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BOR - Papers in Press, published online ahead of print October 15, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.023457
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BIOLOGY OF REPRODUCTION 70, 509–517 (2004)
DOI: 10.1095/biolreprod.103.023457
© 2004 by the Society for the Study of Reproduction, Inc.

Ovary

Follicle Stimulating Hormone-Induced DNA Synthesis in the Granulosa Cells of Hamster Preantral Follicles Involves Activation of Cyclin-Dependent Kinase-4 Rather Than Cyclin D2 Synthesis1

Peixin Yang, and Shyamal K. Roy2

Departments of Obstetrics and Gynecology and Physiology and Biophysics, University of Nebraska Medical Center, Omaha, Nebraska 68198-4515


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although cyclin D2 mRNA synthesis precedes gonadotropin-induced DNA synthesis in quiescent granulosa cells in culture, it is unclear whether a similar mechanism exists for the granulosa cells of growing preantral follicles in cyclic animals. The objective was to evaluate whether the synthesis of cyclin D2 protein was a prerequisite for FSH-induced DNA synthesis in the granulosa cells of intact preantral follicles of cyclic hamsters. Preantral follicles from cyclic hamsters were cultured in the presence or absence of FSH, and cell cycle parameters were examined. FSH stimulated cyclin-dependent kinase (CDK)-4 activity by 2 h and DNA synthesis by 4 h without altering the levels of cyclin D2 in the granulosa cells. The FSH effect was mimicked by epidermal growth factor administered in vivo. Although FSH increased the levels of cyclin D2 mRNA, it also stimulated the degradation of cyclin D2 as well as p27Kip1 and p19INK4 proteins. FSH activation of CDK4 was mediated by cAMP and ERK-1/2. In contrast to granulosa cells in intact follicles, FSH or cAMP significantly increased cyclin D2 protein levels in cultured granulosa cells but failed to induce DNA synthesis. Collectively, these data suggest that granulosa cells of preantral follicles, which are destined to enter the S phase during the estrous cycle, contain necessary amounts of cyclin D2 and other G1 phase components. FSH stimulation results in the formation and activation of the cyclin D2/CDK4 complex leading to DNA synthesis. This mechanism may be necessary for rapid movement of follicles from preantral to antral stages during the short duration of the murine estrous cycle.

follicle, follicle-stimulating hormone, follicular development, granulosa cells, ovary


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Repetitive but controlled division of granulosa cells is essential for ovarian follicular growth. Onset of DNA synthesis requires G1 phase preparation involving various cyclins and their corresponding cyclin-dependent kinases (CDKs) [1]. Activation of cyclins and CDKs is controlled by INK4 and Kip/Cip families of inhibitory proteins [2]. D-type cyclins (D1–D3) are the S phase cyclins in all cells [3], but cyclin D2 dominates in mouse and rat granulosa cells and is up-regulated by FSH and estradiol [4, 5]. In the resting phase of the cell cycle, most of the CDK4/6 exists as INK4 complexes [2]. In quiescent cell populations, activation of the Ras-Raf-MEK-ERK kinase cascade by mitogen(s) results in cyclin D transcription and translation leading to more CDK4 binding to cyclin D [6]. The complex (cyclin D2/CDK4) becomes an active kinase complex on phosphorylation by Cdk-activating kinase (CAK [6]) and sequesters p27kip1 from the cyclin E/Cdk2 complex, thus making the latter active [7]. Active cyclin E/Cdk2, along with cyclin D/CDK4, hyperphosphorylates Rb protein resulting in the release of EF2 and transcription of genes necessary for DNA synthesis and cell division [2]. A stable p27kip-cyclin D/CDK4/6 complex plays an important role in preventing INK4 inhibition of CDK4/6 [8], nuclear translocation [9, 10], and eventual ubiquitination and clearance of the complex [11]. Robker and Richards [5] have demonstrated that FSH, estradiol, or cAMP stimulates cyclin D2 mRNA and protein synthesis in the granulosa cells in in vitro culture or in the ovaries of hypophysectomized rats. Whereas cyclin D2 synthesis is essential for quiescent granulosa cells in the ovaries of hypophysectomized rodents or in in vitro culture to enter the S phase, a prolonged period of quiescence is unlikely for granulosa cells in gonadotropin-intact, cyclic animals because continuous follicular growth is essential for an undisrupted estrous cycle and ovulation.

