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Department of Physiology, Medical College of Georgia (C.L.C., R.S.B.), and Veterans Affairs Medical Center (R.S.B.), Augusta, Georgia 30912; Department of Physiology and Pharmacology, Oregon Regional Primate Research Center (R.L.S.), Beaverton, Oregon 97006; and California National Primate Research Center, University of California (C.A.V.), Davis, California 95616
Address all correspondence and requests for reprints to: Dr. Charles L. Chaffin, Department of Physiology, Medical College of Georgia, 1120 15th Street, Augusta, Georgia 30912. E-mail: cchaffin{at}mail.mcg.edu.
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Introduction |
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The Myc/Max/Mad family of transcription factors is linked closely to proliferation, differentiation, and apoptosis. Members of this family heterodimerize with Max to form complexes capable of binding E box promoter elements and trans-activating target genes (7). The best characterized member of this group is the protooncogene c-myc, which typically facilitates movement of cells into the DNA synthesis (S) phase of the cell cycle (8). In contrast, the Mad proteins (Mad1, Mad3, Mad4, Mxi1, and Mnt) are a group of naturally occurring c-Myc antagonists that compete with c-Myc for access to Max and E box promoter sites and are thus associated with cell cycle arrest and differentiation (9). In this model, Myc/Max heterodimers act to progress the cell cycle, while Mad/Max complexes function as cell cycle repressors. For example, enforced expression of Mad prevents entry into the S phase and can inhibit transformation by overexpressed c-Myc (10). Although there is evidence that c-Myc and Mad gene targets are not entirely overlapping (9), it nevertheless remains possible that the ratio of Myc:Mad dictates whether a given cell will proliferate or differentiate (7). Because exit from the cell cycle may be an important step in the terminal differentiation of many cell types, the interaction between c-Myc and the Mad proteins in the control of the cell cycle is a critical issue.
The transition from a follicle to a corpus luteum in primates is not well understood, especially the early events, which are exceedingly difficult to study. An in vitro model of primate granulosa cell luteinization was developed with which to examine events occurring immediately after the initiation of the periovulatory interval. This model was used to test the hypothesis that a luteinizing dose of gonadotropin induces rapid changes in the expression of the Myc/Max/Mad family consistent with a role for these genes in the terminal differentiation of primate granulosa cells.
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Materials and Methods |
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Preparation of macaque granulosa cells
Granulosa cells were recovered from the filter rinse by a modification of the method previously described (14). Briefly, the cell suspension was centrifuged for 5 min at 300 x g to pellet the red cells; this was then increased to 500 x g for an additional 5 min, resulting in a thin layer of granulosa cells over the red cell pellet. The supernatant was removed, and the layer of granulosa cells was transferred to a 40% Percoll gradient in medium 199 (Sigma-Aldrich, St. Louis, MO) and centrifuged for 30 min at 500 x g. The supernatant was removed, and the granulosa cells were recovered from the surface of the Percoll with a Pasteur pipette and washed once with TL-HEPES/PVA. The cell pellet was resuspended in 1 ml TL-HEPES/PVA and counted on hemocytometer. An additional 14 ml TL-HEPES/PVA supplemented with 5 µg/ml r-hFSH were added to the cell suspension. Cells were placed in a biohazard shipping container and were shipped from the CNPRC to the Medical College of Georgia by overnight delivery at ambient temperature from September to June. Upon receipt, cells were recovered by centrifugation, and viability, as determined by trypan blue exclusion, remained over 85%.
In vitro luteinization of macaque granulosa cells
Granulosa cells were plated overnight at 37 C with an initial seeding density of 5 x 105 viable cells/well in 24-well plates precoated with fibronectin in DMEM/Ham’s F-12 supplemented with 20 mM HEPES, penicillin/streptomycin (50 U/ml), 1% fetal calf serum, and 50 ng/ml hFSH (Sigma-Aldrich, F4021). Preliminary experiments using [3H]thymidine uptake verified that macaque granulosa cells remain proliferative during this initial plating interval in response to 50 ng/ml hFSH (data not presented). After the initial overnight seeding period, media were changed to include either 50 ng/ml hFSH to maintain a proliferative phenotype (controls) or 20 IU/ml human chorionic gonadotropin (hCG; Sigma-Aldrich) to induce luteinization. Cultures were terminated either before medium change (0 h) or 1, 4, 8, 18, or 48 h after the addition of hCG or hFSH. Cell extracts were harvested as described below, and media were retained for the measurement of steroid concentrations using a commercially available RIA kit (Diagnostic Products, Los Angeles, CA).
