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Endocrinology Vol. 142, No. 10 4357-4362
Copyright © 2001 by The Endocrine Society

ARTICLES

Synergism Between FSH and Activin in the Regulation of Proliferating Cell Nuclear Antigen (PCNA) and Cyclin D2 Expression in Rat Granulosa Cells

Talal El-Hefnawy and Anthony J. Zeleznik

Department of Physiology and Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

Address all correspondence and requests for reprints to: Anthony J. Zeleznik, Ph.D., 830 Scaife Hall, University of Pittsburgh School of Medicine, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261. E-mail: zeleznik+{at}pitt.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Follicular development is associated with both proliferation and differentiation of granulosa cells under the control of FSH. We show that regulation of genes involved in cellular proliferation by FSH can be functionally separated from the regulation of genes involved in granulosa cell differentiation by synergistic actions of activin and T. Incubation of undifferentiated rat granulosa cells with FSH, forskolin, activin-A, or T alone did not influence either the expression of the proliferation-associated genes cyclin D2 and proliferating cell nuclear antigen or the differentiation-associated genes P450 aromatase, LH receptor, P450 cholesterol side-chain cleavage enzyme, and 3ß-hydroxysteroid dehydrogenase. However, when granulosa cells were stimulated with either FSH or forskolin in the presence of activin-A, significant increases (P < 0.05) were observed for cyclin D2 and proliferating cell nuclear antigen at both the mRNA and protein levels as well as mRNAs for P450 aromatase, LH receptor, P450 cholesterol side-chain cleavage enzyme and 3ß-hydroxysteroid dehydrogenase. Although T synergized with FSH to increase the expression of mRNAs for P450 aromatase, LH receptor, P450 cholesterol side-chain cleavage enzyme, and 3ß-hydroxysteroid dehydrogenase, it did not interact with FSH to increase the expression of mRNAs for cyclin D2 and proliferating cell nuclear antigen. The differences in the actions of activin and T could provide a cellular mechanism by which FSH-regulated granulosa cell proliferation could be functionally separated from FSH-regulated granulosa cell differentiation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FOLLICULAR DEVELOPMENT BEYOND the early antral stages is absolutely dependent upon FSH (1). Women with mutation in the gene encoding either the ß subunit of FSH or the FSH receptor are represented with developmental arrest of follicles at the preantral stage causing primary amenorrhea and infertility (2, 3). A similar phenotype is seen in FSHß or FSH receptor knockout mice (4, 5). In response to FSH stimulation, a number of proteins involved in the differentiation of the granulosa cell are induced that enable the cells to produce E (P450 aromatase) (6) to become responsive to the midcycle gonadotropin surge (LH receptor) (7) and commence progesterone production following ovulation and luteinization (3ß-hydroxysteroid dehydrogenase and P450 cholesterol side chain cleavage) (8, 9). In addition to the differentiating effects of FSH, the final stages of follicular development are also associated with heightened proliferation of granulosa cells (10, 11). Although progress has been made in understanding the control of granulosa cell differentiation, the control of granulosa cell proliferation is less well understood. The recent finding that the cyclin D2 knockout mouse is infertile owing to a defect in the proliferation of granulosa cells (12) and that FSH and forskolin stimulate cyclin D2 expression in granulosa cells (13) has provided a direct link among gonadotropins, cAMP, and cell cycle regulation.

In addition to cyclin D2, proliferating cell nuclear antigen (PCNA), a proliferation-associated protein that is required for DNA synthesis (14), also appears to be involved in follicular growth. Expression of PCNA in granulosa cells begins upon the formation of a primary follicle, and its level of expression appears to increase during the gonadotropin-dependent stages of preovulatory follicular development (15). In thyroid epithelial cells, the mitogenic actions of TSH are associated with an induction of PCNA expression in a cAMP-dependent manner (16). Whether FSH and cAMP are similarly involved in PCNA expression in the ovary is not known. We initiated the current studies to investigate the regulation of PCNA and cyclin D2 expression in granulosa cells and to compare their regulation with genes associated with granulosa cell differentiation. Using granulosa cells from immature rat ovaries, which are undifferentiated with respect to the absence of LH receptors as well as their ability to produce E and progesterone, we show that activin and FSH synergistically act to increase PCNA and cyclin-D2 and that these markers of proliferation are regulated differently than those involved in granulosa cell differentiation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Unless otherwise noted, all reagents were purchased from Sigma (St. Louis, MO). Human FSH (AFP-4161-B; 3205 IU 2nd IRP FSH/mg, 225 IU 2nd IRP LH/mg), and antiserum to cAMP (lot CV-27) were generously provided by the National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health. Activin-A and IGF-I were provided by Genentech, Inc. (South San Francisco, CA).

