Cellular and Molecular Mechanisms That Mediate Insulin-Dependent ...

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undergo insulin- or phorbol ester-dependent mitosis in vitro. ... c-fos and c-jun, are expressed during GC mitosis, studies were undertaken to determine whether ...
BIOLOGY OF REPRODUCTION 52, 124130 (1995)

Cellular and Molecular Mechanisms That Mediate Insulin-Dependent Rat Granulosa Cell Mitosis' JJ. PELUSO,~>* A.M. LUCIANO,~,*A. PAPPALARDO, and B.A.

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Departments of Obstetrics and ~ynecology*and ~ n a t o m yUniversity ,~ of Connecticut Health Center Farmington, Connecticut 06030 ABSTRACT Rat ovarian follicles are composed of small and large granulosa cells (GC). The present studies demonstrate that small GCs undergo insulin- or phorbol ester-dependent mitosis in vitro. In order to examine the cellular and molecular events that account for insulin's mitogenic action, small GCs were cultured with either insulin, phorbol ester (TPA), or both insulin and TPA. Insulin and TPA increased GC numbers by 21 3% and 20 f 2% over control values, respectively ( p < 0.05). Simultaneous addition of insulin and TPA increased GC numbers by 20 f 3% ( p < 0.05). In a second experiment, small GCs were exposed to control medium, insulin, staurosporine (a protein kinase C [PKC] inhibitor), or both insulin and staurosporine. These studies revealed that insulin induced a 21 f 5% increase in GC numbers and that staurosporine blocked insulin's mitogenic action. These observations suggest that insulin mediates its mitogenic action through a PKC-dependent mechanism. Since the proto-oncogenes, c-fos and c-jun, are expressed during GC mitosis, studies were undertaken to determine whether or not the expression of these two proto-oncogenes products was enhanced by insulin. The expression of c-jos and c-jun proteins was assessed by immunocytochemistry. These studies showed that after 5 h, insulin increased the percentage of cells that stained for c-fos and c-jun by 15 2% and 19 f 4, respectively ( p < 0.05). The expression of these proto-oncogenes was blocked by staurosporine. Both progesterone and 8-br-CAMP,which block insulin-dependent GC mitosis, also inhibited the expression of c-jos and c-jun. Finally, small GCs were cultured with insulin in the presence of sense and antisense oligonucleotides specific for c-fos and c-jun. Under these conditions, insulin induced a 32 f 10% and 24 2 5% increase in cell numbers in the presence of c-fos and c j u n sense oligonucleotides, respectively. Insulin-dependent GC proliferation was completely prevented in the presence of either c-fos or c-jun antisense oligonucleotide. Taken together, these experiments support the concept that insulin stimulates small GC mitosis by activating PKC, which induces the expression of both c-fos and c-jun. Both of these genes play essential roles in GC mitosis.

*

*

INTRODUCTION

The pituitary gonadotropins, FSH and LH, control ovarian follicular growth and steroidogenesis through very complex and ill-defined mechanisms. This is illustrated by the observation that rat granulosa cells (GC) temporarily lose their ability to synthesize steroids in response to gonadotropin stimulation. This transient loss of gonadotropin responsiveness coincides with an increase in GC mitosis [I]. Further, gonadotropins do not stimulate rat GC proliferation in vitro [ 2 ] . In fact, FSH suppresses GC DNA synthesis, while the combined treatment of FSH and insulinlike growth factor-l (IGF-I) restores the rate of DNA synthesis to control values [3]. These observations suggest that gonadotropins may not mediate their mitogenic action directly but rather stimulate the production of various intraovarian growth factors that in turn induce GC mitosis [4,51. Several gonadotropin and/or growth factor treatments have been shown to promote GC DNA synthesis in vitro. However, whether a specific growth factor functions as a GC mitogen is very species-dependent [4-61. With regard to rat GCs, Anderson and Lee [7] have shown that GC proAccepted September 5, 1994. Received May 31, 1994. 'This work was supported in part by NIH Grant I-R55-HD27578-01A2. 'correspondence. FAX: (203) 679-1436. 3Current address: lnstituto Sperimentale Italiano, Lazzaro Spallanzani, Universita degli Studi di Milano, Via Capolago, 2, 20133 Milano, Italy.

