on Ovulation and Luteinization in the Human Ovary

摘要 排卵过程是周期中期促性腺激素激增诱导的急性炎症反应。 白细胞是炎症反应中的关键效应物。然而，白细胞的机制 白细胞在排卵卵泡中的募集和特定作用仍然是难以捉摸的，特别是在人类中。 目前的研究旨在阐明周期中期LH峰如何协调白细胞募集和 在排卵前卵泡细胞中基因的表达，从而使成功的排卵和黄体化， 人类卵巢。 从我们的试点单细胞转录组分析使用人类卵泡吸出物从妇女 通过体外受精，我们发现OSM（抑瘤素-M）只在特定的 类型的白细胞，而它的受体，OSMR和LIFR（白血病抑制因子受体）及其共 受体，IL 6ST（IL 6家族信号转导子）主要存在于颗粒细胞中。同样在 在整个排卵期，从正常周期的妇女中收集优势卵泡的颗粒细胞， 结果发现，hCG刺激后，OSMR mRNA水平明显升高。另外我们 初步数据显示，hCG增加了OSM受体、OSMR和IL 6ST的表达， 人颗粒细胞培养物，模拟OSMR的体内上调。我们的初步数据还显示 OSM可以激活特定的信号通路，刺激孕酮和前列腺素E2 生产，排卵的两个重要介质。 基于这些新的数据，我们假设：1）LH峰诱导OSM的募集， 表达白细胞，同时增加滤泡细胞中其受体（OSMR、LIFR、IL 6ST）的表达 2）白细胞来源的OSM，在结合到排卵前卵泡中的OSM受体后， 细胞，激活特定的信号通路，并调节特定的排卵基因的表达，从而 促进必要的排卵变化。这些假设将首先通过表征表达来检验 白细胞中的OSM及其受体，占主导地位的卵泡细胞中的OSMR，LIFR和IL 6ST的概况 收集整个排卵期的卵泡，并检查参与排卵的调节机制。 OSM受体在人颗粒细胞中的表达（具体目标1）。接下来，我们将描述OSM- 激活人颗粒细胞中的细胞内信号传导途径（特异性目的2）。最后，我们将阐明 白细胞来源的OSM对围排卵过程的影响，通过鉴定OSM调节的下游 基因（RNA-seq分析）和使用人类基因组评估对受影响途径的细胞/生物学影响 颗粒细胞培养。这项拟议的研究将“首次”证明OSM作为一种新的排卵药， 白细胞来源的因子，并确定其在人类卵巢中的具体作用，从而填补了我们的空白差距， 白细胞在排卵过程中的作用。

ABSTRACTS The ovulatory process is the mid-cycle gonadotropin surge-induced acute inflammatory response. Leukocytes are critical effectors in the inflammatory response. However, the mechanisms of leukocyte recruitment and the specific roles of leukocytes in the ovulatory follicle remain elusive, especially in humans. The current study proposes to elucidate how the mid-cycle LH surge coordinates leukocyte recruitment and the expression of genes in preovulatory follicular cells to bring about successful ovulation and luteinization in the human ovary. From our pilot single-cell transcriptome analyses using human follicular aspirates from women undergoing in vitro fertilization, we discovered that OSM (oncostatin-M) is exclusively expressed in specific types of leukocytes, whereas its receptors, OSMR and LIFR (leukemia inhibitory factor receptor) and their co- receptor, IL6ST (IL6 family signal transducer) are present predominantly in granulosa cells. Similarly, in granulosa cells of dominant follicles collected from normally cycling women throughout the periovulatory period, we found that OSMR mRNA levels were markedly increased after hCG stimulation. In addition, our preliminary data showed that hCG increased the expression of OSM receptors, OSMR and IL6ST in primary human granulosa cell cultures, mimicking in vivo up-regulation of OSMR. Our preliminary data also revealed that OSM could activate specific signaling pathways and stimulate progesterone and prostaglandin E2 production, two crucial mediators of ovulation. Based on these novel data, we hypothesized that 1) the LH surge induces the recruitment of OSM- expressing leukocytes, while increasing the expression of its receptors (OSMR, LIFR, IL6ST) in follicular cells of preovulatory follicles, and 2) leukocyte-derived OSM, upon binding to its receptors in preovulatory follicular cells, activates specific signaling pathways, and regulates the expression of specific ovulatory genes, thereby promoting necessary ovulatory changes. These hypotheses will be tested by first characterizing expression profiles of OSM in leukocytes and its receptors, OSMR, LIFR, and IL6ST in follicular cells of the dominant follicles collected throughout the periovulatory period and examining the regulatory mechanisms involved in the expression of OSM receptors in human granulosa cells (Specific Aim 1). Next, we will delineate OSM- activated intracellular signaling pathways in human granulosa cells (Specific Aim 2). Lastly, we will elucidate the impact of leukocyte-derived OSM on the periovulatory process by identifying OSM-regulated downstream genes (RNA-seq analysis) and evaluating cellular/biological impacts on affected pathways using human granulosa cell cultures. The proposed study will demonstrate “for the first time” OSM as a novel ovulatory factor of leukocyte-origin and identify its specific roles in the human ovary, thus filling the gap in our understahopteanding of the role of leukocytes in the ovulatory process.