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BOR - Papers in Press, published online ahead of print August 30, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.052613
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BIOLOGY OF REPRODUCTION 75, 859–867 (2006)
DOI: 10.1095/biolreprod.106.052613
© 2006 by the Society for the Study of Reproduction, Inc.

research-article

Participation of Mitogen-Activated Protein Kinase in Luteinizing Hormone-Induced Differential Regulation of Steroidogenesis and Steroidogenic Gene Expression in Mural and Cumulus Granulosa Cells of Mouse Preovulatory Follicles1

You-Qiang Su 3 6, Mette Nyegaard 4 6 7, Michael Toft Overgaard 4 6 7, Jie Qiao 5 6, and Linda C. Giudice 2 6 7

Department of Obstetrics and Gynecology,6 Stanford University, Stanford, California 94305 Department of Obstetrics, Gynecology, and Reproductive Sciences,7 University of California, San Francisco, California 94143-0132

ABSTRACT

The LH surge induces the terminal differentiation and onset of luteinization in granulosa cells of preovulatory follicles, a process that involves the differential expression of genes essential for steroidogenesis and appears to be mediated by complex signaling pathways. The objective of this study was to investigate whether these processes that commonly occur in mural granulosa cells (MGCs) also occur in cumulus cells, and whether they are mediated by the mitogen-activated protein kinase (MAPK), specifically MAPK3/1 (also commonly known as extracellular signal-regulated kinase 1&2, ERK1/2). The standard superovulation model for premature female mice was used to obtain MGCs and cumulus-oocyte complexes (COCs), and sensitive real-time RT-PCR was used to simultaneously detect the expression levels of transcripts encoding key steroidogenic enzymes in the same sample. We observed significant downregulation of Cyp19a1 and upregulation of Star and Cyp11a1 mRNA expression in both COCs and MGCs after in vivo administration of hCG or in vitro treatment with gonadotropins or 8-Br-cAMP. This differential pattern of steroidogenic gene expression was correlated with the ultimate changes of circulating estradiol (E2) and progesterone (P4) levels after administration of hCG. In vitro, when MGCs and COCs were treated with U0126—a specific inhibitor of MAPK3/1 activation—gonadotropin-induced P4 production, 8-Br-cAMP-induced P4 production, and expression of Star and Cyp11a1 mRNA were significantly downregulated, whereas the levels of E2 and Cyp19a1 mRNA in the same samples were significantly upregulated. We conclude that the surge of preovulatory LH induces the differential expression of transcripts encoding key steroidogenic enzymes essential for E2 and P4 synthesis in both cumulus and MGCs, and this process is mediated by the MAPK3/1-dependent pathway.

cumulus cells, granulosa cells, luteinizing hormone, MAPK3/1, ovulation, signal transduction, steroidogenesis, steroidogenic gene expression

INTRODUCTION

In preovulatory follicles, granulosa cells enter a dramatic remodeling process, terminal differentiation, after the LH surge, which results in the onset of luteinization. This process appears to be mediated by complex intracellular signaling pathways that induce divergent changes in granulosa cells, including the distinct patterns of steroidogenic gene expression and steroidogenesis [1]. Specifically, the expression of Cyp19a1 encoding aromatase is rapidly inhibited [2–4], whereas the expressions of Cyp11a1 and Star (encoding cholesterol side-chain cleavage cytochrome P450 and steroidogenic acute regulatory protein, respectively) are induced [3–7]. Accordingly, this change of gene expression in steroidogenic enzymes leads to the shift of steroidogenesis in granulosa cells from 17ß-estradiol (E2) to progesterone (P4) [8, 9]. This transition of steriodigenesis is a hallmark of granulosa cell terminal differentiation and is essential for ovulation and luteinization.