liferation can be induced in medium supplemented with FSH, platelet-derived growth factor, transforming growth factor p (TGFP), transforming growth factor a , and growth hormone. The importance of FSH and TGFP has also been demonstrated by Dorrington and associates [8]. Dorrington's studies have shown that the combined action of FSH and TGFP is required to stimulate 3~-thymidineincorporation within rat GCs. The synergistic effect of FSH on GC DNA synthesis can be replicated by epidermal growth factor (EGF) [9] or steroidogenesis-inducing protein [lo]. Insulin and IGF-1 also promote rat GC DNA synthesis and mitosis [ l l ,121. Although several gonadotropin/growth factor combinations stimulate rat GC proliferation,'the cellular mechanisms through which these factors mediate their mitogenic action have not been elucidated. Recent studies have shown that the phorbol ester 12-0tetradradecanoyl phorbol 13-acetate (TPA) stimulates rat GC proliferation in vitro [13].TPA is thought to directly activate PKC [14], thereby implicating PKC activation as part of a mitogenic pathway of rat GCs. In rat GCs, TPA promotes the expression of the proto-oncogenes, c-fos [15,16]and cj u n [16]. Since these proto-oncogenes are induced during rat GC mitosis [17],it has been proposed that GC mitogens activate PKC, which in turn promote the expression of c-fos and c-jun products. These proto-oncogene proteins would then dimerize to form AP-I transcription complexes and subsequently activate a gene cascade that ultimately results in mitosis [MI.

MECHANISM OF INSULIN-DEPENDENT GRANULOSA CELL MITOSIS The first series of experiments described herein was designed to determine whether or not insulin mediates its mitogenic action via a PKC-dependent pathway that subsequently promotes the expression of c-fos and c-jun. Further experiments were conducted to determine whether regulatory agents that prevent GC mitosis affect c-fos and c-jun expression. Finally, GCs were exposed to sense and antisense oligonucleotides to c-fos and cjun, respectively, in order to determine whether either of these proto-oncogene products was essential for insulin-dependent GC mitosis. MATERIALS AND METHODS Animals Immature female Wistar rats (22 days of age) were obtained from Charles River Breeding Labs. (Wilmington, MA) and housed under controlled conditions of temperature, humidity, and photoperiod (12L:12D; lights-on at 0700 h). Rats were anesthetized with CO 2 and then cervically dislocated between 0930 and 1000 h when they were between 25 and 29 days of age. This protocol was approved by the Animal Care Committee of the University of Connecticut Health Center. Preparationof Culture Medium RPMI 1640 without phenol red (Gibco Labs., Grand Island, NY) was used in all culture experiments. It was supplemented with penicillin (0.14 g/L), streptomycin (0.27 g/L), HEPES (4.76 g/L), BSA fraction V (2 g/L), sodium selenite (5 ng/ml), transferrin (5 V.g/ml), and sodium bicarbonate (2.2 g/L). The pH was adjusted to 7.4 and the medium was filtered through a 0.2-Rm filter. Depending on the experimental design, insulin (1 M), TPA (2 nM), staurosporine (0.7 nM; Kamiya Biomedical, Thousand Oaks, CA), 8-br-cAMP (0.2 mM), and progesterone (640 nM) were added to the cultures. All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless indicated otherwise. Progesterone was dissolved in ethanol and then diluted in RPMI 1640 to the desired final concentration. GC Isolation and Culture GCs were isolated according to the procedure of Rao et al. [19] with slight modifications. Briefly, ovaries were placed in Medium 199 containing 0.2% BSA, 9.1 mM EGTA at pH 7.4. The follicles within the ovary were punctured with 20gauge needles and then incubated for 5 min at 37°C in 5% CO2:95% air. The ovaries were transferred to Medium 199 containing 0.2% BSA, 2.1 mM EGTA, and 0.5 M sucrose at pH 7.4 and then incubated at 37 0C in 5% C0 2:95% air for 10 min. The ovaries were washed, resuspended in fresh Medium 199 containing 0.2% BSA, and then pressed to release the GCs. The cells were loaded onto the top of a 1545% Percoll gradient (8 ml total volume). The gradient was

