FOLLICULOGENESIS is the culmination of a process of

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1 /99/$03.00/0 Vol. 140, No. 1 Endocrinology Printed in U.S.A. Copyright 1999 by The Endocrine Society Activin from Secondary Follicles Causes Small Preantral Follicles to Remain Dormant at the Resting Stage HIDEKI MIZUNUMA, XIAOWEI LIU, KAZUMICHI ANDOH, YUMIKO ABE, JUNRO KOBAYASHI, KIYOHIKO YAMADA, HIDEMI YOKOTA, YOSHITO IBUKI, AND YOSHIHISA HASEGAWA Department of Obstetrics and Gynecology (H.M., X.L., K.A., J.K., K.Y., H.Y., Y.I.), Gunma University School of Medicine, Maebashi, Gunma , Japan; and Laboratory Animal Science (Y.H.), Kitasato University School of Veterinary Medicine and Animal Science, Towada, Aomori , Japan ABSTRACT The purpose of the present study was to investigate 1) whether activin A can cause primary follicles to become dormant at the resting stage, and 2) the role of the secondary follicle on follicular growth of primary follicles. Preantral follicles ( m in diameter) harvested from adult mice and cultured in in vitro follicle culture system showed a significant increase in size and estrogen and inhibin secretion in response to FSH, but the administration of activin A blocked the effect of FSH. Withdrawal of activin A not only restored the follicular response to FSH but also enhanced the effect of FSH, indicating that the action of activin A is to cause small preantral follicles to become dormant at the preantral stage. To investigate the role of secondary follicles in early folliculogenesis, small preantral follicles FOLLICULOGENESIS is the culmination of a process of follicular growth characterized by morphological changes accompanied by functional development. In the early stage of folliculogenesis, a cohort of primary follicles depart from a dormant pool (1, 2) and then the onset of antrum formation is a critical developmental threshold for folliculogenesis because the secondary follicles are destined to enter further growth or atresia (3). It is generally accepted that the follicular development is induced without FSH in the early stages (1, 2, 4) and that putative intraovarian factors are responsible for early folliculogenesis (1, 2, 5). However, most intraovarian factors are not detected in follicles during early folliculogenesis, thus posing a biological enigma of how folliculogenesis can be triggered. On the other hand, FSH receptors are expressed in granulosa cells during the transition from primordial follicles to growing preantral follicles (6), and indeed isolated preantral follicles with 1 4 layers of granulosa cells show increased DNA synthesis (7) and follicular diameter (8) in response to FSH. Therefore, the intriguing question has been why primary follicles can stay at their dormant stage while others depart from the resting pool despite the fact that they are both equally exposed to circulating FSH. One likely explanation is a difference in sensitivity to FSH among primary follicles, but reported results Received May 15, Address all correspondence and requests for reprints to: Hideki Mizunuma, Department of Obstetrics and Gynecology, Gunma University School of Medicine, , Showa-machi, Maebashi, Gunma , Japan. mizunuma@news.sb.gunma-u.ac.jp. were cocultured with secondary follicle ( m in diameter) in the presence of FSH. The secondary follicle showed a significant increase in follicular diameter as a result of stimulation by FSH, but the small preantral follicles did not increase in size. After removal of the secondary follicle, however, the small preantral follicles commenced follicular growth, indicating that the growth of small preantral follicles is suppressed by the secondary follicle. Administration of the activin binding protein follistatin caused a significant increase in follicular diameter of both small preantral and secondary follicles as a result of stimulation by FSH. These results have suggested that secondary follicles cause primary follicles to become dormant at the resting stage by secreting activin. (Endocrinology 140: 37 42, 1999) have shown that DNA synthesis by FSH stimulation is higher in isolated follicles at the preantral stage than at the antral stage (7), thus tending to discount this explanation. Here we have provided evidence that activin derived from the secondary follicles causes small preantral follicles to remain