(356) The Synergistic Effect of Leukocyte-derived Interleukin-1 Beta and Oncostatin-M on Periovulatory Granulosa Cell Function in the Human Ovary

Yohan Choi¹, James W. Akin², Thomas E. Curry¹, and Misung Jo¹

¹Department of Obstetrics & Gynecology, University of Kentucky College of Medicine, Lexington, KY 40536; ²Bluegrass Fertility Center, Lexington, KY 40509.

Abstract Text:

Mid-cycle gonadotropin-induced ovulation, an essential event in female reproductive physiology, exhibits features of an acute inflammatory response characterized by massive leukocyte infiltration into the dominant follicle. Despite the recognized importance of leukocyte function in ovulation, the precise role of leukocytes and their cytokines during the periovulatory period remains to be understood. In our previous study, we employed single-cell RNA sequencing (scRNA-seq) analysis on follicular aspirates from IVF patients, identifying distinct leukocyte populations and their specific cytokines. Interleukin-1 beta (IL1B) and Oncostatin-M (OSM) were predominantly expressed in leukocytes, while their receptors IL1R1, IL1RAP, OSMR and IL6ST were predominantly detected in granulosa cells. To further investigate their actions and functions, primary human granulosa-lutein cells (hGLCs) were treated with either recombinant human IL1B (rhIL1B), rhOSM, or both in the presence or absence of human chorionic gonadotropin (hCG). Either rhIL1B or rhOSM increased the expression of their receptors in the presence of hCG, while hCG alone had no effect. Co-treatment with both cytokines further elevated mRNA levels of their receptors beyond those induced by either cytokine alone, and this synergistic effect was observed regardless of hCG presence. Western blot analyses showed that rhIL1B and rhOSM activated distinct signaling pathways – MYD88 (IRAK4) and STAT3, respectively – along with the activation of AKT, ERK1/2, and p38MAPK. Notably, co-treatment with both cytokines induced synergistic enhancement across all signaling pathways examined. Functionally, hCG + rhOSM significantly elevated progesterone (P4) production compared to the levels increased by hCG alone. Intriguingly, adding rhIL1B suppressed hCG + rhOSM-induced increases in P4 levels. Corresponding to these findings, hCG + either rhIL1B or rhOSM enhanced STAR mRNA expression – encoding the rate-limiting enzyme in steroidogenesis – relative to hCG alone, whereas their combined treatment abolished this upregulation. Furthermore, these two cytokines individually promoted prostaglandin E2 (PGE2) synthesis and PG synthase (PTGS2) mRNA expression upon hCG stimulation. Notably, co-treatment induced a robust synergistic increase in both PGE2 production and PTGS2 expression. Collectively, these findings highlight functional synergy between IL1B and OSM in regulating their receptor expression, activating multiple signaling cascades, and modulating P4 and PGE2 production, thereby suggesting their coordinated roles in facilitating the ovulatory process in the human ovary. This study is supported by R01HD0960774, R03HD95098, and R01HD115554.