PREANTRAL OVARIAN FOLLICLES IN SERUM-FREE CULTURE - SUPPRESSION OF APOPTOSIS AFTER ACTIVATION OF THE CYCLIC GUANOSINE 3',5'-MONOPHOSPHATE PATHWAY AND STIMULATION OF GROWTH AND DIFFERENTIATION BY FOLLICLE-STIMULATING-HORMONE

Progression of preantral follicle development is essential to further follicle maturation and ovulation, but there are few models for studyi ng the regulation of preantral follicle survival and growth. We have e valuated preantral follicle survival in vivo and in vitro, and have de veloped a serum-free rat follicle culture system that can be used to c haracterize the regulation of preantral follicle growth and differenti ation. Analysis of ovarian cell DNA fragmentation during the first wav e of follicle growth in the infantile rat indicated negligible apoptos is up to day 16 of age. However, a major increase in apoptosis was fou nd by day 18, a time point associated with the appearance of large ant ral follicles. In situ analysis confirmed that apoptotic DNA fragments were limited to antral follicles. Culture of individual preantral fol licles mechanically dissected from ovaries of 12- or 14-day-old rats i n serum-free conditions led to major increases in follicle cell apopto sis, similar to that seen in cultures of antral and preovulatory folli cles. In contrast to antral and preovulatory follicles, treatment of p reantral follicles with gonadotropins or cAMP analogs did not prevent apoptosis. However, treatment with 8-bromo-cGMP or 10% serum suppresse d apoptosis by 75% in cultured preantral follicles. In situ analysis i dentified granulosa cells as the cell type susceptible to apoptosis re gulation. Taking advantage of the ability of the cGMP analog to suppre ss apoptosis, we evaluated the potential of FSH as a growth factor. In the absence of serum, FSH treatment for 48 h did not affect follicle size compared to controls; however, treatment with the cGMP analog tog ether with FSH increased follicle diameter (13%; P < 0.01) and viable cells (2.4-fold; P < 0.01) compared to control values. Immunoblot anal ysis further indicated that the inhibin-cr content of the cultured fol licles was increased by treatment with the combination of FSH and 8-br omo-cGMP, demonstrating the induction of follicle cell differentiation during culture. Therefore, we demonstrated that activation of the cGM P pathway promotes the survival of cultured preantral follicles and th at in the presence of a cGMP analog, FSH is a growth and differentiati on factor for preantral follicles. The present serum-free follicle cul ture model system will be useful in further evaluation of the regulati on of growth and differentiation of preantral follicles.