The dynamic changes in steroidogenesis and steroidogenic gene expression in preovulatory follicles after the LH surge have been studied primarily in mural granulosa cells (MGCs), the subpopulation of granulosa cells that line the follicular wall. Whether similar changes also occur in cumulus cells, the subpopulation of granulosa cells surrounding the oocyte, is largely unknown. Although cumulus cells produce steroid hormones, their steroidogenic ability differs largely from MGCs [10–13], due to their direct proximity to the oocyte and interaction with oocyte-derived paracrine factors [12–16]. Cumulus cells express lower levels of CYP11A1 and CYP19A1 than MGCs and, hence, are less active in steroidogenesis [17, 18]. When cultured in the form of cumulus-oocyte complexes (COCs), they produce predominantly E2 but not P4 [12–16]. However, cumulus cells in ovulated COCs express high levels of CYP11A1 and actively synthesize P4 [15, 19, 20], implicating a dramatic change in their steroidogenic characteristics during the ovulatory process. Given that E2 and P4 produced by cumulus cells may contribute to the control of the local steroidogenic environment of the oocyte to promote oocyte maturation and fertilization [21–27], herein we investigated the dynamic changes in the expression of Star, Cyp11a1, and Cyp19a1 transcripts in COCs both in vivo and in vitro, compared with MGCs.

The mitogen-activated protein kinase 3 and 1 (MAPK3/1, also commonly known as extracellular signal-regulated kinase 1&2 [ERK1/2])-dependent pathway has been found to be involved in several key processes induced by the LH surge in preovulatory follicles. These processes include oocyte meiotic resumption [28–32], cumulus expansion [29, 33], gene expression [30, 34, 35], and the disruption of gap junctional communications within the follicle [28, 31]. This indicates that the MAPK3/1-dependent pathway participates in the induction of the ovulatory process. The MAPK3/1-dependent pathway also has been reported as being involved in the regulation of steroidogenesis in granulosa cells; however, its precise role in this process is controversial. Some studies suggest that activation of MAPK is required for promoting steroidogenesis and steroidogenic gene expression in granulosa cells [36–42], whereas other studies indicate that activation of MAPK3/1 suppresses granulosa cell steroidogenesis and steroidogenic gene expression [38, 43–45]. Notably, most of the previous studies use either human luteinizing granulosa cells derived from patients receiving in vitro fertilization treatment or rat granulosa cells isolated from early antral follicles. Therefore, species-specific variations, as well as some other factors, such as the stage of granulosa cell development, may account for these discrepancies. Because the regulation of steroidogenesis and steroidogenic gene expression is a complex process [46, 47], it is necessary to reevaluate the role of MAPK3/1 in the regulation of steriodogenesis in granulosa cells, especially in cumulus cells, using the standard culture system for mouse MGCs and COCs. Given that both the ovulation and the changes in granulosa cell steroidogenesis are major processes induced by the LH surge in preovulatory follicles and the MAPK3/1 pathway mediates several key events during ovulation, we hypothesized that the MAPK3/1-dependent pathway also participates in LH-induced differential regulation of steroidogenesis and steroidogenic gene expression in granulosa cells. We tested this hypothesis using well-established models for the culture of mouse MGCs and COCs isolated from preovulatory follicles of eCG-primed immature mice.

MATERIALS AND METHODS

Mice

Immature 22- to 24-day-old C57BL/6x129 (B6/129) female mice were used for most of the experiments, except in the experiment determining steroid hormone levels, for which (C57BL/6J)xSJL F1 (B6SJL F1) mice were used. B6/129 mice were housed and bred at Stanford University Research Animal Facility. B6SJL F1 mice were obtained from The Jackson Laboratory. All animal protocols were approved by the Administrative Panel on Laboratory Animal Care at Stanford University, and all experiments were conducted in accordance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals.

Isolation of MGCs and COCs

MGCs and COCs were isolated as described previously [48, 49]. Briefly, MGCs and COCs were released into the culture medium by puncturing the large antral follicles with a pair of 27-gauge needles connected to 1-ml syringes. COCs were aspirated using a mouth-controlled, small-bore glass pipette. After removal of COCs, small pieces of MGC clumps were collected and isolated by centrifugation at 1000 rpm for 5 min. Medium used for cell collection and culture was DMEM/F-12 (Invitrogen Corp., Grand Island, NY) supplemented with 500 nM androstenedione, 2 mM L-glutamine, 25 nM sodium selenite, 5 µg/ml transferrin, 3 mg/ml BSA, 75 mg/l penicillin G, and 50 mg/l streptomycin sulfate. All medium supplements were purchased from Sigma (St. Louis, MO).