Activation of the CDK 4/6 or cdk2 kinases is considered the central biological event at the restriction point [12] when cell cycle progression becomes autonomous. Quelle et al. [13] have shown that overexpression of D-type cyclins accelerates fibroblast proliferation. Further, D-type cyclins preferentially bind to CDK4 to form a holoenzyme [14], the activity of which is stimulated by a variety of growth factors [15, 16]. While cyclin D2 is essential for CDK4 activation, regulated degradation is also essential for its cyclic expression [2]. Besides cyclin degradation, ubiquitination of p19INK4 also influences cyclin D/CDK4(6) complex formation [17]. All these lines of evidence suggest that temporal degradation of cell cycle regulatory protein plays an important role in determining the G1 to S phase transition. However, whether FSH influences the degradation of cell cycle regulatory proteins in granulosa cells of intact preantral follicles is not known. The objectives of the present studies were to reveal (1) whether FSH induction of DNA synthesis in the granulosa cells of intact preantral follicles must involve cyclin D2 protein synthesis before CDK4 activation or if cells contained adequate amounts of cyclin D2 to initiate the DNA synthesis and (2) whether FSH would influence the degradation of p27kip, p19INK, and cyclin D2 in preantral follicles.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Golden hamsters (90–100 g) were purchased from SASCO (Madison, WI) and maintained in a 14L:10D cycle in a climate-controlled facility according to Institutional Animal Care and Use Committee (IACUC) and U.S. Department of Agriculture guidelines. The use of hamsters for this study was approved by the IACUC. Ovine FSH-19 was purchased from the National Pituitary Program (National Institute of Health [NIH]); rabbit anti-cyclin D1-, D2-, and CDK4-IgG were from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit anti-p27Kip1- and p19INK4-IgG and murine epidermal growth factor (EGF) were from BD/Transduction Laboratories (San Diego, CA); serine-tyrosine (dual)-phosphorylated ERK-1/2 antibody was from New England Biolab (Beverley, MA); [3H]thymidine (TdR) (specific activity 40 Ci/mmol) was from Amersham (Arlington Heights, IL); 8-bromo-3',5'-cyclic-AMP (8-Br-cAMP) methyisobutylxanthine (MIX), and mouse monoclonal anti-ß-tubulin antibody were from Sigma Chemical Co. (St. Louis, MO); H-89, a widely used inhibitor of protein kinase A, PD98059, a common inhibitor of MEK-1/2, lactacystin, an inhibitor of proteosome activity, were from Calbiochem (La Jolla, CA); progesterone antibody was a gift from Dr. Donald Johnson, University of Kansas Medical Center (Kansas City, MO); and chemiluminescence WestFemto substrate was from Pierce Chemical Co. (Rockford, IL). All other analytical-grade chemicals were purchased from Fisher Scientific Company (Pittsburgh, PA) or Sigma.

Time Course of FSH Effect on Follicular TdR Incorporation; Levels of Cyclin D2, p27kip1, and p19INK4 Proteins; and Cyclin D2 mRNA and CDK4 Activity

In the first experiment, preantral follicles at stage 6 (preantral follicles with seven to eight layers of granulosa cells and theca [18]) and stage 7 (follicles with incipient antrum [18]) were sonified in 1x RIPA (phosphate-buffered saline [PBS] pH 7.4, containing 0.1% SDS, 1% NP-40, and 0.5% deoxycholate) and a protease inhibitor cocktail (Sigma) and clarified at 26 000 x g at 4°C, and 40 µg of supernatant protein was analyzed by Western blotting using antibody specific to cyclin D1, D2, CDK4, or CDK6 to determine the predominant form of cyclin D or CDK in the hamster granulosa cells. Next, follicles at stages 6 and 7 were cultured for 2, 4, and 6 h in the absence or presence of 25 ng/ml of ovine FSH-19 (NIH) and 1 µCi/ml of [3H]TdR in Dulbecco modified Eagle medium supplemented with ITS+ (final concentration: 6.25 µg insulin, 6.25 µg transferrin, 6.25 ng selenium, and 5.35 µg linoleic acid/ml; Collaborative Research, Bedford, MA), penicillin G, streptomycin sulfate, amphotericin B, and 5% bovine serum albumin/ml at 37°C in a Haereus incubator under 5% CO2 in air [19]. Follicles were retrieved, rinsed thoroughly in ice-cold PBS, pH 7.4, and used for the determination of [3H]TdR incorporation, the levels of cyclin D2, p27kip1 and p19INK4 proteins, and the activity of the cyclin D2/CDK4 complex. The steady-state levels of cyclin D2 mRNA were evaluated by a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) using follicular RNA.

Effect of EGF In Vivo on Follicular ERK-1/2 Phosphorylation, CDK4 Activation, and Cyclin D2 Levels

Proestrous hamsters were injected i.p. with 20 µg EGF, and ovaries were collected after 3 h. Ovarian cyclin D2 and dual-phosphorylated ERK-1/2 levels were determined by Western immunoblotting, while CDK4 activity was determined in the cyclin D2/CDK4 complex.

Effect of 8-Br-cAMP and H89 on FSH-Stimulated Follicular DNA Synthesis, Levels of Cyclin D2 and p27kip1 and p19INK Proteins, and the Activity of CDK4

Follicles were cultured for 2, 4, or 6 h in the absence or presence of FSH and 1 mM MIX, and cAMP accumulated in the medium was measured by radioimmunoassay. Next, follicles were incubated without or with 1 mM MIX, 10 µM or 1 mM 8-Br-cAMP, and 1 µCi/ml [3H]TdR, and the incorporation of [3H]TdR was determined.

In a separate experiment, follicles were cultured with or without 10 µM H89 for 1 h followed by a 6-h culture with H89 and/or 25 ng/ml ovine-FSH-19 and 1 µCi/ml [3H]TdR. Expression of p27Kip1 and p19INK proteins relative to ß-tubulin was determined by Western immunoblotting, and the activity of CDK4 in the cyclin D2/CDK4 complex was determined by kinase assay. Follicular DNA synthesis and cAMP production were measured by [3H]TdR incorporation and cAMP RIA, respectively.

