Primary human ovarian interstitial cells contribute to murine follicle growth through follicle-interstitial paracrine crosstalk

Paracrine signaling is critical for ovarian development and maintenance of non-growing, gonadotropin-independent follicles. During puberty the human ovary develops defined compartments as gonadotropin signaling triggers follicle activation and maturation. Adjacent follicles utilize paracrine signaling but also benefit from nearby interstitial cell populations. Here, we characterized human ovarian interstitial cells and their effects on follicle growth through a cross-species culture assay. Both prepubertal and postpubertal primary ovarian interstitial populations improved follicle growth, and postpubertal cells conferred a significant increase in follicle estradiol production but not maturation rates. In one cohort, interstitial cell-conditioned media was not sufficient for the growth advantage observed with co-culture. To investigate how follicle-interstitial cell crosstalk changes during puberty, single-cell RNA-sequencing was performed on 11 ovarian tissue cryopreservation samples (participants 0.34-22.8 years). Results confirmed the presence of heterogenous interstitial cell populations previously identified in the adult ovary, including abundant theca/stromal cells expressing genes enriched for ECM-related processes. Finally, over 100 proteins were identified in co-culture media using bottom-up proteomics representing the human ovarian interstitial secretome. This study uniquely characterized human ovarian interstitial cells across the pubertal transition and advances our understanding of the human pediatric and adolescent ovarian follicular microenvironment.