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Regular Article |
a Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, California 94305-5317
b Department of Biomedical Sciences (Physiology), Edinburgh University Medical School, Edinburgh, United Kingdom
c Department of Obstetrics and Gynecology, University of Kentucky Chandler Medical Center, Lexington, Kentucky 40536
d Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada
e Department of Physiology, University of California, San Francisco, California 94143-0444
ABSTRACT
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-
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- 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.
activin, apoptosis, developmental biology, follicle, follicular development, FSH, gene regulation, granulosa cells, growth factors, hormone action, inhibin, ovary, signal transduction, theca cells
INTRODUCTION
The finite pool of ovarian follicles is established early in mammalian development. Some of the pool of primordial follicles begin to grow almost immediately after formation. The remaining, resting pool of follicles is gradually depleted as primordial follicles continue to enter the growing pool until the time of reproductive senescence [1]. Once a follicle begins to grow and differentiate, it takes a prolonged time to develop into a preovulatory Graafian follicle. In rats, this process takes approximately 60 days, or 15 estrous cycles [1]. The majority of this time is spent as a slow-growing, preantral follicle. Although multiple studies have been performed to elucidate the hormonal regulation of the development of antral and larger follicles, relatively little is known about the regulation of the growth and development of ovarian follicles before the antral stage.
Müllerian inhibitory substance (MIS), also known as anti-Müllerian hormone, is a member of the transforming growth factor (TGF)-ß family of glycoprotein hormones. This family signals via heterodimeric serine-threonine kinase receptors. The ligand-specific, type II receptor phosphorylates the type I receptor, which subsequently activates the downstream cascade of SMAD signaling proteins [2]. Müllerian inhibitory substance was first identified as a testicular product that induced the regression of the Müllerian duct in males [3]. Subsequently, MIS protein and mRNA were localized to the ovary, although a definitive function of ovarian MIS has not been established.
Müllerian inhibitory substance is expressed by granulosa cells of small, growing follicles [4–6]. Expression levels decline in larger antral follicles and also in atretic follicles [4, 5]. In follicular fluid, the concentration of MIS is higher in small antral follicles than in larger, more differentiated follicles [6]. The MIS type II receptor is expressed by granulosa cells of preantral and small antral follicles, but not by granulosa cells of larger antral follicles or of preovulatory follicles [7, 8]. However, this receptor is also expressed by theca cells of small follicles [9]. The theca cell expression is most marked in preantral and small antral follicles, but unlike the granulosa cell expression, theca cells continue to express the receptor in antral and early atretic follicles [9]. This pattern for expression of MIS and its receptor in small, growing follicles leads to the speculation that MIS has a role in early follicle development.
Although female mice that are deficient in MIS expression are fertile [10], such mice have recently been shown to have an increased number of growing preantral follicles during early adulthood and to undergo earlier reproductive senescence [11], suggesting that MIS plays a role in the regulation of early follicle development. Using a preantral follicle culture model [12, 13], we have explored the role of MIS in regulation of the early development of ovarian follicles by treating cultured preantral follicles with bioactive recombinant MIS [14] and assessing the parameters of growth, differentiation, and apoptosis.
MATERIALS AND METHODS
Hormones and Reagents
Human FSH (ISIAFP-1; 8466 IU/mg) and recombinant activin A were obtained from the National Hormone and Pituitary Distribution Program, NIDDK, National Institutes of Health (NIH; Baltimore, MD). The 8-bromo-cGMP was purchased from Sigma (St. Louis, MO). The TGF-ß1 was purchased from R&D Systems (Minneapolis, MN).
Recombinant MIS
Recombinant bioactive MIS and inactive MIS ligand were obtained from conditioned media collected from HEK-293S cells stably transfected with expression vectors encoding rat MIS cDNA as described previously [14]. Conditioned media were concentrated, and MIS protein content was quantitated by immunoblot analysis. The concentration after filtration was 14 ng/µl. Bioactivity and concentration of the recombinant protein were verified by Müllerian duct regression assay as described previously [14]. This is an organ culture bioassay that verifies the ability of the MIS conditioned medium to induce regression of the Müllerian duct in the female embryonic genital ridge. Inactive MIS, for use as a control, was produced in the same way as bioactive MIS, except that the inactive MIS contained a point mutation resulting in an amino acid change at the cleavage site that prevents the cleavage and, therefore, the bioactivity of the molecule [14]. Müllerian inhibitory substance was used at 200 ng/ml, the lowest dose that reliably induced duct regression, and at 1000 ng/ml, the highest dose possible to include in the small volume of medium used in the follicle cultures. Ovarian fragments cocultured with gonadal ridge effectively induce a magnitude of Müllerian duct regression similar to that from 200 ng/ml of the MIS preparation [5, 14].
