Initiation of growth of baboon primordial follicles in vitro.

Factors that cause some primordial follicles to enter the growth phase while the others remain quiescent are unknown. The hypothesis was tested that primate primordial follicles can survive and initiate growth in vitro in serum-free medium. Superficial pieces of ovarian cortex, containing mostly primordial follicles, were obtained from baboon fetuses during late gestation and cultured for 0, 2, 4, 7, 10 or 20 days in Waymouth MB 752/1 medium supplemented with insulin, transferrin, selenium, linoleic acid, and bovine serum albumin (ITS +). Histological examination of cortical pieces revealed that after 2 and 4 days in culture, the total number of primordial follicles had decreased by 55 and 76% (P < 0.01) respectively, relative to day 0 of culture. This was associated with a sustained, 5- to 8-fold increase in total primary follicles (P < 0.01) beginning on day 2 of culture. There was also a gradual increase in the total number of early secondary and secondary follicles. The average diameter of follicles and oocytes increased gradually throughout culture for all follicular categories (P < 0.01), except secondary follicles and oocytes. Immunohistochemical localization of proliferating cell nuclear antigen (PCNA), a marker for cell proliferation and growth, showed that PCNA was generally absent in primordial follicles on day 0, but was observed after 2 or 4 days in culture in both granulosa cells and oocytes of most growing follicles. Comparison of cortical pieces cultured for 10 or 20 days with ITS + versus 10% fetal bovine serum (FBS) showed a more pronounced decrease in the numbers of primordial follicles and more primary, early secondary and secondary follicles in ITS + compared to FBS-treated cortical pieces (P < 0.01 at 20 days). These results show that primordial follicles from non-human primates can survive and develop to the secondary stage in vitro in serum-free conditions.