Effect of PD98059 on FSH-Stimulated Follicular DNA Synthesis, Levels of Cyclin D2 and p27kip1 and p19INK4 Proteins, and the Activity of CDK4

Follicles were cultured without or with 10 µM PD98059 for 1 h followed by a 6-h culture with PD98059 and/or 25 ng/ml ovine-FSH-19 and 1 µCi/ml [3H]TdR. Expression of p27Kip1 and p19INK proteins relative to ß-tubulin were determined by Western immunoblotting, and the activity of CDK4 in the cyclin D2/CDK4 complex was determined by kinase assay. Follicular DNA synthesis and cAMP production were measured by [3H]TdR incorporation and cAMP RIA, respectively.

Next, follicles were incubated without or with H89 or PD98059 for 1 h, then cultured for 6 h with H89 or PD98059 and/or 25 ng/ml FSH. Activity of ERK-1/2 was determined in the follicular lysate by a kinase assay.

FSH Induction of Cyclin D2 Protein in Quiescent Granulosa Cells in Culture Versus Nonquiescent Cells in Intact Preantral Follicles

Granulosa cells were isolated from preantral follicles at stage 6 by needle puncture, rinsed in DMEM containing 5% fetal bovine serum (FBS), and plated at a density of 10 000 cells/cm2 in a Falcon four-well chambered slide in the presence of 5% FBS in DMEM, and allowed to attach for 24 h. Medium with serum was removed 24 h later, and cells were rinsed twice with fresh serum-free DMEM with ITS+. The culture was continued for 72 h in the absence of serum for growth arrest [20] with medium changed after 48 h. After 72 h, 25 ng/ml FSH, 1% FCS, FSH +10 µM H89, FSH + 10 µM PD98059, or 10 µM 8-Br-cAMP was added, and the culture was continued for an additional 24 h. Cells were processed for immunofluorescence detection and quantification of cyclin D2 levels. Next, intact follicles at stage 6 were isolated from proestrous hamsters, cultured with or without 25 ng/ml FSH and 10 µM 5-bromo-2'-deoxyuridine (BrdU) for 6 h, and processed for cyclin D2 and BrdU immunofluorescence.

Effect of Lactacystin on FSH-Stimulated Follicular DNA Synthesis and Cyclin D2 and p27kip1 and p19INK4 Protein Levels

Follicles were cultured in the absence or presence of 5 µM lactacystin for 1 h, then cultured for 6 h with lactacystin and/or 25 ng/ml ovine-FSH-19 and 1 µCi/ml [3H]TdR. Follicular DNA synthesis and levels of cyclin D2, p27kip1, p19INK4, and ß-tubulin were determined as described previously. Progesterone accumulated in the medium was assayed by RIA to determine whether the lactacystin effect was due to impairment of FSH-FSH-receptor interaction.

Measurement of [3H]TdR Incorporation

This was done as described by Roy and Greenwald [21]. The rate of DNA synthesis was expressed as cpm [3H]TdR incorporated per ng DNA.

Western Immunoblot Detection of Cell Cycle Proteins

This was done as described by Roy and Kole [22] with adjustments for small amounts of follicular protein. Briefly, follicles were sonified in 30 µl ice-cold RIPA containing a protease inhibitor cocktail at 20 W with two to three 10-sec bursts, kept on ice for 30 min, and centrifuged at 15 000 x g at 4°C. Forty micrograms of protein in the supernatants of untreated and treated follicles was subjected to immunoblotting using antibodies specific to cyclin D2, p27Kip1, p19INK4, dual-phosphorylated ERK-1/2, ERK-1/2, or ß-tubulin. The signal was generated using WestFemto chemiluminescence substrate, and the light emitted from the bands was directly captured by a UVP (Upland, CA) gel documentation system. Because the system could record light intensity up to 5 OD, the sensitivity exceeded that of conventional film by several orders of magnitude and completely eliminated the problem of signal saturation, which was common to film densitometry. Images of representative immunoblots were arranged using Adobe Photoshop and Microsoft PowerPoint software.

Measurement of the Activities of Cyclin D2/CDK4 Complex and ERK-1/2

Because cyclin D2/CDK4 complex represented active holoenzyme [2], the activity of CDK4 was determined as cyclin D2/CDK4 immunocomplex as described by Matsushime et al. [23] with modification to suit small amounts of follicular protein. Briefly, 40 µg of follicular protein was clarified with 1 µg of rabbit IgG and 20 µl protein A-agarose in 500 µl of lysis buffer for 4 h at 4°C. After removal of the beads at 10 000 x g for 30 sec, 2 µg of a rabbit polyclonal anti-cyclin D2 (M-20, Santa Cruz Biotechnology) antibody was added to the supernatant, and immunoprecipitation was continued overnight at 4°C. Immune complexes were washed four times with the same lysis buffer and twice with the wash buffer (50 mM HEPES pH 7.5, 1 mM DTT). Thirty microliters of reaction mixture, containing 50 mM HEPES pH 7.5, 1 mM DTT, 10 mM MgCl2, 20 µM ATP, 10 µCi {gamma}-[32P]ATP, and 0.4 µg retinoblastoma (Rb) protein (Santa Cruz Biotechnology), was added to the immunoprecipitate, and the enzyme reaction was continued for 30 min at 30°C. The reaction was stopped by adding an equal volume 2x SDS sample buffer [24] and heating at 95°C for 2 min.

