Mammalian ovaries consist of follicles as basic functional units. The total number of ovarian follicles is determined early in life, and the depletion of this pool leads to reproductive senescence. Each follicle develops to either ovulate or, more likely, to undergo degeneration. The dynamics of ovarian follicle development have interested endocrinologists and developmental biologists for many years. With the advent of assisted reproductive techniques in humans, the possibility of regulating follicle development in vivo and in vitro has gained clinical relevance. In this review, we focus upon key branching points during the development of ovarian follicles as well as factors involved in determining the eventual destiny of individual follicles. We discuss inconsistencies in the literature regarding the definitions of follicle recruitment and selection and propose to name the two major steps of follicle development as initial and cyclic recruitment, respectively. Because some of these disparities have arisen due to differences in the animal systems studied, we also compare the development of the ovarian follicles of both humans and rats. We also review the status of knowledge of several puzzling clinical issues that may provide important clues toward unlocking the mechanisms of follicle development.

Mammalian ovaries consist of follicles as basic functional units. The total number of ovarian follicles is determined early in life, and the depletion of this pool leads to reproductive senescence. Each follicle develops to either ovulate or, more likely, to undergo degeneration. The dynamics of ovarian follicle development have interested endocrinologists and developmental biologists for many years. With the advent of assisted reproductive techniques in humans, the possibility of regulating follicle development in vivo and in vitro has gained clinical relevance. In this review, we focus upon key branching points during the development of ovarian follicles as well as factors involved in determining the eventual destiny of individual follicles. We discuss inconsistencies in the literature regarding the definitions of follicle recruitment and selection and propose to name the two major steps of follicle development as initial and cyclic recruitment, respectively. Because some of these disparities have arisen due to differences in the animal systems studied, we also compare the development of the ovarian follicles of both humans and rats. We also review the status of knowledge of several puzzling clinical issues that may provide important clues toward unlocking the mechanisms of follicle development. (Endocrine Reviews 21: 200 -214, 2000)

In the intracellular death program, heteroand homodimerization of different anti-and pro-apoptotic Bcl-2-related proteins are critical in the determination of cell fate. From a rat ovarian fusion cDNA library, we isolated a new pro-apoptotic Bcl-2 gene, Bcl-2-related ovarian killer (Bok). Bok had conserved Bcl-2 homology (BH) domains 1, 2, and 3 and a C-terminal transmembrane region present in other Bcl-2 proteins, but lacked the BH4 domain found only in anti-apoptotic Bcl-2 proteins. In the yeast two-hybrid system, Bok interacted strongly with some (Mcl-1, BHRF1, and Bf l-1) but not other (Bcl-2, Bcl-xL, and Bcl-w) anti-apoptotic members. This finding is in direct contrast to the ability of other pro-apoptotic members (Bax, Bak, and Bik) to interact with all of the anti-apoptotic proteins. In addition, negligible interaction was found between Bok and different pro-apoptotic members. In mammalian cells, overexpression of Bok induced apoptosis that was blocked by the baculoviralderived cysteine protease inhibitor P35. Cell killing induced by Bok was also suppressed following coexpression with Mcl-1 and BHRF1 but not with Bcl-2, further indicating that Bok heterodimerized only with selective anti-apoptotic Bcl-2 proteins. Northern blot analysis indicated that Bok was highly expressed in the ovary, testis and uterus. In situ hybridization analysis localized Bok mRNA in granulosa cells, the cell type that underwent apoptosis during follicle atresia. Identification of Bok as a new pro-apoptotic Bcl-2 protein with restricted tissue distribution and heterodimerization properties could facilitate elucidation of apoptosis mechanisms in reproductive tissues undergoing hormone-regulated cyclic cell turnover.

Transgenic mice with deletion of the GDF-9 (growth differentiation factor-9) gene are characterized by the arrest of ovarian follicle development at the primary stage. Based on the hypothesis that GDF-9 is important for early follicle development, we isolated rat GDF-9 complementary DNA (cDNA) and generated recombinant GDF-9 protein to study its physiological role. Using bacteria-derived GDF-9-glutathione S-transferase (GST) fusion protein, specific antibodies to the mature form of GDF-9 was generated. Immunohistochemical staining of ovarian sections indicated the localization of GDF-9 protein in the oocyte of primary, secondary and preantral follicles, whereas immunoblotting demonstrated the secretion of GDF-9 by mammalian cells transfected with GDF-9 cDNAs. Recombinant GDF-9 was shown to be an N-glycosylated protein capable of stimulating early follicle development. Growth of preantral follicles isolated from immature rats was enhanced by treatment with either GDF-9 or FSH whereas the combined treatment showed an additive effect. In addition, treatment with GDF-9, like forskolin, also stimulated inhibin-alpha content in explants of neonatal ovaries. In contrast, the stimulatory effects of GDF-9 were not mimicked by amino-terminal tagged GDF-9 that was apparently not bioactive. Thus, the present study demonstrates the important role of GDF-9 in early follicle growth and differentiation. The availability of recombinant bioactive GDF-9 allows future studies on the physiological role of GDF-9 in ovarian development in vivo.