[3H]Thymidine uptake
Granulosa cells were cultured and hormonally treated as described above, and [3H]thymidine uptake was determined at 0, 4, 8, 12, 24, and 48 h post-hCG (n = 3 animals). In brief, [3H]thymidine (2 µCi) was added to cultures 2 h before termination. Unincorporated [3H]thymidine was removed with four rinses of room temperature DMEM/Ham’s F-12, followed by 6-min incubation with trypsin (0.25%)/EDTA (0.1%) at 37 C. The resulting detached cells were pelleted and solubilized in 50 µl 10% sodium dodecyl sulfate for 10 min at room temperature before determining the counts per minute.
RNA analysis
To maximize the amount of information obtained from limited numbers of granulosa cells, an RT-PCT assay was employed. Total RNA was extracted from granulosa cells using TRIzol (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s instructions, and contaminating genomic DNA was removed by treating the extract with ribonuclease-free deoxyribonuclease I (DNase I; Life Technologies, Inc.) for 15 min at room temperature. DNase I was subsequently inactivated by the addition of 1 µl 25 mM EDTA for 15 min at 65 C. RT was carried out for 2 h at 37 C in a 20-µl reaction volume using 10-µl DNase I reaction, single strength RT buffer [50 mM Tris-Cl (pH 8.3), 40 mM KCl, and 6 mM MgCl2], 1 mM dithiothreitol, 25 pmol oligo(deoxythymidine) primer (Promega Corp., Madison, WI), and 200 U of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.); then the reverse transcriptase was heat-inactivated at 94 C for 5 min.
PCR was carried out in a 20-µl volume that included an empirically determined amount of the RT reaction dictated by the specific primer set, 2 µl 10x strength Taq buffer (Roche Molecular Biochemicals, Indianapolis, IN), 1–4 mM MgCl2, 0.8 µl 10 mM deoxy-NTPs, 3 U FastStart Taq (Roche), and the experimental and internal standard primers sets (Table 1
). The reaction was carried out 95 C for 4 min, followed by 94 C for 30 sec, 60 C for 1 min, and 72 C for 1 min for an empirically determined number of cycles. The entire PCR was electrophoresed through a 2% agarose gel stained with 0.1 µg/ml ethidium bromide. Gels were visualized on a UV transilluminator and were photographed using 667 Polaroid film (Fisher Scientific, Fairlawn, NJ), and photographs were analyzed by densitometry (Un-Scan-It, Silk Scientific, Orem, UT). All values were normalized to the internal standard; no apparent changes were observed for either standard after hCG treatment.
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Protein analysis
Granulosa cells were detached from plates with 0.25% trypsin/0.1% EDTA for 6 min at 37 C, pelleted, and snap-frozen. Cell pellets were stored at -80 C until protein isolation. Whole cell protein was isolated by resuspending cells in 100 µl F-buffer (16): 10 mM Tris (pH 7.05), 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 5 mM zinc chloride, 100 mM sodium orthovanadate (Na3VO4), Triton X-100, and mixed protease inhibitors (one tablet/25 ml; Complete Protease Inhibitors, Roche Molecular Biochemicals) for 10 min on ice, followed by vortexing for 45 sec. Lysates were cleared by centrifugation at 15,000 rpm and 4 C for 15 min, and supernatant protein concentrations were determined with a commercially available kit (bicinchoninic acid kit, Pierce Chemical Co., Rockford, IL). Five micrograms of protein were analyzed by SDS-PAGE. After separation, proteins were transferred to a polyvinylidene difluoride membrane, rinsed with PBS, and blocked for 1 h with 5% dry milk at room temperature. The antiproliferating cell nuclear antigen (anti-PCNA) monoclonal (clone PC10, LabVision, Fremont, CA) was added in a 1:200 dilution in the same blocking solution. The secondary antibody was used at a concentration of 1:5000 for 1 h at room temperature. Antibody complexes were visualized using the ECL-Plus kit (Pierce Chemical Co., Rockford, IL) and were densitometrically quantified.