A similar immunoprecipitation protocol was used to determine the activity of ERK, except a kinase assay buffer (25 mM Tris-HCl pH 7.4, 12.5 mM ß-glycerophosphate, 10 mM MgCl2, 0.5 mM EGTA, 0.05 mM NaF, 1 mM DTT, 0.5 mM sodium vanadate, 10 nM okadaic acid, and 1 mM PMSF) was used in the presence of 2 µg of anti-ERK-1/2 antibody. The immunoprecipitate was mixed with 20 µl reaction buffer containing 4 µg of myelin basic protein (Upstate Biotechnology, Upstate, NY), 0.05 nmol PKA inhibitor protein (Calbiochem, La Jolla, CA), 50 µM ATP, and 1.25 µCi {gamma}-[32P]ATP (specific activity 10 Ci/mmol, ICN Biochemicals, Irvine, CA). After 30 min at 30°C, the reaction was stopped with 20 µl 2x SDS gel buffer.

Reaction mixtures for both kinases were resolved in a denaturing 10% polyacrylamide gel, transferred to Optitran membrane, and exposed overnight to phosphor screen, and the signal was quantified in a Cyclone Phosphor imager (Perkin-Elmer, Shelton, CT). The data were presented as picomol phosphate transferred per minute per microgram of protein. To deduce the relationship between the digital light unit (DLU) and 32P cpm, known amounts of {gamma}-[32P]ATP (specific activity, 7000 Ci/mmol) were spotted in triplicates on Optitran membrane, exposed to a phosphor screen for 1–4 h, and digitized to obtain the DLU values. The increases in the DLU values were linear with an increasing amount of 32P cpm and time of exposure. From the DLU values and cpm spotted on the membrane, the amount of 32P cpm per DLU was calculated. Because only the {gamma}-phosphate was radioactive in {gamma}-[32P]ATP and was transferred to the protein during phosphorylation, the cpm was contributed solely by the terminal phosphate. Therefore, the DLU per cpm was converted to DLU per picomol phosphate from the specific activity of {gamma}-[32P]ATP. Next, the DLU values obtained from the phosphorylated protein of the kinase reaction were converted to picomol phosphate transferred to the substrate protein, which was 4 µg in all reaction tubes. Finally, the data were normalized against the amount of follicular protein used for the kinase reaction and the time of reaction and eventually expressed as picomol phosphate transferred per minute per microgram protein.

RT-PCR Detection of Follicular Cyclin D2 Levels

Hamster cyclin D2 cDNA was partially cloned using RT-PCR amplification of hamster ovarian RNA. Follicular RNA was extracted using a RNAeasy micro kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. In our hands, this protocol always yielded RNA without significant genomic DNA contamination. Five hundred nanograms of total ovarian RNA was reverse transcribed using gene-specific reverse primer as described previously [25]. The cDNA was amplified for 30 cycles using cyclin D2-specific forward and reverse primers, and a predicted 223-bp amplicon was cloned in a PCRIITOPO plasmid (Invitrogen, Carlsbad, CA) after verifying the specificity of the product by Southern hybridization [26] and sequencing in an ABI automated sequencer (Eppley DNA Sequencing Core, University of Nebraska Medical Center, Lincoln, NE). The conditions for cyclin D2 PCR were 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C followed by a 10-min extension. Cyclin D2 primers were designed by comparing human ([27], accession no. X68452), xenopus (accession no. X89476), avian ([28], accession no. U28980), and rat ([29], accession no. L09752) cyclin D2 cDNA sequences and selecting a conserved region. The sequences of hamster ß-actin primers were described earlier [30]. The primer sequences for cyclin D2 were 5'-ATGGAGCTGCTGTGCTGTGAGGTGG-3'; (forward primer, nucleotides 1–15 of the reading frame) and 5'-AGACTTCACACACCTCCATC-3' (reverse primer, nucleotides 202–223 of the reading frame). To verify any genomic DNA contamination, control reverse-transcription reaction was done without reverse transcriptase enzyme and used it in the PCR reaction. No PCR product could be identified, indicating either that samples were free of any DNA contamination or that the contamination amount did not contribute to the product formation during the actual RT-PCR reaction.

Next, follicular RNA was reverse transcribed using random hexamers as described [31], and cDNA was amplified for 30 cycles (the reaction was linear from 10 through 50 cycles) using forward and reverse primers for cyclin D2 or ß-actin. The levels of ß-actin were detected to normalize cyclin D2 values and to check the specificity of FSH action on cyclin D2 mRNA expression. PCR product was resolved in 1% agarose containing SYBR green I, and the fluorescence was directly captured by a UVP gel documentation system.