Müllerian inhibitory substance (MIS), also known as anti-Müllerian hormone, is best known as the hormone that regulates the regression of the Müllerian duct in males. In females, MIS is expressed in granulosa cells of preantral and early antral follicles. The specific MIS type II receptor is present in granulosa and theca cells of these small, growing follicles. Because the role of MIS in preantral follicle development is unknown, we have evaluated the effect of MIS on the growth, differentiation, and apoptosis of intact preantral follicles in a serum-free culture system. In this system, treatment with FSH induces an increase in both follicle diameter, cell number, and follicle cell differentiation based on increased inhibin-alpha synthesis. Of interest, treatment with MIS enhances the effect of FSH both on follicle diameter and cell number. Although treatment with activin A also enhances FSH effects on follicle growth, treatment with transforming growth factor (TGF)-ss inhibits the FSH effects on follicle growth. Based on in situ staining of fragmented DNA, MIS was found to have no effect on follicle cell apoptosis, unlike its proapoptotic action on Müllerian ducts. In contrast to MIS and activin, TGF-ss was a potent proapoptotic factor for preantral follicles in culture. Analysis of inhibin-alpha expression of cultured preantral follicles further indicated that in contrast to activin, treatment with MIS did not enhance FSH-stimulated follicle differentiation. Thus, MIS is a unique factor that promotes preantral follicle growth but not preantral follicle cell differentiation and apoptosis.

The stimulatory effects of gonadotropins on antral and preovulatory follicles are well known, but conflicting results have been reported regarding the gonadotropin responsiveness and dependency of preantral follicles. Taking advantage of the relatively uniform development of the first wave of follicles in the postnatal rat ovary, we evaluated the role of endogenous and exogenous gonadotropins on preantral follicle development. Reduction of the high levels of gonadotropins present in juvenile rats by either hypophysectomy (at Day 15) or GnRH antagonist treatment (starting from Day 11 of age) resulted in decreased ovarian weight at Day 19 of age that was associated with a reduced number of developing follicles and increased atresia of remaining follicles. In contrast, treatment with FSHctp (a long-acting FSH agonist) in intact (Days 5-19 of age), hypophysectomized (Days 15-19), or GnRH antagonist-treated (Days 11-19) animals resulted in increased ovarian weight and follicle development as determined histologically and by inhibin-alpha expression. A dose-dependent stimulatory effect of hCG on ovarian weight was seen when animals were cotreated with FSHctp and the GnRH antagonist. At low doses of hCG, augmentation of antral follicle formation occurred, whereas higher doses of hCG led to morphological signs of luteinization. These findings demonstrate the important role of endogenous gonadotropins in preantral follicle development and indicate that preantral follicles are highly responsive to exogenous gonadotropins.

The response of pituitary gonadotropes to gonadotropin-releasing hormone (GnRH) correlates directly with the concentration of GnRH receptors (GnRHR) on the cell surface, which is mediated in part at the level of gene expression. Several factors are known to affect expression of the mouse GnRHR (mGnRHR) gene, including GnRH and activin. We have previously shown that activin augments GnRH-mediated transcriptional activation of mGnRHR gene, and that region ؊387/؊308 appears to be necessary to mediate this effect. This region contains two overlapping cis-regulatory elements of interest: GnRHR activating sequence (GRAS) and a putative SMAD-binding element (SBE). This study investigates the role of these elements and their cognate transcription factors in transactivation of the mGnRHR gene. Transfection studies confirm the presence of GnRH-and activin-response elements within ؊387/؊308 of mGnRHR gene promoter. Competition electrophoretic mobility shift assay experiments using ؊335/ ؊312 as probe and ␣T3-1 nuclear extract or SMAD, Jun, and Fos proteins demonstrate direct binding of AP-1 (Fos/Jun) protein complexes to ؊327/؊322 and SMAD proteins to ؊329/؊328. Further transfection studies using mutant constructs of these cis-regulatory elements confirm that both are functionally important. These data define a novel cis-regulatory element comprised of an overlapping SBE and newly characterized non-consensus AP-1 binding sequence that integrates the stimulatory transcriptional effects of both GnRH and activin on the mGnRHR gene.

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.