Collection of MGCs and COCs after hCG Treatment In Vivo

Mice were initially primed with eCG (National Hormone and Peptide Program, NIDDK, Torrance, CA) for 44–46 h by i.p. injection (5 IU per mouse), then injected with hCG (5 IU per mouse; National Hormone and Peptide Program, NIDDK, Torrance, CA) for various times. Samples of COCs and MGCs at the timepoints of 0, 2, 4, and 8 h after hCG injection were collected by puncturing large antral follicles, whereas COCs at 24 h after hCG injection were collected from the oviduct by gently teasing apart the ampulla and releasing the COC mass into the collection medium. Both COCs and MGCs were finally resuspended in RLT buffer (Qiagen Inc, Valencia, CA) containing 1% of ß-mercaptaethanol (Sigma, St. Louis, MO) for RNA isolation. Blood samples were collected accordingly at each timepoint after hCG treatment and clotted at room temperature for about 1.5 h. Sera were isolated by centrifuging the clotted blood at 4600 rpm for 15 min.

In Vitro Culture and Treatment of MGCs and COCs

Both MGCs and COCs were isolated from eCG-primed 22-day-old mice and were cultured in four-well multi dishes (Nunc, Copenhagen, Denmark) at the density of 1 x 105 MGCs or 60 COCs per 250 µl per well. The following treatments were conducted:

  1. To study the kinetics of steroidogenic gene expression in vitro, MGCs and COCs were treated with 100 ng/ml LH or 1 mM 8-Br-cAMP for 4, 8, 24, and 48 h. For each timepoint after culture, cells were collected in RLT buffer containing 1% of ß-mercaptaethanol for RNA isolation and quantification of levels of transcripts encoding key steroidogenic enzymes.
  2. To investigate the effect of inhibition of MAPK activity on steroidogenesis and steroidogenic gene expression in MGCs and COCs, MGCs and COCs were initially treated with 10 µM U0126 or an equal volume of dimethyl sulfoxide (DMSO; vehicle) for 30 min, then treated with 100 ng/ml LH, 100 ng/ml FSH, or 1 mM 8-Br-cAMP, respectively, and cultured for 48 h. At the end of culture, conditioned media were removed and collected for E2 and P4 assays (see below), and cells were collected for RNA isolation and quantification of levels of transcripts encoding key steroidogenic enzymes.
  3. To test the early response of MGCs and COCs to the inhibition of MAPK activity, MGCs and COCs were initially treated with 10 µM U0126 or an equal volume of DMSO (vehicle) for 30 min, then treated with 100 ng/ml LH or 1 mM 8-Br-cAMP and cultured for only 4 h. At the end of culture, cells were collected for RNA isolation and quantification of levels of transcripts encoding key steroidogenic enzymes.

Analysis of Steroid Hormones

The levels of E2 and P4 in mouse serum were measured using the third-generation Estradiol DA RIA kit and the Progesterone EIA kit, respectively, which were obtained from Diagnostic Systems Laboratories, Inc. (Webster, TX). All serum samples were diluted 7.5-fold (for E2) and 5-fold (for P4) prior to analysis, and the levels are presented as the actual concentration (pg/ml and ng/ml for E2 and P4, respectively). The levels of E2 and P4 in the culture medium were measured using enzyme immunoassay kits obtained from Diagnostic Systems Laboratories, Inc., and are presented as the ratio of the ligand- and/or U0126-treated group to the control group (with only DMSO treatment).