Animals and Ovarian Dissection
All animal experiments were performed in accordance with NIH and institutional guidelines. Sprague-Dawley rats were obtained from Simonsen (Gilroy, CA) and housed under standard conditions. Animals were anesthetized with CO2 and killed by cervical dislocation. Ovaries for follicle cultures were obtained and immediately placed in warmed culture medium and mechanically dissected as described previously [12, 13].
Follicle Culture
To assess the effects of different growth factors on the growth and differentiation of individual follicles, follicles were cultured as previously described [12, 13]. Preantral follicles of 140–150 µm in diameter were dissected from the ovaries of 12-day-old rats under a dissecting microscope using fine needles. Follicles were cultured individually in 96-well dishes lined with polycarbonate membranes in 150 µl of medium overlaid with 75 µl of sterile mineral oil at 36°C in a moist atmosphere of 5% CO2 and 95% air. Basal (i.e., control) medium consisted of -Modified Eagle's Medium supplemented with ITS+ (insulin, 10 mg/L; transferrin, 5.5 mg/L; linoleic acid, 4.7 mg/L; and selenium, 5 mg/L) and Pen/Strep (penicillin, 100 U/ml; and streptomycin, 100 µg/ml); all were obtained from Sigma. Follicle diameter was measured daily using an inverted microscope fitted with an ocular micrometer. The follicle diameter was defined as the average distance between the outer edges of the basement membrane in two perpendicular planes. At the end of the 72-h incubation period, follicles were collected for further analysis of cell number or inhibin- content or were fixed in 4% w/v paraformaldehyde.
Quantification of Viable Follicle Cells
Cell proliferation was verified using the tetrazolium salt-based Cell Proliferation Kit I from Boehringer Mannheim (Indianapolis, IN) [12]. This assay is based on the ability of living cells to metabolize yellow tetrazolium salt to blue formazan crystals that can be solubilized and quantified spectrophotometrically. At the completion of the experiments, replicates of four follicles from each treatment group were moved to single wells containing 100 µl of basal medium. Next, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide labeling reagent was added to the wells and incubated for 4 h at 37°C under 5% CO2. Solubilization solution was then added for 18 h before determination of absorbance at 560 nm. Absorbance levels were standardized to known quantities of granulosa cells.
Immunoblot Analysis of Inhibin- Content of Cultured Follicles
Twenty follicles from each treatment group were collected in Eppendorf tubes at the end of the experiment and kept frozen. Follicles were later thawed in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% SDS, 5 mM EGTA, 0.5 mM MgCl2, 0.5 mM MnCl2, and 0.2 mM phenylmethylsulfonylfluoride) and homogenized with a glass rod. Inhibin- antigen levels in tissue homogenates were determined using immunoblot analysis as previously described [12]. Briefly, samples were fractionated using electrophoresis in a 10% polyacrylamide gel. Proteins were transferred to nitrocellulose membranes, and immunoblotting was performed with a mouse monoclonal antibody to inhibin- (Serotec Limited, Oxford, England) followed by incubation with a biotinylated anti-mouse second antibody and immunofluorescent imaging with the enhanced chemiluminescence (ECL) Western System (Amersham). The ECL incubated blots were exposed to Hyperfilm (Amersham, Piscataway, NJ), which resulted in a distinct band at 41 kDa [12]. Quantification of the radiographic signal was performed using densitometric analysis.
Histological and Apoptosis Analysis
Following culture, follicles were fixed in 4% paraformaldehyde, embedded in agarose, then embedded in paraffin and sectioned (5 µm). In situ labeling of free DNA ends was performed using the Apoptag fluorescein isothiocyanate kit (Oncor, Gaithersburg, MD) according to manufacturer's protocols. Serial sections were used for Apoptag and hematoxylin-and-eosin staining. Approximately 10 follicles from each treatment group were analyzed histologically. (Representative follicles are pictured in Fig. 4.)
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Data Analysis
All experiments were repeated at least three times, and a representative autoradiograph is presented where appropriate. Statistical significance between mean values was determined by ANOVA followed by Neuman-Kuels post-hoc testing and was accepted at the P < 0.05 level.
RESULTS
Because MIS and its receptor are both highly expressed in preantral follicles, we used a preantral follicle culture system [12] to evaluate the role of MIS on early follicle development. First, we examined the role of recombinant MIS on the growth (i.e., the increase in size) of cultured follicles (Fig. 1). Follicles cultured in the absence of FSH or MIS had minimal change in size during the 72-h culture period (basal group). The MIS treatment (200 ng/ml) resulted in a 10-µm increase in follicle diameter (P < 0.05). In addition, FSH (100 ng/ml) treatment resulted in an increase in follicle diameter of 25 µm, consistent with previous studies [12, 13, 15]. A combination of treatment with FSH and MIS (at either 200 or 1000 ng/ml) resulted in an increase in follicle diameter of 40 µm (P < 0.05). As mentioned, MIS that was uncleaved and, therefore, not bioactive was used as a control. Uncleaved MIS (200 ng/ml) had no effect on basal (data not shown) or FSH-stimulated follicle growth (P > 0.05; Fig. 1).