Immunofluorescence Localization of Cyclin D2 Protein and BrdU

Following culture, granulosa cells in monolayer were rinsed twice with ice-cold PBS followed by 10 min fixation in 1% freshly prepared paraformaldehyde in PBS at 4°C. In contrast, cultured intact follicles were fixed in 1% paraformaldehyde for 30 min, then rinsed thoroughly in ice-cold PBS, embedded in agar [18], and impregnated with 20% sucrose; subsequently, 7-µm sections were cut in a Leica automatic cryostat at -18°C. The immunofluorescence staining protocol was similar to that described previously [30] except a cyclin D2-specific antibody was used. For 5'-bromo-2'-deoxyuridine (BrdU) staining, sections were first hydrolyzed with 2 N HCl for 30 min at 37°C, rinsed three times with PBS for 5 min each, and subjected to immunofluorescence detection [30] using a monoclonal anti-BrdU antibody (Sigma). Nuclei were stained with DAPI, mounted with Fluoromount G, and viewed under epifluorescence in a Leica DMR research microscope (North Central Instruments, Plymouth, MN) and photographed with an Optronics Magnafire digital camera. For colocalization of cyclin D2 and nuclei or cyclin D2- and BrdU-positive cells, images were overlapped using Openlab Image analysis software. Capture time was set using follicle sections, which were exposed to preimmune rabbit IgG, to subtract any background autofluorescence emanating from granulosa cells. Digital images were merged to determine colocalization. First, the labeling index of cyclin D2-positive cells in the cross section of a follicle was determined (this was found to be 100%). Then the percentage of granulosa cells in the same section labeled with BrdU (labeling index) with respect to cyclin D2-positive cells was calculated. Further, the average intensity (OD/pixel) of cyclin D2 immunoreactivity for untreated and treated granulosa cells either in monolayer cultures or in follicle sections was determined using NIH Image 1.6 image analysis software.

Determination of cAMP and Progesterone (P) in the Culture Medium

The levels of cAMP were determined using a kit (Biomedical Technologies, Inc., Stoughton, MA) according to the manufacturer's instruction, while the levels of P were determined by specific RIA as described previously [32, 33]. All samples were assayed at the same time to avoid interassay variation. The coefficient of intraassay variation was 7%.

Statistical Analysis

All cultures, immunoblotting, kinase assays, and immunofluorescence localization were repeated at least three times using follicles from different hamsters, and representative images were presented. The quantitative immunofluorescence data for each group of cultured granulosa cells reflected an average of at least 10 cells for each replicate. For follicle sections, signals were digitized from five follicles for each untreated and each treated group. All quantitative data were analyzed using two-way ANOVA with Scheffe's post hoc test. For time course of FSH effect on [3H]TdR incorporation, comparison was done both within a given time and across time to determine whether FSH stimulated DNA synthesis at specific time points and whether [3H]TdR incorporation increased over time relative to untreated controls. The level of significance was P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of FSH on Follicular DNA Synthesis, G1/S-Phase Antigen Levels, and CDK4 Activity

The objective was to determine whether FSH augmentation of cyclin D2 levels was necessary for DNA synthesis in the granulosa cells of intact preantral follicles. Although thecal cells were present in intact preantral follicles, their contribution to the overall [3H]TdR incorporation or CDK4 activity was expected to be insignificant because FSH would not affect thecal cells. Further, no DNA synthesis in thecal cells attached to preantral follicles could be induced by either EGF or cAMP (unpublished results). Therefore, increases in [3H]TdR incorporation or enzyme activities would indicate granulosa cell contribution. Cyclin D2 was found to be the primary form of cyclin D in hamster granulosa cells, and no CDK6 protein could be detected in the hamster granulosa cells (data not shown). Therefore, all subsequent studies focused on cyclin D2 and CDK4. Follicular DNA synthesis in response to FSH increased significantly by 4 h and reached a plateau by 6 h (Fig. 1A). [3H]TdR incorporation for the untreated group reflected basal levels of follicular DNA synthesis, which was already initiated by endogenous mitogen(s). Concurrent with DNA synthesis, the activity of CDK4 increased significantly within 2 h of FSH exposure and further by 6 h (Fig. 1B, bottom panel). However, no change in cyclin D2 protein levels relative to untreated follicles was noted during the period of increased DNA synthesis (Fig. 1B, middle panel), indicating that activation of CDK4 rather than cyclin D2 synthesis was the first event in FSH-induced DNA synthesis in the granulosa cells of intact hamster preantral follicles. In contrast to protein, cyclin D2 mRNA levels increased noticeably by 2 h after FSH administration and continued increasing up to 6 h (Fig. 1B, top panel), suggesting that proteosomal degradation might play an important role in maintaining the steady-state levels of cyclin D2 protein in the granulosa cells of intact preantral follicles. The increase in cyclin D2 mRNA levels in untreated groups by 4 and 6 h reflected the time-dependent resumption of transcription in granulosa cells once follicles were placed in a favorable condition following isolation from the ovary. Increases in the activity of CDK4 and DNA synthesis corresponded to decreases in the levels of p27kip1 and p19INK4 by 4 h after FSH administration (Fig. 1B, middle panel), confirming that p27kip1 and p19INK4 negatively influence S-phase progression, and the reduction in their levels would be necessary for CDK4 activation. The levels of total CDK4 protein in the granulosa cells did not change regardless of FSH exposure (data not shown), indicating that the increase in CDK4 activity was not due to increase in the synthesis of CDK4 protein.



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  		FIG. 1. A) Time course of FSH effect on follicular [3H]TdR incorporation. Preantral follicles were incubated with FSH and [3H]TdR for a specified time and analyzed. B) Temporal effect of FSH on the induction of cyclin D2 and ß-actin mRNA (top panel: Sybr green-stained PCR gel), levels of cyclin D2, p27kip1, p19INK4, and ß-tubulin proteins (middle panel; Western immunoblots) and phosphorylated retinoblastoma (pRb) protein reflecting CDK4 activity in cyclin D2/CDK4 immunocomplex (bottom panel: phosphorimage) in follicular cells. C) Effect of 20 µg EGF in vivo on ovarian cyclin D2 expression (top panel: immunoblot), levels of dual phosphorylated ERK-1/2 (middle panel: immunoblot), and phosphorylated retinoblastoma (pRb) protein reflecting cyclin CDK4 activity (bottom panel: phosphorimage). MM-142 cell lysate was used as a positive control for cyclin D2. Bars with same letters are not significantly different from each other

Because EGF is a potent mitogen for hamster granulosa cells [34], it was of interest to determine whether EGF increased cyclin D2 levels in ovarian cells to activate CDK4. Ovarian cyclin D2 levels did not change in response to EGF (Fig. 1C, top panel), but phosphorylation of ERK-1/2 (Fig. 1C, middle panel) and activation of the cyclin D2/CDK4 complex (Fig. 1C, bottom panel) were evident, suggesting that activation of CDK4 in nonquiescent ovarian cells might not require cyclin D2 synthesis.

