A Platform of High-throughput Ex Vivo Mammalian Ovulation Screening to Discover Novel Ovulatory Signaling Pathways and Identify Non-hormonal Contraceptive Targets

Jiyang Zhang^1,2, Pawat Pattarawat^1,2, Jeff Pea^2,3, John Proudfoot^2,4, Qiang Zhang⁵, Brittany A. Goods^2,6, Francesca E. Duncan^2,3 and Shuo Xiao^1,2

¹Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Environmental and Occupational Health Sciences Institute (EOHSI), Rutgers University, Piscataway, NJ 08854, USA; ²Ovarian Contraceptive Discovery Initiative (OCDI), Northwestern University, Chicago, IL 60611, USA; ³Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA; ⁴Discoverybytes, Newtown, CT 06470, USA; ⁵Gangarosa Department of Environmental Health, Rollins School of Public Health, Emory University, Atlanta GA 30322, USA; ⁶Thayer School of Engineering, Dartmouth College, Hanover, NH 03755, USA

Abstract Text:

Introduction: Ovulation is critical to women’s menstrual cycles, fertility, and contraception. Anovulation is a leading cause of female infertility and other ovarian disorders such as polycystic ovary syndrome (PCOS) and luteinized unruptured follicle (LUF) syndrome. There are currently limited effective and scalable models of follicle development and ovulation. We previously demonstrated that a 3D hydrogel encapsulated in vitro follicle growth (eIVFG) system faithfully recapitulates essential ovarian functions, including follicle development, hormone secretion, ovulation, and gene regulatory pathways.

Objective: We used eIVFG to develop a tiered high-throughput ex vivo mammalian ovulation platform to screen a large number of small-molecule compounds from the Bioactive Compound Library and Protease Inhibitor Library from Selleckchem and the ReFRAME library from Calibr to discover novel ovulatory signaling pathways and non-hormonal contraceptive targets.

Methods and

Results: The ex vivo screening pipeline consists of three tiers: Tier 1 for examining follicle rupture and progesterone (P4) secretion at high dose of 10 µM; Tier 2 for dose-response analysis; and Tier 3 for assessing follicle development and estradiol (E2) secretion. In Tier 1, multi-layered secondary mouse follicles were cultured with follicle-stimulating hormone (FSH) to promote follicle development from the preantral to preovulatory stage. Fully grown preovulatory follicles were treated with 1.5 IU/mL human chorionic gonadotropin (hCG) to induce ovulation, while simultaneously being exposed to each candidate compound at 10 µM with DMSO as the vehicle control. Follicle rupture, a key step of ovulation, was assessed 16 hours post-hCG. Follicles that failed to ovulate were cultured for additional 48 hours to evaluate P4 secretion using ELISA. A total of 1,340 compounds were screened in Tier 1, among which 81 compounds inhibited ≥70% follicle rupture without affecting P4 secretion. In Tier 2, we performed a dose-dependence analysis of the 81 compounds at 0.1, 0.3, 1, 3, and 10 µM to determine their potency in blocking ovulation. A total of 35 compounds exhibited a dose-responsive inhibition of ovulation without impacting P4 production. These 35 hit compounds were further tested in Tier 3 to assess their effects on follicle development and E2 secretion. Our screen identified 20 positive hit compounds that blocked ovulation in a concentration-dependent manner without disrupting normal follicle development and hormone secretion, with 1.5% overall positive hit rate. These hit compounds were further validated with fresh compounds from independent sources. Based on the annotation of these 20 hit compounds, they target multiple established ovulatory genes such as EGFR, MMP2, and MMP9, as well as new genes that have not been associated with ovulation, including FADS1, ALOX5, STAT1/3, DDR2, and CCR1. For instance, the pathway involving cyclooxygenase-2 (COX2) that converts arachidonic acid (AA) into prostaglandin is known to regulate ovulation, and COX2 inhibitors block ovulation. Our tiered screening results revealed that compounds targeting other components of the AA pathway also inhibited ovulation, including compounds targeting FADS1 which converts fatty acids into AA, and compounds targeting ALOX5 that converts AA into leukotrienes. Follow-up studies revealed significant up-regulation of Alox5 in follicular cells, and follicles secreted more leukotrienes upon ovulation. Ongoing studies include in-depth analysis of these targets for understand their mechanisms in regulating ovulation.

 


Conclusion: In summary, our study demonstrates that the tiered ex vivo ovulation screening platform is a powerful model to discover novel ovulatory signaling pathways and identify non-hormonal contraceptive targets. (This study is supported by Gates Foundation and NIH/NIEHS R01ES032144)