Müllerian inhibiting substance (MIS/AMH), produced by granulosa cells of growing follicles, is an important regulator of folliculogenesis and follicle development. Treatment with exogenous MIS in mice suppresses follicle development and prevents ovulation. To investigate the mechanisms by which MIS inhibits follicle development, we performed single-cell RNA sequencing of whole neonatal ovaries treated with MIS at birth and analyzed at postnatal day 6, coinciding with the first wave of follicle growth. We identified distinct transcriptional signatures associated with MIS responses in the ovarian cell types. MIS treatment inhibited proliferation in granulosa, surface epithelial, and stromal cell types of the ovary and elicited a unique signature of quiescence in granulosa cells. In addition to decreasing the number of growing preantral follicles, we found that MIS treatment uncoupled the maturation of germ cells and granulosa cells. In conclusion, MIS suppressed neonatal follicle development by inhibiting proliferation, imposing a quiescent cell state, and preventing granulosa cell differentiation.

Significance This study aims to identify drugs that activate the Mullerian inhibiting substance pathway to be used for contraception or other applications in women’s health. We describe a high-throughput screening pipeline to identify small molecules that activate the Mullerian inhibiting substance type 2 receptor (MISR2) and validate their activity in bioassays. We identify five compounds from a repurposed drug library that specifically induce MISR2 signaling, trigger regression of the Mullerian duct, and inhibit follicle activation. We test these compounds in vivo and show that they can repress folliculogenesis in mice and rats in an Misr2 -dependent manner. These drugs may represent a class of ovarian regulators that inhibit preantral follicle activation and growth.

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