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centrifuged at 200 x g for 5 min and, starting from the top, 12 fractions of 666 Il1 were collected. The cells in each fraction were washed and resuspended in RPMI 1640. Small GCs were typically collected in fractions 3-4, while larger GCs were isolated from fractions 5-7. Cells were plated in 35-mm dishes or on 8-chamber Lab-tek slides (Nunc Inc., Naperville, IL) and cultured for up to 24 h in a 5% CO 2 / air atmosphere. GC ProliferationAssessed by In Situ Cell Counting To assess the capacity of GCs to undergo mitosis, 2 to 3 x 105 GCs from the different fractions were added to 35mm dishes in 1 ml of RPMI 1640 with or without insulin as indicated. After 2 h, any unattached GCs were removed and the medium was replaced. An aliquot of these unattached GCs was stained with trypan blue, and cell number and viability were assessed. The remaining unattached GCs were replated and cultured for an additional 22 h in the presence of insulin. After culture, cells were harvested by gently scraping the dish with a diSPO cell scraper (Scientific Products, McGraw Park, IL), and cell number and viability were reassessed. To determine the proliferative capacity of those GCs that attached to the culture dish within the first 2 h of culture, the number of GCs, observed within five different large (160ptm 2) grids within the original 35-mm dish, was recorded. Each of these large grids was divided into 100 smaller grids, allowing for the precise location of each GC. The large grids were located at the ends of the horizontal and vertical axes and at the center of the dish. At this plating density, 20-30 cells per large grid were generally observed. After 24 h of culture, the GCs in each large grid were recounted. Cell proliferation was expressed as the percentage increase in cell number over the 2-h control value. For each experiment, 100-150 GCs were counted. Each of these experiments was replicated two to three times. The results obtained by counting cells in situ were similar to those obtained by harvesting the cells and counting them in a hemacytometer [13]. For the present experiments the method of counting cells was chosen to eliminate cell harvesting. Immunocytochemical Detection of Proto-Oncogene Proteins The localization of the c-jun and c-fos proteins was carried out as described previously [17]. All antibodies and reagents for the immunocytochemical detection of c-fos and c-jun proteins were supplied by Oncogene Science (Uniondale, NY). The c-fos antibody was a rabbit affinitypurified polyclonal antibody that was raised against the peptide SGFNADYEASSSRC, which corresponds to residues 4 to 17 of human c-fos. The c-jun antibody was a rabbit affinity-purified polyclonal antibody raised against the peptide TPTPTQFLCPKNVTD, amino acids 73 to 87, in the N-terminal region of the c-jun.

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Fraction Number FIG. 1. The effect of insulin and TPA on the proliferation of GCs isolated from Percoll gradient fractions 3 through 7. GCs were collected from immature rat ovaries and separated by Percoll gradient centrifugation (for details see Materials and Methods). The cells were then plated in 35-mm culture dishes in the presence of either insulin or TPA. After 2 h, the medium was removed and the number of cells present within five large grids was counted. Twenty-four hours later, the number of cells within these five grids was again counted and the percentage change in cell number calculated. This experiment was repeated three times. In this and subsequent graphs, GC mitosis is expressed as a mean percent change + SE.

Small GCs were plated in Lab-tek chamber slides and exposed to various regulatory factors (see Results and figure legends for experimental details). After 5 h of culture, GCs were fixed in 10% phosphate buffered formalin for 5 min at room temperature, washed, incubated with 1% Triton X for 5 min, and stained for c-fos and c-jun, respectively, according to instructions provided with Oncogene Science's immunohistochemistry system (Uniondale, NY). This detection system is an avidin-biotin peroxidase-based system that uses diaminobenzidine tetrahydrochloride as a substrate. Cells that expressed c-fos and c-jun were detected by the presence of a reddish-brown precipitate. To assess the specificity and number of false positives, the primary antibody was replaced with normal preimmune rabbit serum and GCs stained by means of the avidin-biotin peroxidase-based system. In the absence of the primary antibodies, 3 ± 1% (n = 36) of the GCs stained. For each treatment, c-fos and c-jun expression was presented as the net percentage change from control treatment (i.e., % stained in a given treatment minus % stained in the control). These experiments were replicated four to six times on separate days, and mean ± SE for each treatment was calculated. Effect of Antisense Oligonucleotides on GC Mitosis Oligonucleotides specific to the 5' end of c-fos and c-jun were synthesized in both the sense and antisense orientation as outlined by McDonnell et al. [20]. The following oligonucleotides were used: c-fos Sense 5'-ATG ATG TTC TCG GGC TTG-3'