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To compare the role of MIS in follicle development with other members of the TGF-ß family, we next performed experiments evaluating the effect of MIS, activin A, and TGF-ß on FSH-stimulated follicle growth (Fig. 2). Again, control follicles had little change in follicle diameter during the 72-h culture period, whereas FSH stimulated follicle growth. Treatment with either MIS (200 ng/ml) or activin (200 ng/ml) augmented the effects of FSH by approximately 60% (P < 0.05). In contrast, treatment with TGF-ß (1 ng/ml) suppressed the FSH-stimulated increase in follicle diameter to near basal levels.
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To demonstrate that changes in follicle diameter represent increases in cell number, we also performed an analysis of the viable cell number in follicles at the end of the studies using the tetrazolium assay (Fig. 3). The FSH treatment increased follicle cell number by 56% compared to the basal group (P < 0.05). The addition of MIS (200 ng/ml) or activin (200 ng/ml) increased FSH-stimulated cell division by 28% and 35%, respectively (P < 0.05). In contrast, TGF-ß treatment (1 ng/ml) did not affect the cell number compared to FSH treatment alone (P > 0.05). This is in contrast to the TGF-ß suppression of the FSH-induced increase in follicle diameter. In combination, these data suggest an effect of TGF-ß on theca cell division; however, further studies are necessary to clarify this point.
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Because the action of MIS in the developing male is to promote apoptosis of the Müllerian duct, we also evaluated the effect of MIS on follicle cell survival (Fig. 4). As we have previously shown in this culture system [12], follicles have a very low rate of staining for apoptotic DNA fragments in the absence of hormonal treatment. In the present study, FSH had minimal or no effect on this basal rate. The addition of MIS or activin to FSH treatment also had no effect on the low level of staining for DNA fragmentation. However, the addition of TGF-ß (1 ng/ml) to FSH treatment resulted in a marked increase in staining, signifying increased fragmentation of DNA consistent with an increase in apoptosis. Histologically, the theca layer in the follicles cultured with MIS appeared to be thicker and healthier than those from the FSH-treated group (Fig. 4). Further studies are necessary to clarify the role of MIS in theca cell growth and granulosa cell-theca cell interaction.
Next, we determined the effect of MIS, activin, and TGF-ß treatment on the differentiation of cultured follicles. Inhibin- expression increases with increasing maturity of ovarian follicles [16], and it has been used as a marker for differentiation of granulosa cells and follicles [12, 13, 17]. Using densitometric analysis of films from Western blots, we demonstrated that inhibin- antigen is present at low levels in control preantral follicles after 72 h of culture (Fig. 5). In contrast, treatment with FSH increased inhibin- expression by approximately fourfold. Activin treatment enhanced FSH-induced inhibin- expression by 300%. However, MIS treatment (200 ng/ml) had no effect on FSH-stimulated inhibin- expression. Likewise, the addition of TGF-ß did not affect FSH-stimulated follicle differentiation as indicated by inhibin- expression (P > 0.05).
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DISCUSSION
The present study demonstrated that MIS treatment enhances preantral follicle growth by increasing both follicle size and cell number. We have also shown that MIS, in contrast to TGF-ß, does not promote granulosa cell apoptosis. In contrast to activin, MIS is not a differentiation factor for preantral follicles.
To our knowledge, the present study is the first to evaluate direct effects of MIS on intact ovarian follicles. Treatment of preantral follicles with exogenous MIS enhances both basal and FSH-stimulated follicle growth. Mutated MIS that is not bioactive did not have this effect, nor did conditioned media from cells not expressing MIS. The increase in follicle diameter induced by MIS is also accompanied by an increase in follicle cell number. This is in contrast to the MIS inhibition of granulosa cell division that has been reported for granulosa cells from large antral follicles of rats [18] and the suppression of epidermal growth factor (EGF)-induced proliferation of granulosa cells obtained from Graafian follicles during in vitro fertilization procedures in humans [19]. However, in histological analysis, maximal expression of MIS mRNA has been associated with populations of follicle cells that are the most rapidly dividing [20]. It is possible that the effects of MIS vary with the stage of follicle development. Additionally, effects of growth factors on granulosa-luteal cells in monolayer culture may not reflect the effects on intact follicles, in which theca cell-granulosa cell interaction can occur. The presence of MIS receptors on both theca and granulosa cells highlights this possibility. The magnitude of MIS-induced follicle growth is comparable to that of activin, which is a growth factor with recognized ovarian effects [21]. However, TGF-ß, a closely related family member, does not induce preantral ovarian follicle growth.