dormant and proposed a novel hypothesis that a local decline of activin as a result of atresia of the secondary follicles initiates early folliculogenesis in response to circulating FSH and GH. Chemicals Materials and Methods Recombinant human FSH (rhfsh) was obtained from Organon (Oss, The Netherlands). Recombinant human activin A (rh activin A) was prepared as described previously (8). Recombinant human GH (rhgh) was provided by Serono Laboratories, Inc. (Geneva, Switzerland). Recombinant human follistatin (rhfs) with 288 amino acids was obtained from the National Hormone and Pituitary Program, NIDDK. TGF- 1 was obtained from Sigma Chemical Co. (St. Louis, MO). All other chemicals were of analytical grade or the highest quality commercially available. Follicle culture Preantral follicles were prepared as described previously (8). Briefly, ovaries were removed aseptically from the 56-day-old female mice and placed in 15-mm diameter Falcon plastic Petri dishes (FALCON 3037, Becton Dickinson and Co., Franklin Lakes, NJ) filled with (Gibco BRL, Tokyo, Japan) at room temperature. After removing the surrounding tissue, the ovaries were microdissected using two 27-gauge needles attached to 1 ml syringes under the stereomicroscope and small preantral follicles ( m in diameter) with 1 or 2 layers of granulosa cells around the oocyte and an intact basal lamina with theca cells and 37

2 38 ACTIVIN CAUSES PREANTRAL FOLLICLE TO REMAIN DORMANT Endo 1999 Vol 140 No 1 secondary follicles ( m in diameter) with several layers of granulosa cells with an intact basal lamina with theca cells were mechanically isolated. To test the effect of FSH, activin A, TGF-, and GH on follicles, 10 small preantral follicles were transferred into a Falcon plastic Petri dish filled with 1 ml serum free DMEM supplemented with 6.25 g/ml of insulin, 6.25 g/ml of transferrin, 6.25 ng/ml of selenious acid, 5.35 g/ml of linoleic acid, 0.15% BSA, 15 mm HEPES, 45 g/ml of penicillin G, 350 g/ml of streptomycin, and 1.75 g/ml of Amphotericin B, and were cultured in a humidified chamber with 5% CO 2 in the air at 37 C for 4 8 days. To investigate the role of the secondary follicle on the growth of the small preantral follicle, four small preantral follicles were cocultured with one large preantral follicle in the same conditioned medium. Hormones were added to the same conditioned medium on day 0 in the indicated concentrations. Animal care was in accordance with the institutional guidelines of the Gunma University School of Medicine. Measurements and statistics Two-dimensional maximum and minimum lengths of each follicle were measured daily with an inverted microscope. The mean diameter of the follicle was calculated by averaging these two measurements. Inhibin and estradiol concentrations were measured as markers of follicular development by double antibody RIA as described previously (9, 10). Differences between means of follicular diameters were analyzed by the -square method followed by Scheffe s F test. Differences between means of hormone levels were analyzed by a nonparametric method by using the Kruskall-Wallis test. Results Small preantral follicles obtained from 56-day-old mice, m in diameter, with 1 or 2 layers of granulosa cells around the oocyte and an intact basal lamina with theca cells, showed a significant granulosa cell proliferation and an increase in follicular diameter (Fig. 1) in the presence of FSH, GH, and TGF- in a dose-dependent manner (Fig. 2A). The maximal response was attained at a dose of 100 miu/ml of FSH, 10 miu/ml of GH, and 100 ng/ml of TGF-, respectively. The growth of small preantral follicles was accompanied by a significant increase in estrogen and immunoreactive (IR)-inhibin secretion (Fig. 2B). However, activin A did not show any remarkable changes in follicular diameter or in FIG. 1. Morphological changes in a preantral follicle of an adult mouse cultured for 4 days in the presence of the culture medium alone or 100 miu/ml of FSH, 10 miu/ml of rhgh, 100 ng/ml of TGF-, and 100 ng/ml of activin A. estrogen or IR-inhibin secretion at doses of ng/ml, respectively (Figs. 1 and 2). To examine whether the inhibitory action of activin A is atretogenic, small preantral follicles, m in diameter were cultured with activin A for 4 days and then transferred into a control medium or medium containing