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c-fos Antisense 5'-GAA GCC CGA GAA CAT CAT-3' c-jun Sense 5'-ATG ACT GCA AAG ATG GAA-3' c-jun Antisense 5'-TC CAT CTT TGC AGT CAT-3' The oligonucleotides were synthesized and reverse-phase cartridge-purified by Integrated DNA Technologies, Inc. (Coralville, IA). In these experiments, small GCs were isolated, plated in 35-mm dishes, and exposed to insulin for 2 h. The medium was removed and replaced with medium that was supplemented with insulin and either a sense or an antisense oligonucleotide. Both sense and antisense oligonucleotides were used at a concentration of 40 ptg/ml. The number of 2 GCs observed within five different large (160-[Lm ) grids within the 35-mm dish was recorded after 2 and 24 h of culture. These experiments were replicated three times and the data were pooled and analyzed by a Student's t-test. StatisticalAnalysis When appropriate and unless otherwise stated, the data were analyzed by either a two-way or one-way ANOVA followed by Student-Newman-Keuls multiple range test. Regardless of the statistical test, only p values - 0.05 were considered to be significant. RESULTS Regardless of cell size or fraction number, about 20% (i.e., 0.5-0.6 x 105 cells/ml) of the GCs failed to attach within the first 2 h of culture. The viability of these nonattached GCs was poor. Less than 5% of the small nonattached GCs collected from fractions 3-4 were viable. Since

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virtually all of these cells were dead, their ability to undergo insulin-dependent mitosis was not studied. In contrast, 42 + 1% of the large nonattached GCs within fractions 5-7 were viable. Insulin did not produce an increase in the number of nonattached large GCs but rather a decline in cell viability to 19 4% (n = 8;p < 0.05). GCs that were isolated in fractions 3 to 7 after Percoll gradient centrifugation and attached to the culture dish within 2 h were exposed to either insulin or TPA in order to assess their mitogenic capacity. Both insulin and TPA increased the number of GCs present within fraction 3 by 30-40%.

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This percentage gradually decreased with increasing fraction number, with less than 5% of the GCs within fraction 7 dividing (Fig. 1). On the basis of these observations, only GCs collected from fractions 3 and 4 were used in subsequent studies. Since these GCs were smaller (- 50 pLm 2 in area; 8 ILm in diameter) than those collected in fractions 5 to 7 (- 75 pum2; 11 }xm in diameter), these GCs were referred to as small GCs. These findings were similar to those obtained from eCG-primed immature rats [19, 21]. Both insulin and TPA increased small GC mitosis by 20%. The combined treatment of insulin and TPA did not increase the percentage of GCs undergoing mitosis compared to treatment with either mitogen alone (Fig. 2). Further, staurosporine blocked insulin-dependent GC proliferation (Fig. 3). Since mRNA and protein levels of c-fos and c-jun are increased during gonadotropin-induced GC proliferation [17], studies were undertaken to determine whether or not insulin promoted the expression of these proto-oncogene products in vitro. A pilot study indicated that maximum expression of c-fos and c-jun proteins occurred 5 h after insulin exposure. Both proto-oncogene products were localized within the nucleus of many small GCs after insulin treatment (compare Fig. 4, A or B, with Fig. 4C, the negative control). Insulin induced a 15-20% increase in the number of small GCs that express c-fos and c-jun products. This induction was prevented by staurosporine (Fig. 5). Similarly, two regulatory factors that block insulin/TPA-dependent GC mitosis (i.e., 8-br-cAMP [13] and progesterone [unpublished observation]), also prevented insulin from stimulating c-fos and c-jun protein expression (Fig. 6). To determine whether c-fos and c-jun were necessary in order for insulin to induce mitosis, small GCs were treated

FIG. 4. Immunocytochemical detection of c-fos and c-jun expression within small GCs exposed for 5 h to insulin. The presence of these proto-oncogenes was revealed by a reddish-brown precipitate. Note the intense staining within the nuclei of cells stained for c-fos (A) and c-jun (B). When the primary antibody is replaced with normal rabbit serum (i.e., negative control), the reddish-brown precipitate is rarely observed (- 3% of the GCs) (C). All photographs are shown at x229.

