Increased stiffness mimicking ovarian aging induces a fibroinflammatory response in follicles and impairs oocyte quality

Reproductive aging in females is characterized by decreased ovarian reserve and oocyte quality. With aging, both mouse and human ovaries become pro-fibrotic and stiff. However, whether follicles sense and respond to microenvironmental stiffness and affect folliculogenesis and oocyte quality independent of other aging-related factors is unknown. To address this question, we cultured mouse secondary follicles in alginate hydrogels that reproduce the stiffness of young and reproductively old mice. RNA-sequencing revealed that follicles respond rapidly to increased stiffness and exhibit enrichment in genes related to inflammation and extracellular matrix remodeling. Long-term culture in stiff hydrogels resulted in reduced follicle survival, granulosa cell viability, estradiol synthesis, and oocyte quality. To begin to determine how stiffness is transmitted within the follicle, we examined transzonal projections, which mediate granulosa cell–oocyte communication and nutrient exchange. In stiff conditions, the number of transzonal projections decreased. Our findings demonstrate that follicles are highly mechanosensitive and that stiffness alone can trigger hallmarks of ovarian aging, including reduced follicle growth, reduced oocyte quality, and a fibroinflammatory phenotype potentially integrated to the oocyte via TZPs. Overall design: RNA-seq profiling of ovarian secondary follicles isolated from pre-pubertal (12-13days) CB6F1 mice and cultured in 0.5% and 2% alginate beads in groups of 5 follicles per bead. Follicles were than isolated and processed for sequencing at 3h, 6h, 12h and 24h of in vitro culture.