Targeting Proprotein Convertases and Follicle Rupture to Identify Non-hormonal Contraceptive Candidates

Abstract ID 97333 Poster Board 086 Traditional female contraceptives are based on steroid hormones such as progestin, which block ovulation but also cause undesired side effects, including hormone-related cancers, depression, stroke, and obesity. There is an urgent need to develop novel non-hormonal contraceptives. We previously utilized single-mouse follicle RNA sequencing (RNA-seq) analysis to identify critical ovulatory genes and signaling pathways using an established 3D ex vivo ovulation system. The results of RT-qPCR, RNA-seq analysis, and in situ hybridization revealed that Pcsk3 (Furin), Pcsk5, and Pcsk6, three members of the proprotein convertase (PC) family, were continuously upregulated in mural granulosa cells of ovulating follicles, suggesting their possible roles in contributing to ovulation. Herein, we used the ex vivo ovulation system to test three PC inhibitors: Furin Inhibitor I, Furin Inhibitor II, and Proprotein Convertase Inhibitor (PCI). All three PC inhibitors dose-dependently inhibited the rupture of ovulating follicles, with PCI having the most inhibitory potency. In addition, PCI did not affect follicle survival or hormonal secretion of estradiol (E2) and progesterone (P4). RT-qPCR results showed that PCI did not alter key genes involved in follicle maturation, luteinization, and ovarian steroidogenesis. In situ zymography revealed that PCI inhibited gelatinase activation which critically regulates follicle rupture through proteolysis and extracellular matrix (ECM) remodeling. Using a more complex ex vivo ovarian explant culture model, we were able to validate the inhibitory effect of PCI on follicle rupture. A preliminary in vivo experiment using a mouse superovulation model indicated that the treatment of PCI via intraperitoneal (IP) injection suppressed ovulation in vivo. Furthermore, the Genotype-Tissue Expression (GTEx) Portal database reveals that Pcsk5 has the most abundant expression level in human ovaries compared to all other tissues (e.g., uterus, breast, heart, and liver). Taken together, these results highlight the hormone-independent roles of PCs in regulating follicle rupture, suggesting an attractive druggable target for the development of non-hormonal female contraceptives. Funded by the Bill & Melinda Gates Foundation.