Granulosa cell culture
All procedures were approved by the University of Pittsburgh Institutional Animal Use and Care Committee. Immature (23–25 d old) female rats were purchased from Taconic Farms, Inc. (Germantown, NY). Undifferentiated granulosa cells (i.e. no FSH stimulation) were collected from the ovaries by puncturing follicles with a 25-gauge hypodermic needle and cells were expressed into Medium 199 (M199, Life Technologies, Inc., Gaithersburg, MD) containing 10% FBS. The cells were seeded into 6-well (~106 cells/well) or 12-well (~5 x 105 cells/well) tissue culture plates and allowed to attach overnight. The next morning, medium and unattached cells were removed and replaced with fresh M199 that did not contain serum or other protein supplements. Twenty-four hours later, medium was replaced by fresh M199 without serum containing the appropriate stimulatory agents. Forty-eight hours after addition of hormones, tissue culture medium was collected, boiled for 10 min to inactivate phosphodiesterases, and stored at -20 C for subsequent RIAs. Total RNA was prepared from the cell monolayers using RNAzol B (Tel-Test, Inc., Friendswood, TX) according to the manufacturer’s directions.

mRNA analysis
Samples of total RNA (1–5 µg) were analyzed for mRNAs for cytochrome P450 aromatase, LH receptor (LHr), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), P450 cholesterol side-chain cleavage enzyme (P450SCC), PCNA and cyclin D2 by RNase protection assay according to the instructions provided by the supplier (Ambion, Inc., Austin, TX). Antisense RNA probes were prepared using [32P]UTP from the following cDNA inserts: P450 aromatase (bp 1034–1295) (17); cyclin D2 (bp 1072–1210) (18); cyclophilin, (bp34–142) (19) PCNA (bp 204–456) (20), rat LHr (bp 1–622) (21), P450SCC (bp 18–816) (22), and 3ß-HSD (bp 453–932) (23). Following electrophoresis (5% acrylamide containing 8 M urea), gels were dried and exposed to x-ray film for 16–96 h. Densitometric analysis of protected RNA fragments was performed using NIH Image (version 1.61). Densitometric signals from individual bands were divided by the respective density for cyclophilin to correct for differences in gel loading and/or selectivity of the mRNA changes.

Western immunoblotting
Granulosa cells were scraped into TE buffer (50 mM Tris-HCl, pH7.4, 1.0 mM EDTA) supplemented with 20 µg/ml phenylmethylsulfonylfluoride and 1 µg/ml Leupeptin. Whole-cell lysates (20 µg/lane) were separated on 12% SDS discontinuous polyacrylamide gels, and the resolved proteins were electrophoretically transferred to nitrocellulose membranes. Anti-PCNA immunoblotting was performed using an anti-PCNA monoclonal antibody (no. PC 10; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a final concentration of 1 µg/ml. Anti-cyclin D2 immunoblotting was performed using a polyclonal antibody (no. C-17; Santa Cruz Biotechnology, Inc.) at a concentration of 1 µg/ml. Chemiluminescent detection was accomplished using the BM chemiluminescence Western blotting kit (Roche Molecular Biochemicals, Indianapolis, IN) with the appropriate horseradish peroxidase conjugated secondary antibodies iluted to 1:1000 according to the manufacturer’s directions.

RIA
The cAMP concentrations in culture medium were analyzed by RIA using 125I- cAMP-TME (2-0' monosuccinlyl cAMP tyrosine methyl ester) and anti-cAMP in accordance to the instructions provided by the National Hormone and Pituitary Program.