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with insulin. After a 2-h incubation, culture medium was supplemented with either a sense (control) or antisense oligonucleotide to c-fos or c-jun, respectively. After 24 h of culture in the presence of either c-fos or cjun sense oligonucleotide, insulin stimulated a 20-30% increase in small GC mitosis. In contrast, exposure to either antisense oligonucleotide attenuated insulin's mitogenic capability (Fig. 7). DISCUSSION The present study demonstrates that 30-40% of GCs isolated in fraction 3 undergo mitosis in response to insulin or TPA. While this increase in GC numbers may appear unremarkable, several factors must be considered. First, unlike transformed cell lines, GCs have a limited mitogenic capacity [22]. Second, fraction 3 is composed of approximately 70% small GCs. Since only small GCs undergo insulin-induced mitosis, the maximum mitogenic response (MMR) is reduced to 70%. Third, the cell viability of these Percoll fractions is - 60%. Finally, about 70% of freshly isolated GCs are in the Go/G 1 phase of the cycle [23]. This further reduces the MMR, since insulin and IGF-1 only act on G0/G, cells in facilitating their transition into the S-phase of the cell cycle [24]. Given these considerations, the MMR can be estimated using the following equation: MMR (% increase) = 100% of initial value x % viable cells x % small cells x % cells in Go/Gj. Thus, the MMR is estimated

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to be about 29%, which indicates that both insulin and TPA induce a near MMR for GCs within fraction 3. The mitogenic response to insulin and TPA diminishes for GCs collected in the higher fractions. Failure to observe an MMR in the higher fractions could indicate that the large GCs that predominate in the higher fractions are not capable of undergoing mitosis. This putative loss of "mitotic competence" correlates with the fact that these larger GCs are more differentiated as indicated by their increased ability to secrete estradiol-173 and progesterone (P4) [19, 21]. However, it could be argued that there are two populations of viable large GCs: those that attach and those that do not attach to the culture dish within 2 h. The data generated through use of the in situ cell counting procedure clearly demonstrate that large GCs that attach do not undergo mitosis in response to insulin. Similarly, insulin does not promote mitosis in unattached large GCs. Collectively, these studies support our conclusion that large GCs do not undergo insulin-dependent mitosis. It is possible that the mitogenic stimuli could change with increasing GC size, since these fractions contain many GCs that are in the S- and G2/M-phases of the cell cycle [25]. For example, large GCs may proliferate in response to other growth factors, such as FSH/TGF3 [8] or EGF/TGF3 [9], since these treatments stimulate GC DNA synthesis. If non-insulin-like growth factors can stimulate mitogenic pathways in large GCs, their mechanism most likely would not include the activation of PKC, since the mitogenic response to TPA also decreases for GCs isolated from fractions 5 and greater.