FSH (Fig. 3). Small preantral follicles cultured with activin A did not show a significant increase in size or estradiol or immunoreactive (IR)-inhibin secretion, but after withdrawal of activin A they commenced follicular growth as well as estradiol and IRinhibin secretion as a result of stimulation by FSH, indicating that the inhibitory action of activin A is reversible and causes small preantral follicles to remain dormant. The effect of activin A on small preantral follicular growth as stimulated by potent extra and intraovarian stimulators such as FSH, GH and TGF-, respectively, is shown in Fig. 4. Activin A blocked the effect of FSH and GH but did not block the effect of TGF-. The inhibitory action of activin A on FSH and GH was restored by the administration of the activin binding protein, follistatin (FS). To investigate the role of secondary follicles on follicular growth of small preantral follicles, small preantral follicles were cocultured with a secondary follicle in the presence of FSH (Fig. 5). Small preantral follicles, m in diameter, showed a significant increase in follicular diameter as a result by stimulation of 10 miu/ml of FSH (Fig. 5B), but they did not increase in size in the presence of the secondary follicles. After removal of the secondary follicle, the small preantral follicles commenced follicular growth. Administration of FS at a dose of 2 ng/ml, however, caused a significant increase in follicular diameter of both small preantral and secondary follicles, demonstrating that activin is involved in the inhibitory action of secondary follicles. Discussion Activin A, a gonadal protein, is a homodimer composed of two -subunits of inhibin, which is a heterodimer glycoprotein consisting of an -subunit and one of two -subunits ( A and B) (11). There is increasing evidence that activin A locally modulates granulosa cell function. Activin A enhances FSH-induced aromatase activity (12 14), the number of LH binding sites (15), and progesterone production (11 15) in cultured granulosa cells obtained from estrogentreated immature rats. In addition, activin A stimulates FSHinduced inhibin production (15) and follistatin (FS) messenger RNA (mrna) (16) in rat granulosa cells in vitro. One of the mechanisms of activin A action is to stimulate FSH receptor formation (17, 18), and indeed increased FSH mrna levels because of activin A treatment have been reported (13, 14). Thus most studies conducted on cultured granulosa cells obtained from estrogen-treated immature rats have proposed the stimulatory effect of activin A on granulosa cell function. However, Gonzalez-Manchon et al. (19) have shown that activin A inhibited the growth of Chinese hamster ovary (CHO)-k1 cell line and Woodruff et al. (20) have demonstrated the possibility that activin A may have an atretogenic action on follicular development according to an in vivo study using 25-day-old rats. More recently, we have shown that activin A promotes the folliculogenesis

3 ACTIVIN CAUSES PREANTRAL FOLLICLE TO REMAIN DORMANT 39 FIG. 2. Changes in the follicular diameter of small preantral follicles (A) and estradiol and immunoreactive (IR)-inhibin secretion from follicles (B) cultured with each of recombinant human FSH (rhfsh), rhgh, TGF-, and activin A at indicated doses. Experiments were repeated three to five times for each reagent. The number of follicles for each point was In this and the following figures, the data shown are the mean and SEM. *,P 0.05; **, P 0.001, and ***, P vs. control on the same culture day, respectively. FIG. 3. Changes in follicular diameter of primary follicles after withdrawal of Activin A from the culture medium. Activin A causes primary follicles to become dormant and have an increased responsiveness to FSH. Primary follicles were pretreated either with activin or the control medium for 4 days. The culture medium was replaced at day 4 by the medium indicated. The dose of FSH and activin A was 100 miu/ml and 100 ng/ml, respectively. A, Changes in follicular diameter. B, Estradiol and inhibin secretion. **, P and ***P vs. either of activin A 3 activin A or control 3 control. a, P and b, P of small preantral follicles from 11-day-old female mice but antagonizes the effect of FSH on preantral follicular growth in adult mice (8), clearly indicating that the action of activin A is age dependent. The inhibitory action of activin A on cellular growth has been evidenced in other tissues. For example, it has been shown that activin inhibits: 1) testosterone formation in the Leydig cells derived from normal mature rats (21); 2) both expression of GH and growth of somatotropic cells (22); and 3) initiation of DNA synthesis in rat hepatocyte (23) and 4) vascular endotherial cell growth (24). To examine whether the inhibitory action of activin A is