Statistics
Results were assessed for statistical significance by ANOVA followed by comparison of group means with Fishers least significant difference analysis (StatView v4.5, Abacus Concepts, Berkeley, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Interaction between FSH and activin-A on PCNA and cyclin D2 expression in undifferentiated granulosa cells
The effect of FSH in the absence or presence of activin-A (100 ng/ml) on PCNA and cyclin D2 expression in undifferentiated granulosa cells was assessed by Western immunoblotting. As shown in Fig. 1AGo, PCNA expression was not increased following 48 h of treatment with either FSH (1–100 ng/ml) or activin (100 ng/ml) alone. However, when granulosa cells were costimulated with FSH and activin, levels of PCNA were significantly increased (P < 0.05) at the 100 ng/ml concentration of FSH. As shown in Fig. 1BGo, forskolin (FSK) (10 µM) alone did not influence PCNA expression and cotreatment of undifferentiated granulosa cells with 10 µM FSK and 100 ng/ml activin significantly elevated (P < 0.05) immunoreactive PCNA levels. The regulation of cyclin D2 expression by FSH and activin was qualitatively similar to that of PCNA (Fig. 2Go).



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  		Figure 1. PCNA expression in cultured rat granulosa cells following 48-h incubation with FSH and FSK in the absence or presence of activin-A. Granulosa cells were treated for 48 h in the presence or absence of activin-A (100 ng/ml) with or without 1, 10, or 100 ng/ml FSH (A) or 10 µM FSK (B). Fifty micrograms of total cell protein from each group were resolved on SDS-PAGE and transferred onto nitrocellulose membranes ands analyzed for PCNA by immunoblotting as described in Materials and Methods. Densitometric quantification of the PCNA signal (fold increase relative to the absence of FSH and activin) is presented in the bar panels. Each bar represents the mean ± SEM from four to six separate groups of granulosa cells. *, Statistical significance (P < 0.05) vs. FSH alone.

 


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  		Figure 2. Cyclin D2 expression in cultured rat granulosa cells following 48-h incubation with FSH and activin-A. Granulosa cells were treated for 48 h in the presence or absence of activin-A (100 ng/ml) with or without 1, 10, or 100 ng/ml FSH. Fifty micrograms of total cell protein from each group were resolved on SDS-PAGE and transferred onto nitrocellulose membranes and analyzed for cyclin D2 by immunoblotting as described in Materials and Methods. Densitometric quantification of the cyclin D2 signal (fold increase relative to the absence of FSH and activin) is presented in the bar panels. Each bar represents the mean ± SEM from six separate groups of granulosa cells. *, Statistical significance (P < 0.05) vs. FSH alone.

 
Specificity of the action of activin upon FSH-induced PCNA and cyclin D2 mRNA expression
Using RNase protection assays, we examined the interactions of FSH and activin on mRNAs for PCNA and cyclin D2. In addition, we investigated the regulation of LHr, P450scc, 3ß-HSD, and P450 aromatase mRNAs, which were used as markers of granulosa cell differentiation. As shown in Fig. 3Go, similar to results seen at the protein level, neither FSH nor activin alone was effective in stimulating mRNAs for cyclin D2 or PCNA. Likewise, mRNA for cytochrome P450 aromatase, 3ß-HSD, P450scc, and LHr were not induced by either FSH or activin alone. Following 48-h incubation with FSH (100 ng/ml) and activin (100 ng/ml), significant increases (P <= 0.01) in mRNAs for cyclin D2 and PCNA were observed. Likewise, robust inductions (P < 0.01) were also observed in mRNA for LHr as well as the steroidogenic enzymes P450 aromatase, P450scc, and 3ß-HSD in granulosa cells following 48-h combined FSH and activin treatment (Fig. 3Go).



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  		Figure 3. Ribonuclease protection assay for cyclin-D2, PCNA, LHr, P450scc, 3ß-HSD, aromatase, and cyclophilin mRNA in granulosa cells following 48-h culture with or without FSH (100 ng/ml) in the presence or absence of activin-A (100 ng/ml), IGF-1 (50 ng/ml), T (10 ng/ml), E2 (100 ng/ml), or FBS (10%). Total RNA was isolated from cultured granulosa cells following 48-h incubation with the designated treatments. The + represents the inclusion of 100 ng/ml hFSH. Specific antisense cRNAs were used to hybridize to the corresponding mRNAs, and the protected fragments were resolved on urea-polyacrylamide gels, dried, and exposed to x-ray film. Data are representative of experiments from three separate groups of granulosa cells.