Statistical analysis
Data were tested for heterogeneity of variance with Bartlett’s 
2 test and were subsequently logarithm transformed (log + 2) before analysis by one-way (PCNA) or two-way ANOVA with one repeated measure. Individual means were compared using a Newman-Keuls test. Data are expressed as a percentage of the 0 h (pre-hCG) value. Differences were considered significant when P < 0.05, and all values are presented as the mean ± SEM.
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Results |
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). In control (FSH only) cultures, progesterone was not markedly induced even after 48 h. Similarly, increased expression of the progesterone receptor (PR) gene is related to luteinization of primate granulosa cells (18). The addition of hCG to culture medium resulted in the increased expression of PR mRNA within 4–8 h, whereas increased expression of PR mRNA was not evident in control cultures (Fig. 1B).
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). Within 4 h of hCG treatment, PCNA was reduced to 50% of that in 0 h samples (P < 0.05). Interestingly, PCNA was transiently increased (P < 0.05) 8 h post hCG before declining (P < 0.05) to 30% of 0 h values by the culmination of the culture interval (48 h). The tk-1 gene is induced by active transcription factor E2F before S phase entry (20), and thus serves as an excellent gene marker of G1 to S phase transition. Before hCG (0 h), levels of tk mRNA were highest and declined between 1–4 h post-hCG (Fig. 2). Importantly, a transient increase in tk mRNA occurred 8 h post-hCG, after which mRNA levels declined between 18–48 h. In control cultures, levels of tk mRNA did not appreciably change over time.
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The expression of c-Myc mRNA was lowest before (0 h) and 1 h after hCG administration and tended to increase 4 h (P = 0.10) and 8 h (P = 0.06) later. Levels of c-Myc returned to 0 h values by 18 and 48 h after hCG (Fig. 4A). In contrast, the expression of Max mRNA was highest before hCG treatment and declined by 5-fold (P < 0.05) within 1 h (Fig. 4B). Max mRNA remained suppressed at 4 h post hCG but returned to values equivalent to 0 h between 8 and 48 h. Similarly, Mad1 mRNA was highest before hCG treatment and declined significantly (5-fold; P < 0.05) within 1 h of hCG treatment (Fig. 4C) before returning to 0 h levels at 8 h. The expression pattern for Mad4 mRNA was similar to that for Mad1, except that the levels of Mad4 did not recover until 18 h post hCG (Fig. 4D). Levels of mRNA were highest before hCG treatment before declining significantly (P < 0.05) at 4 h and increasing again at 8–48 h. Mxi1 mRNA tended to be reduced (P = 0.08; 2.5-fold) 8 h after hCG treatment (Fig. 4E).
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In contrast to Myc-regulated pathways, p53 mediates cell cycle arrest by enhancing a large number of target genes (22). To determine whether control of macaque granulosa cell proliferation during luteinization is due principally to the Myc/Mad family or if other relevant pathways are involved, p53 mRNA was measured. Addition of hCG to culture medium caused a marked, but transient, decline in p53 mRNA levels by 4 h (Fig. 5). Control cultures did not have appreciable changes in p53 mRNA. To determine whether p53 mRNA correlates with function, a p53-dependent target gene, the wild-type p53-inducible phosphatase (wip1), was used (23). The expression of wip1 was regulated in a manner identical to p53 (Fig. 5).
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Discussion |
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Hormonal control of rhesus monkeys, i.e. COS, has proven to be an enormously useful tool with which to examine the proliferation and luteinization of granulosa cells. In monkeys undergoing COS, more than 50% of granulosa cells obtained before an in vivo ovulatory stimulus are proliferative, whereas less than 15% remain so at 12 and 36 h post hCG (2). However, in primates [but not rats (25)], cyclin D2 mRNA increases 12 h post hCG, suggesting that early periovulatory events (i.e. <12 h) may not be aimed entirely at suppressing the cell cycle. Although these data clearly indicate that a rapid reorganization of cell cycle machinery occurs within the first 12 h after an ovulatory stimulus, this time interval remains completely unexplored. The in vitro model of macaque granulosa cell luteinization that was established in the current study displays several features in common with the in vivo setting: 1) these cells are proliferative and synthesize estrogen in response to treatment with FSH in vitro (current study and Chaffin, C. L., unpublished observations); 2) treatment of proliferating granulosa cells with hCG in vitro causes a rapid accumulation of progesterone in the culture-medium, indicative of luteinization; and 3) the expression of PR mRNA increases after hCG treatment in vitro, a critical aspect of luteinization. Thus, events and temporal relationships between key markers of luteinization appear to be intact and reflective of the in vivo situation.