Possible Involvement of Protein Kinase A (PKA) and ERK-1/2 in FSH-Induced Activation of G1/S-Phase Events and CDK4 Activity

The objectives were to verify that FSH action on follicular DNA synthesis was mediated by cAMP and to examine whether PKA and ERK-1/2 participated as downstream signaling molecules. We used H89 as an appropriate blocker of PKA activity because it was used widely to study the role of PKA in granulosa cells [35]. Similarly, PD98059 had been widely used to inhibit MEK-1/2 activation of ERK [36]. FSH significantly (P < 0.05) stimulated follicular cAMP production by 2 h, and the synthesis increased further by 6 h (data not shown). Inhibition of phosphodiesterase activity by MIX resulted in a significant increase in follicular TdR incorporation, which did not increase further by simultaneous administration of 8-Br-cAMP (data not shown), indicating that once the mitogenic stimulus reached the optimal level, cells became refractory to additional stimulus.

FSH-induced follicular DNA synthesis was completely attenuated by H89 or PD98059 (Fig. 2A), which corresponded to a parallel decline in the activity of CDK4 (Fig. 2B, bottom panel) and an inhibition of the FSH-induced ERK activity (Fig. 2C) without influencing follicular cAMP production (Fig. 2D). Most interestingly, either H89 or PD98059 completely reversed FSH-induced reduction of p27kip1 and p19INK4 protein levels (Fig. 2B, top panel). These results suggest that FSH stimulation of CDK4 activation, down-regulation of cell cycle inhibitory proteins, and DNA synthesis in the granulosa cell of intact hamster preantral follicles most likely involved PKA and ERK activation.



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  		FIG. 2. Effects of H89 or PD98059 on FSH-induced (A) follicular TdR incorporation, (B) down-regulation of p27kip1 and p19INK4 proteins, and increase in the activity of CDK4, (C) increase in the activity of ERK-1/2, and (D) follicular cAMP production. Follicles were preexposed to the inhibitors for 1 h before FSH administration, and the culture was continued for an additional 6 h. Bars with same letters are not significantly different from each other

Effect of FSH on the Synthesis of Cyclin D2 Protein in Quiescent Granulosa Cells in Cultures and in Granulosa Cells Within Intact Preantral Follicles

Because the synthesis of cyclin D2 protein was shown to be a prerequisite for quiescent rat granulosa cells in culture to enter the S phase [5], it was of interest to examine whether FSH would induce cyclin D2 protein synthesis in growth-arrested hamster granulosa cells via cAMP-ERK activities and whether cyclin D2 synthesis would be associated with DNA synthesis during a 24-h exposure period. Further, we were also interested to know if all granulosa cells within intact preantral follicles expressed cyclin D2 regardless of their entry into the S phase or if FSH stimulation resulted in cyclin D2 synthesis in a subpopulation of granulosa cells that showed the induction of DNA synthesis. Relatively low levels of cyclin D2 protein persisted in serum-starved (untreated) granulosa cells after 96 h of culture (Fig. 3A, top and bottom panels). FSH, cAMP, or serum significantly up-regulated cyclin D2 immunoreactivity within 24 h of administration, and the effect of FSH was inhibited by H89 or PD98059 (Fig. 3A, top and bottom panels). Most notably, despite the increase in cyclin D2 levels, no incorporation of [3H]TdR in the DNA was evident for cultured granulosa cells exposed to FSH or 8-Br-cAMP for 24 h (data not shown). These results indicate that whereas cyclin D2 synthesis was a prerequisite for cultured granulosa cells to enter the S phase, increases in cyclin D2 levels alone might not necessarily reflect the onset of DNA synthesis, and accumulation of other essential factors was needed. This notion was furthered by the fact that despite the absence of any difference in cyclin D2 content in the granulosa cells of intact preantral follicles cultured with or without FSH, not all cells incorporated BrdU (Fig. 3B, top and bottom panels). However, the BrdU labeling index increased significantly within 6 h of FSH exposure (Fig. 3B). Further, all granulosa cells within intact preantral follicles contained cyclin D2 (Fig. 3B, top panel). Therefore, it was evident that granulosa cells in growing preantral follicles contained all necessary G1/S-phase proteins, including cyclin D2, to rapidly enter the S phase when stimulated by FSH, and the mechanism involved PKA and ERK activities.