 
The requirement for activin on FSH stimulation of mRNAs for cyclin D2, PCNA, P450 aromatase, P450scc, and LHr could not be substituted by 48-h treatment with IGF-1 (50 ng/ml), E (100 ng/ml), or 10% serum (Fig. 3Go). When FSH was combined with T, an induction in LHr mRNA as well as the steroidogenic enzyme mRNAs was observed. However, T did not exert a synergistic action on FSH stimulation of mRNAs for either cyclin D2 or PCNA (Fig. 3Go). IGF-1 in the presence of FSH caused only a weak induction of 3ß-HSD (Fig. 3Go) but appeared to amplify the stimulatory effects of FSH plus T on mRNAs for LHr, P450 aromatase, 3ß-HSD, and P450scc but not cyclin D2 or PCNA (Fig. 4Go).



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  		Figure 4. Ribonuclease protection assay for cyclin-D2, PCNA, LHr, P450scc, 3ß-HSD, aromatase, and cyclophilin mRNA in granulosa cells following 48-h culture with FSH (100 ng/ml) and T (10 ng/ml) in the presence or absence of IGF-I (50 ng/ml). Total RNA was isolated from cultured granulosa cells following 48-h incubation with the designated treatments. Specific antisense cRNAs were used to hybridize to the corresponding mRNAs, and the protected fragments were resolved on urea-polyacrylamide gels, dried, and exposed to x-ray film. Data are representative of experiments from three separate groups of granulosa cells.

 
The failure of T to augment FSH-stimulation of mRNAs for cyclin D2 and PCNA did not reflect an underlying inhibitory effect of T as addition of T did not interfere with the ability of FSH plus activin to augment these mRNAs (Fig. 5Go). Also shown in Fig. 5Go, similar to its effects on PCNA and cyclin D2 protein expression, FSK in the absence of activin did not stimulate mRNAs for PCNA and cyclin D2 and inclusion of activin in the culture permitted FSK to increase mRNAs for PCNA, cyclin D2, and P450 aromatase. The inclusion of phosphodiesterase inhibitor (IBMX) did not interact with FSH to induce mRNAs for cyclin D2 or PCNA (Fig. 5Go).



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  		Figure 5. Ribonuclease protection assay for aromatase, CD2, PCNA, and cyclophilin mRNA in granulosa cells following 48-h culture in the presence of hormonal/cAMP-induction treatment. Total RNA was isolated from cultured granulosa cells following 48-h incubation with the designated treatments. The + represents the inclusion of activin-A or hFSH at 100 ng/ml. Specific antisense cRNAs were used to hybridize to the corresponding mRNAs, and the protected fragments were resolved on urea-polyacrylamide gels, dried, and exposed to x-ray film. The figure is representative of experiments from three separate groups of granulosa cells.

 
Effect of activin and T on FSH and FSK-stimulated cAMP production
Figure 6Go illustrates cAMP concentrations in culture media following 48-h stimulation of undifferentiated granulosa cells. FSH, but not activin, increased cAMP production above control levels (P < 0.05 for FSH vs. control). In the presence of either activin or T, FSH-induced cAMP accumulation was further increased, compared with that seen in the presence of FSH alone (P < 0.05). As expected, the IBMX (100 µM) also increased FSH-induced cAMP accumulation. Likewise, FSK alone stimulated cAMP production to a comparable extent as FSH plus activin, yet FSK or IBMX-FSH treatment alone did not stimulate mRNAs for cyclin D2, PCNA, or aromatase unless activin was also present (Fig. 5Go).



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  		Figure 6. FSH and FSK-induced cAMP accumulation from granulosa cells following 48-h incubation in the presence or absence of activin (100 ng/ml), T (10 ng/ml), or IBMX 0.1 mM. The media from 48-h treatment of granulosa cells following the indicated treatments were collected and analyzed for cAMP concentrations by RIA. FSH was included at a concentration of 100 ng/ml and FSK at a concentration of 10 µM. Each bar represents the mean ± SEM from three separate groups of granulosa cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous reports by others have shown that activin and FSH interact to stimulate DNA synthesis in cultured rat granulosa cells (24) and increase the growth of intact follicles in vitro (25). Our findings demonstrate that activin and FSH interact dramatically in undifferentiated granulosa cells to increase mRNA and protein content for cyclin D2 and PCNA, both of which play major roles in cell cycle progression. The D-cyclins initiate proliferation predominantly during the early G1 phase of the cell cycle and are thought to play an essential role in the initiation of cellular proliferation in response to extracellular signaling (26, 27). Indeed, cyclin D2 is obligatory for normal ovarian functions because female mice with a disrupted cyclin D2 gene are infertile owing to a defect of FSH-stimulated proliferation of granulosa cells (12). Further, Robker and Richards (13) demonstrated that FSH induces cyclin D2 expression in ovaries of immature rats as well as in cultured granulosa cells. We extend these observations by demonstrating that FSH-stimulated cyclin D2 expression is dramatically increased by activin.

