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*

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.

Critical roles of the guanylyl cyclase B receptor in endochondral ossification and development of female reproductive organs

“…Nppc and Npr2 genes are expressed in granulosa cells of follicles and thecalinterstitial cells of rat ovaries (30,32). It has been reported that 8-bromo-cGMP can protect ovarian follicles from atresia and promote growth (33). In combination with observations that no corpora lutea and no estrus cycling were seen in null mice, the CNP͞GC-B͞cGMP system appears essential for the growth and maturation of ovarian follicles.…”

Essential role of follicle stimulating hormone in the maintenance of caprine preantral follicle viability in vitro

“…Furthermore, a two-way exchange may occur between the oocyte and granulosa cells and a direct action of FSH on oocytes produces compounds whose diffusion into the granulosa cells is necessary for their proliferation and maturation (Méduri et al, 2002). Other authors observed that FSH promotes an increase in follicular diameter and proliferation of granulosa cells (rats: McGee et al, 1997;mouse: Nayudu & Osborn, 1992;Cortvrindt et al, 1996Cortvrindt et al, , 1997Cortvrindt et al, , 1998bovine: Wandji et al, 1996;Saha et al, 2000;caprine: Silva et al, 2004;ovine: Cecconi et al, 1999;human: Roy & Treacy, 1993;Wright et al, 1999). In addition, FSH was found to stimulate antrum formation and steroidogenesis in granulosa cells (Nayudu & Osborn, 1992;Boland et al, 1993;Ralph et al, 1995Ralph et al, , 1996Wandji et al, 1996;Abir et al, 1997;Gutierrez et al, 2000;Wu et al, 2000;Mao et al, 2002;Mitchell et al, 2002;Kreeger et al, 2005).…”

Regulation of Oocyte and Cumulus Cell Interactions by Intermedin/Adrenomedullin 2

“…Receptor-binding Assay, cAMP, and Estradiol Production by Cultured Granulosa/Cumulus Cells-Granulosa/cumulus cells were obtained from ovaries of immature 21-day-old rats that had been implanted with diethylstilbestrol for 4 days by puncturing antral follicles with fine gauge needles; these cells were cultured in L15 medium with 0.1% BSA as described previously (60). For the receptor-binding assay, cells were resuspended in ice-cold binding buffer (20 mM Tris-HCl (pH 7.4), 2 mM MgCl 2 , and 0.1% BSA) in siliconized microcentrifuge tubes with 0.06 g of […”