Treatment of proliferative (i.e. nonluteinized) granulosa cells results in a marked reduction in PCNA expression within 4 h of hCG, whereas an unexpected increase in PCNA is observed 8 h post hCG. Importantly, PCNA expression is very low 18 and 48 h after hCG treatment, supporting the hypothesis that these cells are terminally differentiated by 18–24 h after an ovulatory stimulus (27). Although rat granulosa cells increase bromodeoxyuridine uptake and PCNA expression within the first 4 h after an ovulatory stimulus (4, 5, 28), Robker and Richards (25) suggested that proliferation of rat granulosa cells is arrested within 4 h of hCG administration in vivo. Thus, issues relating to cell cycle arrest of luteinizing rat granulosa cells have not yet been clearly elucidated. Other models show a similar burst of proliferation during terminal differentiation; for example, NIH-3T3 L1 cells display a transient increase in [3H]thymidine uptake before achieving a final adipocyte phenotype (29), suggesting that this may a generalized feature of differentiating cells.
Unlike transformed cell models (e.g. 3T3 L1 cells) in which the cell cycle can be synchronized, granulosa cells (in vivo and in vitro) are spread throughout the cell cycle, making it difficult to determine whether post-hCG proliferation represents cells entering into a new round of cell division or those in late G1 already destined to divide. The parallel expression of tk and PCNA along with increased [3H]thymidine uptake suggest not only that E2F transcription factor is active 8–12 h post hCG, but also that a fraction of cells traverse the S phase in response to hCG. Further, preliminary evidence suggests that a nearly 30% increase in the number of granulosa cells occurs by 24 h after treatment with hCG in vitro compared with that in control cultures (Chaffin, C. L., and R. L. Stouffer, unpublished observations). It is intriguing to hypothesize about the nature of the granulosa cells that undergo hCG-induced proliferation. These could be less mature granulosa cells that undergo a phase of catch-up growth, or perhaps this period of proliferation is indicative of a heterogeneous population of granulosa cells, and by extension, cell cycle arrest during luteinization could consist of a two-stage decline. Although the consequences of this proliferative burst on subsequent luteal formation and function are not currently understood, overexpression of Mad1 blocks differentiation of 3T3 cells into mature adipocytes (30). It is possible that the proliferative burst during luteinization of primate granulosa cells has a similar function in the completion of terminal differentiation and the formation of a corpus luteum.
The mechanisms responsible for the proliferative burst in granulosa cells before terminal differentiation appear to be linked to changes in the expression of immediate early genes such as c-Myc. The central status of c-Myc in the cell cycle is well established, and it has been hypothesized that the ratio of Myc to Mad is a key determinant in the decision of a given cell to proliferate or differentiate (31). Ayer et al. (31) reported that Mad/Max complexes are present as early as 2 h after the initiation of differentiation of monocytes and completely replace Myc/Max complexes by 24–48 h. Similarly, mRNA levels of c-Myc, Mad1, and Mad4 are out of phase in differentiating 3T3 cells (32). Further, levels of c-Myc and PCNA are increased transiently after an ovulatory stimulus to rats undergoing superovulation, suggesting that c-Myc regulates granulosa cell progression into the S phase during the periovulatory interval (4, 5). In primate granulosa cells after a luteinizing dose of gonadotropin, the relative ratio of c-Myc to Mad is increased before (and during) the increase in PCNA and tk expression as well as [3H]thymidine uptake, whereas Mad1 and Mad4 are more abundant during cell cycle arrest (18–48 h after hCG). It is noteworthy that, in contrast to other cell systems (32), significant overlap exists in the expression and regulation of Mad1 and Mad1 in primate granulosa cells. It is possible that this reflects the brief interval between the luteinizing hCG stimulus and terminal differentiation [
24 h in primates (27)]. Nevertheless, it is not currently known whether the different Mad proteins have redundant or unique functions that temporally overlap in the ovarian follicle. It is interesting to note that a marked increase in [3H]thymidine uptake also occurs 12 h after replenishment of FSH; this may be caused by the sudden pulse of mitogenic stimulus (FSH) to the cells. However, this increase in FSH-mediated proliferation is not accompanied by changes in the ratio of Myc to Mad, suggesting that in primate granulosa cells, this family of transcription factors may function specifically during luteinization. Future studies will be aimed at understanding the individual or collective actions of Mad family members on primate granulosa cell proliferation and differentiation.
