A phenotypic in vitro assay of mouse oocyte maturation identifies reversible blockers of meiotic progression for non-hormonal contraceptive discovery.

Abstract

Study question: Can oocyte maturation be adapted to a phenotypic assay within a drug screening pipeline to identify compounds that block meiotic progression for female non-hormonal contraceptive drug discovery?

Summary answer: A complex phenotypic assay of mouse oocyte maturation identifies potent compounds that reversibly block meiotic progression at specific stages.

What is known already: During oocyte maturation, prophase I-arrested oocytes resume meiosis, undergo germinal vesicle (GV) breakdown, and complete meiosis I with extrusion of the first polar body. Oocyte maturation is critical for generating a fertilizable gamete, and thus, blocking meiotic progression is a promising target for non-hormonal contraception. Moreover, this process can be recapitulated in vitro, providing a powerful phenotypic assay for drug screening.

Study design size duration: Oocytes were collected from CD-1 mice following hyperstimulation and in vitro matured for 14-16 h in the presence of a single dose (10 µM) of compounds from a bioactive compound library. Incubation in dimethyl sulfoxide (DMSO) only or 10 µM milrinone, a known phosphodiesterase 3A (PDE3A) inhibitor, was used as vehicle and positive control, respectively. Primary screening and subsequent concentration-response testing were performed in duplicate.

Participants/materials setting methods: Oocytes were incubated with compounds during IVM to identify compounds with inhibitory effects on meiotic progression. We were specifically interested in compounds that maintained arrest at prophase of meiosis I (GV-intact) or prevented extrusion of the first polar body. Brightfield images were taken before and after IVM to assess maturation status based on morphological criteria. Using this platform, we screened a subset of 818 compounds from the compound library based on annotated target and structural diversity. A hit was defined as a compound that inhibited meiotic progression by ≥80%. Hits were validated through an independent source of compound and concentration responsiveness. Hit compounds were also tested in a prolonged culture treatment to confirm maintenance of meiotic arrest. A counterscreen assay was performed on compounds that maintained arrest at prophase of meiosis I to rule out phenotypes due to inhibition of PDE3A activity.

Main results and the role of chance: From our primary screen, 29 hits were identified that blocked meiotic maturation. Following hit validation via independent sources and concentration-response, 18 compounds were confirmed. Five compounds resulted in arrest at prophase of meiosis I, but only one was not based on PDE3A inhibition. Thirteen compounds enabled resumption of meiosis but not polar body extrusion, and 12 of these maintained arrest during prolonged culture. Thus, using this screening pipeline, we identified a total of 13 compounds for an overall confirmed hit rate of 1.6%. Using this phenotypic assay, we also compared the activity of structural analogs, enabling the establishment of preliminary structure-activity relationships. Additionally, washout of compound-treated oocytes allowed for normal meiotic resumption in 33% and 50% of oocytes arrested at GV and GV breakdown stages, respectively, demonstrating the potential for reversibility.

Large scale data: N/A.

Limitations reasons for caution: Although this screening platform can identify potent compounds, their protein targets and potential mechanisms of action are unknown. As such, further studies are required to deconvolute targets and generate more specific compounds. Given that our phenotypic oocyte screening assay is based on the mouse model, validation using human oocytes will also be necessary for translational consideration. Lastly, all experiments were conducted on denuded oocytes and do not account for activity within an intact cumulus-oocyte complex.

Wider implications of the findings: Through our screening pipeline, we identified compounds that potently inhibit meiotic progression. Beyond furthering our understanding of oocyte maturation, we highlight potent compounds as promising starting points toward novel drug candidates or in novel target identification for female non-hormonal contraception.

Study funding/competing interests: This work was supported by the Gates Foundation [INV-003385]. Under the grant conditions of the Foundation, a Creative Commons Attribution 4.0 Generic License has already been assigned to the Author Accepted Manuscript version that might arise from this submission. The authors have no conflict of interest to disclose.