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The present study is the first attempt to elucidate the cellular mechanism involved in insulin-dependent GC proliferation. These initial studies suggest that insulin mediates its action through a PKC-dependent pathway. This concept is supported by the observation that TPA does not have an additive effect on the number of GCs that undergo insulindependent mitosis. Thus, both mitogens stimulate the same population of "mitotically competent" GCs. Further, staurosporine blocks insulin-dependent proliferation when used at a concentration that specifically inhibits PKC activity in several different cell types. Insulin also stimulates a 15-20% increase in the percentage of GCs that express c-fos and cjun. This increase approximates the percentage of GCs that undergo insulindependent mitosis. It is interesting that staurosporine not only blocks insulin-dependent GC mitosis but also attenuates insulin's ability to induce c-fos and c-jun protein expression. These findings suggest that PKC activation induces functional AP-1 transcription complexes (i.e.,fos/jun heterodimers andjun/jun homodimers) within those GCs that eventually undergo mitosis. The antisense studies support this concept by demonstrating that both c-fos and cjun proteins are essential to insulin's mitogenic action. The importance of c-fos and cjun is further emphasized by the finding that pathways that negatively regulate GC mitosis do so in part by suppressing the expression of these proto-oncogene products. The site at which each of these negative regulators inhibits the expression of c-fos and cjun is unknown and could be at either the nuclear or cytoplasmic level. It is likely that progesterone's action is nuclear. The progesterone receptor is a transcription factor that when ligand activated generally decreases the levels of c-fos and cjun mRNAs [26]. In avian oviductal cells, this inhibitory action on c-jun expression is mediated through a DNA sequence located within the cjun promoter [27]. These published reports support the idea that progesterone has a nuclear site of action. However, progesterone increases intracellular concentrations of cAMP and downregulates agonist-induced inositol phosphate metabolism in human term placenta [28]. If progesterone has a similar action within GCs, then progesterone could mediate its action indirectly by stimulating cAMP synthesis. Thus, more experiments must be conducted to assess the mechanism by which progesterone inhibits insulin's mitogenic action. As previously mentioned, cAMP negatively influences insulin-dependent proto-oncogene expression (present study) and GC mitosis [13, 29, 30]. Cyclic AMP could mediate its anti-mitogenic action by stimulating progesterone synthesis [31], which in turn would block insulin-dependent mitosis. This is unlikely since cAMP prevents GC mitosis in the presence of aminoglutethimide (AG), which inhibits progesterone synthesis (i.e., a 29 + 3% increase in GC numbers for insulin + AG vs. a 5 + 2% increase for insulin + AG + cAMP treatment; p < 0.05; unpublished observations). It is also unlikely that cAMP directly interacts with either the c-fos

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FIG. 7. The effect of sense and antisense oligonucleotides on the insulin-dependent proliferation of small GCs. GCs were exposed to insulin for 2 h and then treated with either sense or antisense oligonucleotides for c-fos and c-jun for an additional 22 h. GC proliferation was assessed as described in Materials and Methods and expressed as the mean percent increase + SE. This experiment was repeated three times.

or c-jun promoter to inhibit their transcription, since agents that increase intracellular levels of cAMP (i.e., FSH, 8-brcAMP, forskolin) either have no effect or increase mRNA levels of c-fos [15, 16] and c-jun [16] in cultured rat GCs. A third possibility could be that cAMP interferes with insulin's ability to interact with its receptor and stimulate inositol phospholipid metabolism [32]. This is also unlikely, since 8-br-cAMP blocks TPA-induced GC mitosis [13]. The most likely site of cAMP's action appears to be at or distal to PKC activation. In other systems TPA activates Ras [33]. Ras then activates Raf-1, which in turn stimulates the sequential activation of two mitogen-activated protein kinases, p4 4mapk and p42 mapk, and ultimately the gene cascade that leads to mitosis [34]. The results of several studies have also shown that Ras/Raf-1 activation enhances the transcription activity of c-jun by inducing phosphorylation of its transcriptional activation domain [35, 36]. This would result in the increased expression of both c-fos and c-jun [37]. Data presented from three separate laboratories demonstrate that cAMP interferes with the ability of Ras to activate Raf-1 [38-40], thereby blocking the mitogen-activated kinase cascade and subsequent mitosis. The possibility that cAMP has a similar mode of action in GCs remains to be investigated. In summary, it appears that insulin stimulates rat GC mitosis by activating PKC, which stimulates a series of kinases that ultimately leads to the expression of the proto-oncogene products, c-fos and c-jun. These proto-oncogene products play an essential role in the mitogenic pathway, presumably by forming AP-1 transcription complexes that in turn induce the mitogenic gene cascade. Finally, factors that inhibit GC mitosis do so in part by attenuating the insulin-induced expression of c-fos and c-jun.

