Methods of separation and in-vitro culture of pre-antral follicles from mammalian ovaries.

A variety of procedures to isolate intact pre-antral follicles have been developed in our laboratories based on different concentrations of collagenase and DNase in a simple salt solution containing glucose as an energy substrate. The enzyme mixture effectively separates follicles of different sizes derived from ovaries of hamster, mouse, rat, pig and human. While no damage of the basal lamina is apparent for the hamster and human follicles, some loss is present in the other species. Follicles were classified into several stages or classes based on their diameter, the number of granulosa cell layers and the presence of an antral cavity. Pre-antral hamster and human follicles cultured in the presence of follicle stimulating hormone for up to 168 h developed an antral cavity with intact germinal vesicle oocytes.