PCNA is a nuclear protein crucial to eukaryotic cell cycle, DNA replication, and DNA repair and is commonly used as a histological marker for hyperplastic cells. In addition to its function as a processivity factor for DNA polymerase {delta}, PCNA may also influence the G1 and S phases of the cell cycle by its ability to form complexes with cyclin D, cyclin-dependent kinases as well as the cyclin-dependent kinase inhibitor p21 (28). With respect to ovarian function, the expression of PCNA in granulosa cells correlates with follicular growth; moreover, it diminishes in atretic follicles and its expression is reduced or eliminated in the nonproliferating corpus luteum (15, 29). To the best of our knowledge, this is the first report that PCNA expression, at both the mRNA and protein levels, is regulated by FSH and activin in undifferentiated granulosa cells. Moreover, activin also synergizes with FSK to induce PCNA expression suggesting that this stimulation, at least in part, is cAMP dependent. In this regard, the PCNA promoter of both mice and humans contains a cAMP response element that is essential for stimulus-induced transcription (30, 31).

Analysis of cAMP production revealed that although activin alone did not increase cAMP levels, it surprisingly amplified cAMP production in response to FSH. One possible explanation for this effect is that activin has been shown to increase FSH receptor mRNA in undifferentiated granulosa cells (32). However the finding that activin also amplified forskolin-stimulated cAMP production indicates that there must also be actions of activin distal to the FSH receptor. Whether activin regulates the activity of G-proteins and/or phosphodiesterases remains a question for future investigation. The question that does arise is whether the synergistic effects of activin on FSH-stimulated PCNA and cyclin D2 are due solely to amplified production of cAMP. This does not appear to be the case as, in agreement with a previous report (33), we found that T augmented the cAMP response to FSH to an extent similar to that of activin (Fig. 5Go). However, as noted above, although T interacted with FSH to increase mRNA for P450 aromatase and other differentiation-specific mRNAs, it did not do so for PCNA or cyclin D2. In addition, although 10 µM FSK alone stimulated cAMP production to an extent similar to that of FSH plus activin, FSK had no effect on either PCNA or cyclin D2 mRNAs unless activin was also included in the incubation medium. The effects of activin appears to be specific as other potential local modulators of FSH action such as E2, T, and IGF-I did not substitute for activin with respect to the expression of either cyclin D2 or PCNA mRNAs. These observations are consistent with unpublished findings that T, E2, and IGF-I can not synergize with FSH to stimulate DNA synthesis in undifferentiated granulosa cells (S.G. Hillier, unpublished observation).

The molecular mechanism that underlies activin’s synergism of FSH-mediated gene expression is not known. One possibility is that the FSH and activin signaling pathways converge at the promoters of individual genes to regulate their expression. In this regard, Zang and Derynck (34) have shown recently that the TGF-ß, through the Smad signaling pathway, requires the presence of a functional cAMP response element to activate the transcription of the Ig {alpha} constant region gene. The results of our present study indicate that a number of mRNAs (PCNA, cyclin D2, P450 aromatase, P450SCC, LHr, and 3ß-HSD) are synergistically elevated by FSH and activin. If the functional synergy between FSH and activin lies at the promoters of individual genes, it would be expected that each of the many genes that exhibit this synergism would have similar regulatory sequences. Indeed, the Smad DNA binding element 5'-AGAG-3' (35) is present in the promoter regions of PCNA, cyclin D2, P450 aromatase, and the LHr (GenBank analysis). Alternatively, the Smads may also play a more global role in gene expression by modulating the activity of transcriptional corepressors (35), which could explain the large number of mRNAs increased by FSH and activin in the current study. However, mRNA for cyclophilin was not induced by FSH and activin (Figs. 3Go and 4Go) indicating that a ubiquitous effect of activin upon transcription and/or mRNA stability does not appear to account for our findings. In addition, we did not observe any stimulatory effects of FSH and activin, alone or in combination, on a Rous sarcoma virus promoter-driven ß-galactosidase reporter gene transiently expressed in rat granulosa cells with an adenovirus vector (data not shown).