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REFERENCES 1. Orly J, Sato G, Erickson GF. Serum suppresses the expression of hormonally induced functions of cultured granulosa cells. Cell 1980; 20:817-827. 2. Greenwald G, Terranova P. Follicular selection and its control. In: Knobil E, Ewing LL, Greenwald GS, Markert CL, Pfaff DW, (eds.), The Physiology of Reproduction. New York: Raven Press; 1988: 387-446. 3. Adashi EY, Resnick CE, Svobada ME, Van Wyk J. Somatomedin-C synergizes with follicle-stimulating hormone in the acquisition of progestin biosynthetic capacity by cultured rat granulosa cells. Endocrinology 1985; 116:2135-2142. 4. Adashi EY, Resnick CE, Hernandez ER, Hurwitz A, Roberts CT, Leroith D, Rosenfeld R Insulin-like growth factor I as an intraovarian regulator: basic and clinical implications. Ann NY Acad Sci 1991; 626:161-168. 5. May J, Schomberg D. The potential relevance of epidermal growth factor and transforming growth factor-alpha to ovarian physiology. Semin Reprod Endocrino] 1989; 7:1-11. 6. Gospodarowicz D. Fibroblast growth factor: involvement in early embryonic development and ovarian function. Semin Reprod Endocrinol 1989; 7:21-28. 7. Anderson E, Lee GY. The participation of growth factors in simulating the quiescent, proliferative and differentiative stages of rat granulosa cells grown in a serum-free medium. Tissue &Cell 1993; 25:49-72. 8. Dorrington J, Chuma A, Bendell J. Transforming growth factor 3 and folliclestimulating hormone promote rat granulosa cell proliferation. Endocrinology 1990; 123:353-359. 9. Bendell J, Dorrington J. Epidermal growth factor influences growth and differentiation of rat granulosa cells. Endocrinology 1990; 127:533-541. 10. Khan SA, Shum LW, Teerds K, Dorrington JH. Steroidogenesis-inducing protein interacts with transforming growth factor-1 to stimulate DNA synthesis in rat granulosa cells. Mol Cell Endocrinol 1992; 89:97-103. 11. Bley M, Simon J, Saragueta P, Baranao J. Hormonal regulation of rat granulosa cell deoxyribonucleic acid synthesis: effects of estrogens. Biol Reprod 1991; 44:880888. 12. Bley MA, Sim6n JC, Est6vez AG, Jim6nez de Asfia L, Baraiiao JL. Effect of folliclestimulating hormone on insulin-like growth factor-I-stimulated rat granulosa cell deoxyribonucleic acid synthesis. Endocrinology 1992; 131:1223-1229. 13. Peluso l, Pappalardo A, White BA. Control of rat granulosa cell mitosis by phorbol ester-, cyclic AMP-, and estradiol-17 beta-dependent pathways. Biol Reprod 1993; 49:416-422. 14. Nishizuki Y. The family of protein kinase C for signal transduction. JAMA 1989; 262:1826-1933. 15. Pennybacker M, Herman B. Follicle-stimulating hormone increases c-fos mRNA levels in rat granulosa cells via a protein kinase C-dependent mechanism. Mol Cell Endocrinol 1991; 80:11-20. 16. Ness JM, Kasson BG. Gonadotropin regulation of c-fos and cjun messenger ribonucleic acids in cultured rat granulosa cells. Mol Cell Endocrinol 1992; 90:1725. 17. Delidow BC, Lynch JP, White BA, Peluso J. Regulation of proto-oncogene expression and deoxyribonucleic acid synthesis in granulosa cells of perifused immature rat ovaries. Biol Reprod 1992; 47:428-435. 18. Angel P, Karin M. The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim Biophys Acta Rev Cancer 1991; 1072:129-157. 19. Rao IM, Mills TM, Anderson E, Mahesh VB. Heterogeneity in granulosa cells of developing rat follicles. Anat Rec 1991; 229:177-185 20. McDonnell S, Kerr L, Matrisian L. Epidermal growth factor stimulation of stromelysin mRNA in rat fibroblasts requires induction of proto-oncogenes, c-fos and c-jun and activation of protein kinase C. Mol Cell Biol 1990; 10:4284-4293.