Although MIS treatment promotes apoptosis of the cells of the Müllerian duct, it does not result in apoptosis of granulosa cells of cultured preantral follicles. Apoptosis can occur in the present system, however, because treatment with TGF-ß promotes widespread granulosa cell apoptosis in cultured preantral follicles. The effects of MIS on the Müllerian duct are thought to be paracrine, because the MIS receptor is not expressed on the epithelial duct cells themselves but, rather, on the mesenchymal cells surrounding the duct [22]. The presence of MIS receptor on both theca and granulosa cells may play a key role in the absence of the apoptotic effect in the follicle. Histological examination of the cultured follicles does suggest that the theca layer is affected by treatment with MIS (Fig. 4). Future studies are necessary to fully characterize the role of MIS in theca cell growth and granulosa cell-theca cell interaction and the mechanisms of MIS action in the ovarian follicle.
Müllerian inhibitory substance does not enhance the differentiation of preantral follicles, as demonstrated by the lack of regulation of inhibin- subunit expression by MIS. Although to our knowledge no reports of MIS effects on intact preantral follicles have appeared, MIS treatment has inhibited the expression of markers for differentiation in most other systems of organ and tissue culture. In cultured fetal rodent ovaries (that do not yet contain follicle structures), MIS suppresses the expression of aromatase [23], whereas in differentiated granulosa cells in monolayer culture, MIS suppresses phosphorylation of the EGF receptor and progesterone production [19]. Treatment with MIS also inhibits branching of fetal lung ducts in culture [24]. Interestingly, in female mice that do not express MIS, inhibin levels are elevated during early adulthood [11].
The phenotypes of MIS knock-outs and transgenic animals overexpressing MIS are interesting yet perplexing in light of our findings in isolated rat follicles. In female transgenic mice that chronically express MIS, a reduced number of follicles are formed and rapidly lost very early during infantile life, leading to the eventual formation of abnormal gonads [25]. To our knowledge, detailed analysis of the growth of these follicles has not been reported, but this phenotype may be secondary to effects of MIS before the follicles are formed. Conversely, in MIS knock-out mice, a normal number of primordial follicles are found early in life [17]. However, the primordial pool of follicles is depleted more rapidly than in wild-type controls. During young adulthood, a larger number of growing and atretic preantral follicles are present compared to control animals, yet litter sizes and ovulation rates seem to be unchanged. The number of follicles during the latest stages of development are not affected by the absence of MIS, although serum levels of FSH are reduced. Thus, the main abnormality in the MIS null mice appears to involve the dynamics of the pool of small, growing follicles. The increased number of follicles seen in these young knock-out mice could result from an increased rate of entry into the growing pool, increased duration of time in the growing pool, or decreased rate of loss from the pool. To our knowledge, no reports of MIS or MIS receptor expression in primordial follicles have appeared, so a direct effect on the initiation of follicle growth is unlikely. In the null mice, the number of atretic follicles are increased by a magnitude similar to the number of healthy preantral follicles. Therefore, it seems to be possible that the follicles spend an increased time in the growing pool or, put another way, grow more slowly in the null mice. The direct growth-promoting action of MIS on isolated follicles supports this possibility. Further studies are needed to clarify the role of MIS in follicle growth and atresia in rats and mice.
The unique properties of MIS in promoting preantral follicle growth while inhibiting follicle differentiation may also have an effect on the interaction of larger follicles as well. When a MIS-affected follicle does ultimately differentiate, it should be larger and contain more granulosa cells than an unaffected follicle. This follicle should, therefore, have a greater capacity for producing estrogen and angiogenic factors, which in turn should provide a competitive advantage compared to the other follicles in its cohort. Thus, it is possible that aspects affecting follicle selection and dominance occur much earlier during follicle development than the antral stage.
Clearly, the development of preantral follicles is a complex and regulated process. Factors regulating early follicle development and survival seem to be different in smaller follicles than in more differentiated antral and preovulatory follicles. Increased understanding about the regulation of this pool of small, growing follicles may have a great impact on the ability to manipulate fertility as well as the timing of menopause.
FOOTNOTES
First decision: 22 February 2000.
1 Supported by National Institutes of Health grant HD31398 and Specialized Cooperative Centers Program in Reproduction Research, Wellcome Trust, and the Royal Society. E.A.M. was supported by National Institute of Child Health and Human Development grant K12-HD-0084908, cofunded by the American Society for Reproductive Medicine through the Reproductive Scientist Development Program. 
2 Correspondence: Elizabeth A. McGee, University of Pittsburgh, Magee Women's Research Institute, 204 Craft, Pittsburgh, PA 15218. FAX: 412 641 5290; rsieam{at}mail.magee.edu
Accepted: August 28, 2000.
Received: January 19, 2000.
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60 µm