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  		FIG. 3. A) Immunofluorescence localization of cyclin D2 protein in granulosa cells harvested from preantral follicles at stage 6 and, after plating for 24 h in the presence of 10% FBS, cultured without serum for 72 h for growth arrest (quiescence). Cells were then stimulated with FBS, 8-br-cAMP, and FSH with or without H-89 or PD98059 for 24 h and stained for cyclin D2 protein (top panel). Quantitative values of immunofluorescence are presented in the bottom panel. B) Sections of preantral follicles at stage 6 showing granulosa cells (top panel). Bottom panel represents the quantitative data of the cyclin D2 immunosignal and BrdU labeling index corresponding to the top panel. Because 100% cells were cyclin D2 positive, the percentage (labeling index) of BrdU-positive cells relative to cyclin D2-positive cells was calculated. Green fluorescence in photographs B–D and F–H represents cyclin D2; red fluorescence in photographs A, C, F, and G represents nucleus; and red fluorescence in photographs D and H represents BrdU. (C and G) Overlapped images depicting the presence of cyclin D2 in all cells (red + green = orange-yellow). (D and H) Overlapped images depicting BrdU-positive nuclei. Bars in the microphotographs = 10 µm. Bars in the graphs with same letter are not significantly different from each other

Degradation of Cell Cycle Proteins by FSH in Granulosa Cells

Degradation of cell cycle antigens by the ubiquitin-proteosomal pathway is essential for the normal cell proliferation cycle. Whether FSH influenced proteosomal degradation of cyclin D2 remains unknown. Further, steady levels of cyclin D2 despite an increase in mRNA levels in the granulosa cells of intact preantral follicles would suggest that FSH might have stimulated the rate of cyclin degradation. Therefore, a block in ubiquitin-proteosomal activity should lead to accumulation of cyclin D2 in the granulosa cells of intact follicles. Indeed, significant accumulation of cyclin D2 protein in follicular cells was noted within 6 h when lactacystin, an inhibitor of protein ubiquitination [37], was added to the culture containing FSH (Fig. 4A, top panel). Lactacystin alone had no effect on the levels of cyclin D2 levels (Fig. 4A, top panel). However, lactacystin inhibition of FSH-induced cyclin D2 degradation failed to support FSH-induced follicular DNA synthesis (Fig. 4B, bottom panel). These results suggest that FSH does accelerate cyclin D2 degradation as a necessary mechanism to maintain DNA synthesis. Lactacystin did not affect FSH-stimulated follicular progesterone production (Fig. 4B, bottom panel), which doubled by 24 h (data not shown), thus ruling out any possible adverse effect of lactacystin on FSH-FSH-receptor interaction and subsequent cAMP generation.



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  		FIG. 4. Effect of lactacystin (Lac) on (A) FSH-induced degradation of cyclin D2 protein and (B) DNA and progesterone synthesis during 6 h of follicle culture. Follicles were preexposed to 5 µM Lac for 1 h before FSH administration. Lactacystin inhibited FSH-induced degradation of cyclin D2 protein as well as DNA synthesis in follicular cells (A) without affecting FSH-induced steroidogenesis (B). Bars with same letter are not significantly different from each other


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of the present studies suggest that in contrast to growth-arrested granulosa cells in culture, cells in growing preantral follicles of cyclic hamsters contain adequate amounts of cyclin D2, which form active complexes with CDK4 on FSH stimulation and initiate DNA synthesis. Further, the downstream mechanism of FSH regulation of CDK4 activation and subsequent DNA synthesis in the granulosa cells of intact preantral follicles appear to involve PKA and ERK-1/2 activities. FSH also regulates proteosomal degradation of cyclin D2, p27Kip1, and p19INK, which appears to be an important requirement for granulosa cell DNA synthesis, via cAMP and ERK signaling. However, to maintain a functional amount of cyclin D2 during the S phase, FSH also induces cyclin D2 mRNA synthesis. Robker and Richards [5] have demonstrated that FSH stimulation of DNA synthesis in cultured rat granulosa cells or in hypophysectomized rat ovaries is preceded by an increase in cyclin D2 mRNA levels without any change in p27kip levels. However, the results of the present studies demonstrate that the amount of cyclin D2 critical for a rapid activation of CDK4 already exists in the granulosa cells of intact preantral follicles of cyclic hamsters, and the formation of cyclin D2/CDK4 complex may be a first event in FSH-stimulated DNA synthesis. The increase in cyclin D2 content without the induction of DNA synthesis in granulosa cells cultured with FSH for 24 h suggests that granulosa cells, even those waiting at the G1/S-phase boundary, exit the cell cycle if deprived of FSH support for a prolonged period. Thereafter, FSH can induce the synthesis of some important cell cycle regulatory proteins within a relatively short time (e.g., 24 h), but it cannot induce the full complement of factors that are essential for the initiation of DNA synthesis. That is why granulosa cells in growing preantral follicles maintain a steady high level of cyclin D2 in vivo regardless of serum FSH levels, along with other cell cycle components, and rapidly enter the S phase on FSH stimulation so that successful folliculogenesis can continue within the specified period of the estrous cycle. It is likely that endogenous endocrine or paracrine factors keep granulosa cells prepared for entering the S phase on a mitogenic stimulus, such as the FSH surge. Increased DNA synthesis by granulosa cells of preantral follicles following the preovulatory gonadotropin surge has been reported [38]. The rapid induction of DNA synthesis may explain the rapid growth of preantral follicles to preovulatory class during the short duration of the estrous cycle.