The low levels of mRNA for PCNA and the absence of mRNAs for LHr and P450 aromatase in unstimulated granulosa cells (Figs. 3Go and 4Go) are consistent with the low rate of proliferation in the absence of cellular differentiation that is characteristic of preantral follicular growth. During gonadotropin-independent preantral folliculogenesis, other mechanisms appear to regulate granulosa cell proliferation in the absence of concomitant cellular differentiation. An attractive candidate is growth differentiation factor-9 (GDF-9) because GDF-9 stimulates thymidine incorporation but inhibits FSH-stimulated differentiation in granulosa cells harvested from preantral follicles (36). In addition, GDF-9 deficient mice manifest an arrest of preantral follicular growth, a reduction in PCNA staining in granulosa cells and premature expression of LHr, P450scc, and P450 aromatase (37). In contrast to preantral follicular development, FSH-dependent preovulatory follicular development is associated with accelerated proliferation as well as the differentiation of granulosa cells. Our findings that activin and FSH synergize to regulate PCNA and cyclin D2 expression as well as markers of granulosa cell differentiation is consistent with both a shortening of the G1 phase of the cell cycle as well as the differentiation that occurs during the final stages of preovulatory follicle development. Our results are consistent with previous findings that both T and activin interact with FSH to regulate the expression of P450 aromatase and other mRNAs associated with granulosa cell differentiation that accompanies preovulatory follicular development (38, 39). Our preliminary observation that IGF-I did not markedly enhance FSH-mediated expression of differentiation-associated mRNAs but amplified the effects of FSH plus T (Fig. 4Go) is consistent with the notion that IGF-I may regulate mRNA stability rather than transcription in granulosa and Leydig tumor cells (40, 41).

The physiological significance of our observations is underscored by the developmental link among the expression of activin in the ovary, the follicular requirement for FSH, and the accelerated proliferation and differentiation of granulosa cells that occurs during the later stages of antral follicular development. On the basis of in situ hybridization studies, large preantral and early antral follicles express the ß activin/inhibin subunit but not the {alpha} subunit (42). Presumably these follicles would be under the influence of activin B because they would lack the ability to produce inhibin (which requires the {alpha} subunit). Further, a recent preliminary study by McGee et al. (43) demonstrated that the expression of Smad2 and Smad3, which are downstream targets of the activin receptor (44), is highest in granulosa cells of preantral follicles and declines in the granulosa cells of preovulatory follicles and corpora lutea. The actions of T to augment FSH-dependent differentiation but not proliferation could provide a mechanism by which the differentiated state of granulosa cells is maintained in the absence of proliferation (i.e. after luteinization).

In summary, our results indicate that mRNAs involved in granulosa cell proliferation (cyclin D2 and PCNA) are regulated differently than those involved in granulosa cell differentiation owing to differences between activin and T in their synergy with FSH. Thus, development-dependent changes in the production of activin (42) by the follicle could provide a cellular mechanism by which gonadotropic regulation of granulosa cell proliferation could be functionally separated from gonadotropic regulation of granulosa cell differentiation.


   Acknowledgments
 
We acknowledge Dr. D. Segaloff, Dr. I. Mason, Dr. S. G. Hillier, and Dr. C. J. Sherr for providing cDNAs for this study and Dr. Zourab Bebia and Ms. Lynda Little-Ihrig for technical assistance.


   Footnotes
 
This work was supported by NIH-HD-16842 (to A.J.Z.).

Abbreviations: FSK, Forskolin; 3ß-HSD, 3ß-hydroxysteroid dehydrogenase; GDF-9, growth differentiation factor-9; IBMX, 3-isobutyl-1-methylxanthine; LHr, LH receptor; PCNA, proliferating cell nuclear antigen; P450SCC, P450 cholesterol side-chain cleavage enzyme.

Received March 30, 2001.

Accepted for publication June 22, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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