One of the surprising findings from the current study is the dramatic regulation of max gene expression during terminal differentiation. Max has typically been reported to be constitutive (7), although there is some evidence that Max mRNA levels are under hormonal control in endocrine-sensitive organs such as rat endothelial cells (33). The transient suppression of Max mRNA observed in primate granulosa cells after hCG suggests that control of this gene is a facet of granulosa cell differentiation. Importantly, the nadir of Max expression occurs in close temporal association with the increase in the relative ratio of Myc/Mad. The reduction of Max 1–4 h post hCG may be a mechanism by which differentiating granulosa cells partially blunt the potentially apoptotic actions of high levels of Myc unopposed by Mad (34). Alternatively, the suppression of Max may actually sensitize differentiating granulosa cells to Myc by creating competition between Myc and Mad for access to limiting levels of Max. Further studies will be aimed at determining the functional consequences of Max regulation.
Although the proliferative burst of granulosa cells appears driven in part by reduced levels of Mad mRNAs, the observation that treatment of macaque granulosa cells with hCG results in a transient down-regulation of p53 mRNA indicates that other cell cycle control pathways are involved. Although the limited samples available for the current study preclude analysis of p53 protein function, the parallel expression of a p53-dependent target gene (wip1) strongly suggests that p53 mRNA correlates with function in macaque granulosa cells. Further, preliminary evidence indicates that p53 protein is localized to nuclei of macaque granulosa cell (Chaffin, C. L., and R. L. Stouffer, unpublished observations), supporting the presence of active p53. Although the function of p53 in the process of luteinization is not yet clear, it may contribute to cell cycle arrest after the proliferative burst. Interestingly, p53-regulated Wip1 is involved in cell cycle suppression (23) and may be an important mediator of p53 actions in the ovary. Whether the transient repression of p53 is a cause or a consequence of post-hCG cell cycle characteristics is not known.
One of the notable findings from the current study is the expression of multiple cell cycle suppressors in granulosa cells before hCG. In growing macaque follicles, only a fraction of cells are proliferative at any one time; thus, it is not yet known whether the mRNAs for Mad1, Mad4, Mxi1, p53, and wip1 are 1) translated into functional proteins, 2) expressed in the actively proliferating fraction of nonluteinized granulosa cells, or 3) contribute to cell cycle arrest of that percentage of granulosa cells present in the growing follicle that are not actively dividing (50%; Ref. 2). However, some of these gene products are coexpressed with c-Myc in other cell types, notably Mxi1 and Mad3 (30, 32, 35, 36, 37). These proteins could possibly fulfill roles other than cell cycle control, for example, as (anti-)apoptotic factors. Therefore, the role of Myc/Max/Mad in cell cycle control is far more complex than currently realized. Further studies are clearly needed to establish the cellular colocalization of markers of cell cycle progression, luteinization, and apoptosis during follicular maturation.
In summary, the acquisition of a luteal phenotype by primate granulosa cells in vitro is preceded by a proliferative burst driven in part by a transient increase in the ratio of c-Myc to Mad1, Mad4, and Mxi1 as well as p53 and the p53-dependent wip1, indicating that multiple pathways are involved in cell cycle control during terminal differentiation. It is hypothesized that this proliferative burst is a necessary component of terminal differentiation of primate granulosa cells.
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Acknowledgments |
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Footnotes |
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Abbreviations: COS, Controlled ovarian stimulation; CNPRC, California National Primate Research Center; DNase, deoxyribonuclease; hCG, human chorionic gonadotropin; PCNA, proliferating cell nuclear antigen; PR, progesterone receptor; PVA, polyvinylalcohol; r-hFSH, recombinant human FSH; tk, thymidine kinase; TL, tyrode lactate.
Received July 1, 2002.
Accepted for publication December 17, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Nature 326:515–517[CrossRef][Medline]
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) or 50 ng/ml hFSH (
). Media and cells were harvested before (0 h) and 1, 4, 8, 18, or 48 h after the addition of hCG. A, Levels of progesterone were determined by RIA at the indicated time points; B, induction of PR mRNA during luteinization of primate granulosa cells. GAPDH was used as an internal control. Data are the mean ± SEM (n = 3/time point). CTRL, Control (FSH only). *, Significantly different (P < 0.05) from 0 h; #, significantly different from time-matched FSH controls.
