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Hydrogel-encapsulating culture systems support the consistent growth of ovarian follicles from various species, such as mouse, non-human primate, and human; however, further innovations are required for the efficient production of quality oocytes from early-stage follicles. In this report, we investigated the coculture of mouse ovarian follicles with mouse embryonic fibroblasts (MEFs), commonly used as feeder cells to promote the undifferentiated growth of embryonic stem (ES) cells, as a means to provide the critical paracrine factors necessary for follicle survival and growth. Follicles were encapsulated within alginate hydrogels and cocultured with MEFs for 14 days. Coculture enabled the survival and growth of early secondary (average diameter of 90-100 μm) and primary (average diameter of 70-80 μm) follicles, which developed antral cavities and increased in diameter to 251-347 μm. After 14 days, follicle survival ranged from 70% for 100-μm follicles to 23% for 70-μm follicles. Without MEF coculture, all follicles degenerated within 6-10 days. Furthermore, 72%-80% of the oocytes from surviving follicles underwent germinal vesicle breakdown (GVBD), and the percentage of metaphase II (MII) eggs was 41%-69%. Medium conditioned by MEFs had similar effects on survival, growth, and meiotic competence, suggesting a unidirectional paracrine signaling mechanism. This advancement may facilitate the identification of critical factors responsible for promoting the growth of early-stage follicles and lead to novel strategies for fertility preservation.

Embryonic Fibroblasts Enable the Culture of Primary Ovarian Follicles Within Alginate Hydrogels