Abstract
Progression of preantral follicle development is essential to further follicle maturation and ovulation, but there are few models for studying the regulation of preantral follicle survival and growth. We have evaluated preantral follicle survival in vivo and in vitro, and have developed a serum-free rat follicle culture system that can be used to characterize the regulation of preantral follicle growth and differentiation. Analysis of ovarian cell DNA fragmentation during the first wave of follicle growth in the infantile rat indicated negligible apoptosis up to day 16 of age. However, a major increase in apoptosis was found by day 18, a time point associated with the appearance of large antral follicles. In situ analysis confirmed that apoptotic DNA fragments were limited to antral follicles. Culture of individual preantral follicles mechanically dissected from ovaries of 12- or 14-day-old rats in serum-free conditions led to major increases in follicle cell apoptosis, similar to that seen in cultures of antral and preovulatory follicles. In contrast to antral and preovulatory follicles, treatment of preantral follicles with gonadotropins or cAMP analogs did not prevent apoptosis. However, treatment with 8-bromo-cGMP or 10% serum suppressed apoptosis by 75% in cultured preantral follicles. In situ analysis identified granulosa cells as the cell type susceptible to apoptosis regulation. Taking advantage of the ability of the cGMP analog to suppress 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 together with FSH increased follicle diameter (13%; P < 0.01) and viable cells (2.4-fold; P < 0.01) compared to control values. Immunoblot analysis further indicated that the inhibin-alpha content of the cultured follicles was increased by treatment with the combination of FSH and 8-bromo-cGMP, demonstrating the induction of follicle cell differentiation during culture. Therefore, we demonstrated that activation of the cGMP pathway promotes the survival of cultured preantral follicles and that in the presence of alpha cGMP analog, FSH is a growth and differentiation factor for preantral follicles. The present serum-free follicle culture model system will be useful in further evaluation of the regulation of growth and differentiation of preantral follicles.
Highlights
OVARIAN FOLLICLES, consisting of an oocyte surrounded by inner layers of granulosa and outer layers of thecal cells, represent the basic functional unit of the ovary
Because previous data [17] indicated that apoptosis of preovulatory follicles can be suppressed by treatment with cGMP analogs and agents that stimulate endogenous cGMP production (IL-1 and sodium nitroprusside (SNP)), we evaluated the effect of treatment with a cGMP analog, IL-1, and SNP on preantral follicles
We have demonstrated the ability of FSH to promote the growth and differentiation of preantral follicles and have shown that preantral follicles differ from antral follicles both in their in vivo propensity to undergo apoptosis as well as in the regulation of their survival in vitro
Summary
OVARIAN FOLLICLES, consisting of an oocyte surrounded by inner layers of granulosa and outer layers of thecal cells, represent the basic functional unit of the ovary. The use of enzymatically dispersed ovaries has been described [7,8,9,10,11,12], follicle structures in these cultures did not uniformly include basement membranes and thecal layers, and disruption of the three-dimensional follicular architecture may alter follicle responses to hormonal stimuli. Using these models, studies dealing with a potential stimulatory effect of FSH on preantral follicle growth have been conflicting [4, 7, 8, 13].
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