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 pr...