21. Sanbuissho A, Lee GY, Anderson E. Functional and ultrastructural characteristics of two types of rat granulosa cell cultured in the presence of FSH or transforming growth factor alpha (TGF-alpha). J Reprod Fertil 1993; 98:367-376. 22. Hirshfield A. Development of follicles in the mammalian ovary. Int Rev Cytol 1991; 124:43-101. 23. Hirshfield AN, Flickinger GL, Ben-Rafael Z. Flow cytofluorometric analysis of granulosa cell proliferation in rats. J Reprod Fertil 1988; 84:231-238. 24. Campisi J. Growth factors, proto-oncogenes and the control of cell proliferation: lessons from the fibroblast. In: Hirshfield A (ed.), Growth Factors and the Ovary. New York, NY: Plenum Press; 1989: 61-74. 25. Rao I, Allsbrook W, Conway B, Martinez J, Beck J, Pantazis C, Mills T, Anderson E, Mahesh V. Flow cytometric analysis of granulosa cells from developing rat follicles. J Reprod Fertil 1991; 91:521-530. 26. Schuchard M, Landers JP, Sandhu NP, Spelsberg TC. Steroid hormone regulation of nuclear proto-oncogenes. Endocr Rev 1993; 14:659-669. 27. Subramanian M, Harris SA, Rasmussen K, Spelsberg TC. Rapid down-regulation of c-jun proto-oncogene transcription by progesterone in the avian oviduct. Endocrinology 1993; 133:2049-2054. 28. Rivera J, Bernal AL, Cano A. Progesterone increases basal 3',5'-cyclic adenosine monophosphate formation and down-regulates the agonist-induced inositol phosphates generation in human term placenta. Acta Endocrinol (Copenh) 1992; 127:499-503. 29. Yong EL, Baird DT, Yates R, Reichert LJ, Hillier SG. Hormonal regulation of the growth and steroidogenic function of human granulosa cells. J Clin Endocrinol &Metab 1992; 74:842-849. 30. Yong EL, Baird DT, Hillier SG. Mediation of gonadotrophin-stimulated growth and differentiation of human granulosa cells by adenosine-3',5'-monophosphate: one molecule, two messages. Clin Endocrinol (Oxf) 1992; 37:51-58. 31. Amsterdam A, Rotmensch S, Ben-Ze'ev A. Coordinated regulation of morphological and biochemical differentiation in a steroidogenic cell: the granulosa cell model. Trends Biol Sci 1989; 14:376-382. 32. Farese RV, Konda TS, Davis JS, Standaert ML, Pollet RJ,Cooper DR Insulin rapidly increases diacylglycerol by activating de novo phosphatidic acid synthesis. Science 1987; 236:586-590. 33. Bruder JT, Heidecker G, Rapp UR. Serum-, TPA-, and Ras-induced expression from Ap-1/Ets-driven promoters requires Raf-1 kinase. Genes & Dev 1992; 6:545556. 34. Lenormand P, Pages G, Sardet C, L'Allemain G, Meloche S, Pouyss6gur J. Map kinases: activation, subcellular localization and role in the control of cell proliferation. Adv Second Messenger Phosphoprotein Res 1993; 28:237-244. 35. Binetruy B, Smeal T, Karin M. Ha-Ras augments c-jun activity and stimulates phosphorylation of its activation domain. Nature 1991; 351:122-127. 36. Smeal T, Binetruy B, Mercola DA, Birrer M, Karin M. Oncogenic and transcriptional cooperation with Ha-Ras requires phosphorylation of c-jun on serines 63 and 73. Nature 1991; 354:494-496. 37. Ransone LJ, Verma IM. Nuclear proto-oncogenes fos and jun. Annu Rev Cell Biol 1990; 6:539-557. 38. Cook SJ, McCormick F. Inhibition by cAMP of Ras-dependent activation of Raf. Science 1993; 262:1069-1072. 39. Wu J, Dent P, Jelinek T, Wolfman A, Weber MJ, Sturgill TW. Inhibition of EGFactivated MAP kinase signaling pathway by adenosine 3',5'-monophosphate. Science 1993; 262:1065-1068. 40. Chen J, lyengar R. Suppression of Ras-induced transformation of NIH 3T3 cells by activated Gat. Science 1994; 263:1278-1281.