4 40 ACTIVIN CAUSES PREANTRAL FOLLICLE TO REMAIN DORMANT Endo 1999 Vol 140 No 1 FIG. 4. The effect of activin A on follicular diameter (A) and estradiol and IRinhibin secretion (B) stimulated with FSH (100 miu/ml), GH (10 miu/ml) and TGF- (100 ng/ml) in the presence or absence of follistatin (FS). Activin A at a dose of 100 ng/ml blocked the stimulatory effect of FSH and GH, but not the effect of TGF-. Administration of 100 ng of rh-fs reversed the inhibitory action of activin A. **, P and ***, P vs. either of rhfsh or rhgh., P 0.001, and, P vs. either of rhfsh activin A, rhgh activin A or TGF- activin A. a, P and b, P reversible, we cultured small preantral follicles with activin A for 4 days and then transferred them into a control medium or medium containing FSH. Woodruff et al. (20) have shown that intrabursal injection of activin A resulted in minimal follicular development with an increase in the number of atretic follicles in all size classes, but our present study has shown that the inhibitory action of activin A on small preantral follicles is reversible because small preantral follicles pretreated with activin A showed a significant increase in size and hormone secretion in response to FSH after withdrawal of activin A (Fig. 3). In addition, it has been shown that pretreatment of small preantral follicles with activin A enhances the response to FSH, suggesting that the inhibitory action of activin A is not atretogenic but instead causes small preantral follicles to remain dormant. The discrepancy between the two studies may be attributed to several factors. First, Woodruff et al. (20) used 25-day-old rats, whereas we employed adult mice. Our previous study has shown that the action of activin A on small preantral follicles in immature and adult mice is paradoxical (8), and our unpublished results showed that the paradoxical action take places around the age of 28 days, when the pubertal gonadotropin surge has ended (Liu et al., unpublished data). Second, the follicular size defined by Woodruff et al. (20) as preantral follicles was 350 m and included much larger follicles than what was used in this study. Studies on size-frequency analysis have revealed that follicles at an early antral stage ( m) are much more susceptible to atresia than follicles at the preantral or preovulatory stage (25). Next, we studied the effect of activin A on small preantral follicular growth as stimulated by potent extra and intraovarian stimulators such as FSH, GH, and TGF-, respectively (1, 2, 7). Activin A blocked the effect of FSH and GH, but the inhibitory action of activin A was restored by the administration of the activin binding protein, follistatin (FS) (Fig. 4). These findings suggest for the first time that activin A can hold follicular growth at the preantral stage despite small preantral follicles being exposed to extraovarian factors, FSH and GH. Interestingly, activin A did not modify the effect of the action of the putative intraovarian factor, TGF-, thus being compatible with the generally accepted concept that primary follicles can initiate their own growth as a result of stimulation by intraovarian factors (1, 2, 4). But most putative intraovarian factors are hardly detectable in early preantral follicles at the resting stage (5, 26, 27), suggesting the possibility that these intraovarian factors are less involved in triggering the initial growth of primary follicles. Inhibin -A and -B mrna, as well as most putative

