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引起的急性炎症反应。 白细胞是炎症反应中的关键效应者。然而，白细胞的作用机制 白细胞在排卵滤泡中的募集和具体作用仍然难以捉摸，尤其是在人类中。 目前的研究建议阐明周期中期的黄体生成素高峰是如何协调白细胞募集和 排卵前卵泡细胞中促排卵和黄体生成基因的表达 人类卵巢。 来自我们的试验性单细胞转录组分析，使用的是来自女性的人卵泡抽吸物 在进行体外受精的过程中，我们发现OSM(抑癌蛋白-M)在特定的 白细胞的类型，而其受体，OSMR和LIFR(白血病抑制因子受体)及其共同作用。 受体IL6ST(IL6家族信号转导)主要存在于颗粒细胞中。同样，在 正常周期妇女围排卵期优势卵泡颗粒细胞 期间，我们发现hCG刺激后OSMR基因表达水平显著升高。此外，我们的 初步研究表明，hCG可促进OSM受体、OSMR和IL6ST的表达。 人颗粒细胞培养，模拟体内OSMR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E_2 生产，排卵的两个重要中介。 基于这些新的数据，我们假设：1)黄体生成素激波诱导OSM- 表达白细胞，同时增加其受体(OSMR、LIFR、IL6ST)在滤泡细胞中的表达 2)白细胞来源的OSM与其在排卵前卵泡中的受体结合 细胞，激活特定的信号通路，调节特定排卵基因的表达，从而 促进必要的排卵改变。这些假设将通过第一个特征化的表达来检验 OSM在白细胞中的分布及其受体、OSMR、LIFR和IL6ST在优势滤泡细胞中的分布 收集整个排卵期的卵泡，并检查参与 OSM受体在人颗粒细胞中的表达(特异靶1)接下来，我们将描述OSM- 激活人颗粒细胞的细胞内信号通路(特定目标2)。最后，我们将澄清 白细胞来源的OSM通过识别OSM调节的下游对围排卵期的影响 基因(RNA-seq分析)和评估细胞/生物对受影响途径的影响 颗粒细胞培养。拟议中的研究将“首次”证明OSM是一种新的排卵器 白细胞来源的因子，并确定其在人卵巢中的特定作用，从而填补我们的 了解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.