This is the first report to suggest that FSH reduces the levels of p27kip1 and p19INK4 and influences cyclin D2 synthesis as well as degradation in the granulosa cells of intact preantral follicles during the early phase of the cell cycle. Matsushime et al. [39] have demonstrated by pulse-chase analysis of metabolically labeled, CDK4-bound cyclin D1 that the cyclin D1/CDK4 holoenzyme complex exists in a dynamic equilibrium, with the bound regulatory subunits being replaced continuously by newly synthesized cyclins. FSH-stimulated progesterone production in the presence of lactacystin indicates that inhibition of DNA synthesis is not due to alteration in follicular response to FSH. Similar to cyclins, the central role of p27kip1 in mammalian cell cycle regulation is well established [40]. It has been demonstrated that p27kip1 abundance in cells is regulated by transcriptional [41], translational [42], and proteolytic mechanisms [43], and phosphorylation at threonine 187 by Cdk2 is necessary for proteosome-mediated degradation of p27kip1 [44]. In contrast to p27kip1, information of p19INK4 degradation is less clear. Although we do not know at present whether the decrease in p27kip1 and p19INK4 protein levels in the granulosa cells is due to phosphorylation-related degradation, the suppression of gene transcription, or both, it is clear that the mechanism involves cAMP and ERK signaling. Further studies are in progress to shed light on this intriguing issue.

In corroboration with our previous findings [45], the results of the present study suggest that cAMP is involved in the activation of ERK and DNA synthesis in ovarian granulosa cells. Increased DNA synthesis in the presence of MIX indicates that inhibition of hydrolysis elevates the levels of cAMP, resulting in its availability to many cells and in subsequent stimulation of DNA synthesis. Once the optimum stimulation is achieved, granulosa cells become refractory to further stimuli. This is further evident from the failure of H89 to affect basal DNA synthesis with or without FSH. Although H89 at a 10-µM dose level is widely used to determine the involvement of PKA in biological functions, it is important to recognize that this dose can also suppress highly purified forms of other kinases, such as mitogen- and stress-activated protein kinase 1, AMP-activated protein kinase, protein kinase B{alpha} (PKB), serum- and glucocorticoid-induced kinase (SGK), and p70 ribosomal protein S6 kinase [46] in a reaction tube. Therefore, the results of the H89 effect require careful interpretation. Although PKB and SGK-1 have been shown to influence granulosa cell survival [47, 48], the presence or the cell cycle regulatory roles of other kinases in the granulosa cells are not known. Further, continuation of basal DNA synthesis during 6 h of follicle culture and the increase in cAMP production in the presence of H89 + FSH rule out any possible compromise with cell survival. We also need to recognize that only a portion of the 10-µM dose is likely to cross the basal lamina and enter the granulosa cells in a multilayered follicle structure during the relatively short culture period. On the other hand, PD98059 at a 50-µM dose level had no inhibitory influence on any of the kinases indicated previously; however, it blocks ERK-1/2 activation by preventing the activation of MEK-1 rather than affecting MEK-1 activity [49]. Therefore, it is likely that the results obtained with H89 or PD98059 reflect suppression of the activity of the respective targeted enzymes.

Inhibition of FSH-induced follicular DNA synthesis by PD98059 provides strong evidence for the involvement of ERK-1/2 in this process. Although not directly linked to any granulosa cell function, phosphorylation of ERK-1/2 as early as 5 min after 100 ng/ml FSH administration followed by a dephosphorylation after 2 h has been reported for porcine granulosa cells in monolayer culture [50]. On the other hand, a decline in FSH-induced ERK-1/2 phosphorylation after 30 min has been demonstrated for immortalized pig granulosa cells in culture [51]. In contrast to these findings, high ERK-1/2 activity as late as 6 h after FSH administration has been observed for hamster preantral follicles (Fig. 3C), and the increase correlates well with follicular DNA synthesis. ERK-1/2 activity has been shown to be involved in cell proliferation as well as differentiation [52]; however, sustained ERK activity has been suggested to be essential for the reentry of cells in the cell cycle [53]. Therefore, it can be speculated that whereas ERK-1/2 activity for a shorter duration is adequate for inducing differentiation-related functions in granulosa cells, FSH-induced sustained activation of ERK-1/2 is necessary for CDK4 activation and DNA synthesis. Further studies are in progress to examine this hypothesis in detail.

In summary, the results of the present studies demonstrate that granulosa cells in growing preantral follicles are poised at the G1/S-phase boundary with adequate amounts of cyclin D2. FSH, via cAMP, leads to phosphorylation and activation of ERK-1/2, which promotes the formation and activation of cyclin D2/CDK4 complex, resulting in DNA synthesis. Further, FSH appears to stimulate proteosomal activity that constantly removes cyclin D2; however, to keep granulosa cell DNA synthesis uninterrupted, FSH also induces cyclin D2 transcription and translation.


   ACKNOWLEDGMENTS
 
We thank Dr. JinRong Wang for his help in immunofluorescence staining.


   FOOTNOTES
 
1 This work was supported by grants HD28165 and HD38468 to S.K.R. from the National Institute of Child Health and Human Development and a Lalor Foundation postdoctoral fellowship to P.Y. Back

2 Correspondence: Shyamal K. Roy, BH4030, Departments of OB/GYN and Physiology and Biophysics, University of Nebraska Medical Center, 984515 Nebraska Medical Center, Omaha, NE 68198-4515. FAX: 402-559-6164; skroy{at}unmc.edu Back

Received: 19 September 2003.

First decision: 4 October 2003.

Accepted: 14 October 2003.


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