5 ACTIVIN CAUSES PREANTRAL FOLLICLE TO REMAIN DORMANT 41 FIG. 5. Changes in follicular diameter of small preantral follicles cultured with a secondary follicle. Small reantral follicles cultured in a control medium or medium containing 2 ng/ml of follistatin did not show significant increase in size (A and C). Primary follicles cultured in a medium containing 10 IU/ml of FSH showed a significant increase in size (B). Primary follicles cocultured with a secondary follicle did not show an increase in size in the presence of 10 IU/mlof FSH, but they showed a significant increase after removal of the secondary follicle (D). When primary follicles are cocultured with a secondary follicle in the presence of FSH and follistatin, they showed a significant increase in size (E). Open circles represent changes in follicular size of primary follicles and closed ones represent those of secondary follicles. **, P and ***, P vs. day 0. intraovarian factors, are not detected in primordial or primary follicles and are first seen in the granulosa cells of secondary follicles (28); therefore, it is presumed that secondary follicles neighboring the primary follicles secrete activin and elicit inhibitory actions on primary follicles by the paracrine pathway. To test this hypothesis, we cultured small preantral follicles with a secondary follicle in the presence of FSH (Fig. 5). The secondary follicle showed significant growth as a result of stimulation by FSH, whereas small preantral follicles did not increase their size until the secondary follicle was removed, indicating that the growth of small preantral follicles was inhibited by the presence of the large preantral follicle (Fig. 5D). Administration of FS, however, caused a significant increase in follicular diameter of both small preantral and secondary follicles, thus clearly demonstrating that activin is involved in the inhibitory action of secondary follicles. In conclusion, although follicular development is induced without FSH in its early stage (3, 4, 6), the present study has confirmed that small preantral follicles are susceptible to FSH and GH, and that these extraovarian factors are potent stimulators that can initiate the first step of folliculogenesis. Due to an inhibitory control by activin, however, small preantral follicles can stay at the dormant stage despite the fact they are exposed to FSH and GH. Moreover, the present study has suggested for the first time that a local decline of activin as a result of atresia of secondary follicles triggers the early folliculogenesis to depart from the dormant pool, thus regulating the size of a cohort of primary follicles to enter further growth. This novel function of the secondary follicles has not only given a possible answer to the biological enigma of what controls the first step of folliculogenesis, but also sheds light on how the size of the cohort of growing follicles is determined. Acknowledgments We are grateful to Dr. Y. Eto (Ajinomoto Company, Kawasaki, Japan) for his kind gift of rh-activin A. We also thank Miss Y. Hayashi and Miss T. Ishihara for their assistance. References 1. Tonetta SA, dizerega GS 1989 Intragonadal regulation of follicular maturation. Endocr Rev 10: Gougeon A 1996 Regulation of ovarian follicular development in primates: facts and hypotheses. Endocr Rev 17: Hsueh AJ, Billig H, Tsafriri A 1994 Ovarian follicle atresia: a hormonally controlled apoptotic process. Endocr Rev 15: Kumar TR, Wang Y, Lu N, Matzuk MM 1997 Follicle stimulating hormone is required for ovarian follicle maturation but not male fertility. Nat Genet 15: Giudice LC 1992 Insulin-like growth factors and ovarian follicular development. Endocr Rev 13: Richards JS 1980 Maturation of ovarian follicles: actions and interactions of pituitary and ovarian hormones on follicular cell differentiation. Physiol Rev 60: Roy SK, Greenwald GS 1989 Hormonal requirements for the growth and differentiation of hamster preantral follicles in long-term culture. J Reprod Fertil 87: Yokota H, Yamada K, Liu X, Kobayashi J, Abe Y, Mizunuma H, Ibuki Y 1997 Paradoxical action of activin A on folliculogenesis in immature and adult mice. Endocrinology 138: Mizunuma H, Andoh K, Obara M, Yamaguchi M, Kamijo T, Hasegawa Y, Ibuki Y 1994 Serum immunoreactive inhibin levels in polycystic ovarian disease (PCOD) and hypogonadotropic amenorrhea. Endocr J 41: Takagi T, Mizunuma H, Andoh K, Obara M, Yamaguchi M, Hasegawa Y, Ibuki Y 1994 Changes in serum immunoreactive inhibin during ovulation induction Endocr J 41: Burger HG, Igarashi M 1988 Inhibin: definition and nomenclature, including related substances. Endocrinology 122: Hutchinson LA, Findlay JK, de-vos FL, Robertson DM 1987 Effects of bovine

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