Analysis of Steady-State mRNA Levels of Steroidogenic Genes

Isolation and purification of total RNA were accomplished using RNeasy Micro Kits (Qiagen). In vitro transcription was carried out at 37°C for 1 h using the Omniscript RT Kit (Qiagen), in combination with oligo-dT and random hexamer primers purchased from Invitrogen Corp. (Grand Island, NY). Real-time PCR was then conducted to quantify the steady-state mRNA levels of Star, Cyp11a1, and Cyp19a1 using QuantiTect SYBR Green PCR Kits (Qiagen) on the Mx4000 Multiplex Quantitative PCR system (Stratagene, La Jolla, CA). To avoid potential contamination of genomic DNA in the RNA samples, one RNase-free DNase digestion step was incorporated in the RNA isolation process, and primers were designed to amplify a specific fragment spanning two exons. A dissociation curve analysis was performed at the end of the amplification process in order to evaluate the specificity of the PCR products. The same PCR products also were evaluated by agarose gel electrophoresis to confirm that only one unique PCR product was amplified for each gene of interest. Primers used in real-time PCR are shown in Table 1. To determine the efficiency of each gene-specific PCR, standard curves were generated from real-time PCR using a dilution series of cDNA derived from MGCs isolated from large antral follicles after priming with eCG for 44–46 h. Similar PCR efficiencies were observed among the four pairs of primers (97.4%–103.2%). The threshold cycle (Ct) was used to determine relative expression levels, with the housekeeping gene Rpl19 used as the internal control. The expression levels of transcripts are presented as the ratio of treated groups to controls.


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  		TABLE 1. Primers used for real-time PCR.

Statistical Analysis

All experiments were repeated independently at least three times. Data are presented as mean ± SEM. Statistical analysis was conducted using StatView software (SAS Institute, Inc., Cary, NC). Data were first compared by ANOVA, followed by Fisher protected least significant difference post hoc test. P < 0.05 was considered significantly different.

RESULTS

Differential Synthesis of E2 and P4 In Vivo after Administration of hCG

As indicated in Figure 1A, there was a pattern of biphasic E2 production in mouse serum after administration of hCG in vivo. A transient increase in the levels of E2 was detected at 2 h after hCG injection. After the peak at 2 h, the level of E2 decreased over time. At 8 h after hCG administration, the level of E2 in serum was almost undetectable. In contrast to the sharp decrease of serum E2 level at 4–8 h after hCG injection, the levels of P4 at the same timepoints were still significantly higher than the basal level (0 h of hCG injection) after the initial increase at 2 h after hCG injection (Fig. 1B). Despite the initial significant increase in E2 level at 2 h, the ratio of E2:P4 gradually declined over time.


Figure 75060701
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  		FIG. 1. Dynamic levels of E2 (A) and P4 (B) in serum after administration of hCG. At least four mice were used at each timepoint. *P < 0.05 compared to control (time 0) group.

Differential Expression of Transcripts Encoding Key Steroidogenic Enzymes in MGCs and COCs In Vivo after hCG Administration

A time-dependent elevation of steady-state levels of Star and Cyp11a1 mRNA was observed in both MGCs (Fig. 2A) and COCs (Fig. 2C) in vivo after receiving hCG treatment. A significant increase in Star mRNA levels was noted at 4 h after hCG administration in MGCs and COCs, whereas that of Cyp11a1 mRNA began at 8 h after hCG injection. In contrast to the gradual increases of Star and Cyp11a1 mRNA levels, the levels of Cyp19a1 were only transiently elevated in both MGCs and COCs, followed by a rapid decline (Fig. 2, B and D). In both types of cells, the level of Cyp19a1 increased at 2–4 h after hCG injection and dramatically decreased thereafter.


Figure 75060702
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  		FIG. 2. Dynamic changes in the expression of transcripts encoding key steroidogenic enzymes in MGCs (A and B) and COCs (C and D) after administration of hCG in vivo. Mural granulosa cells and COCs were collected at indicated times after hCG injection and the steady-state levels of Star and Cyp11a1 (A, C, E) and Cyp19a1 (B, D, F) mRNA in each individual sample were detected by real-time RT-PCR. Three mice were used at each time point in one experiment, and the same experiment was repeated three times. The mRNA level of each gene was normalized to the level of Rpl19. In A–D, data are presented as the ratio of normalized mRNA levels in the hCG-treated groups to that in the control (time 0) group. In E–F, data are presented as the ratio of the normalized mRNA level in MGCs to that in COCs at each corresponding time point. *P < 0.05, compared to control (time 0) group.

Changes in steroidogenic gene expression were more dramatic in COCs than in MGCs. Before administration of hCG, expression of Star and Cyp11al mRNAs in COCs was extremely low. The levels of these two transcripts in MGCs were 57.3- and 111.1-fold higher than in COCs (Fig. 2E). After treatment with hCG, the differences between MGCs and COCs in the expression of both transcripts were gradually minimized. By 8 h after hCG injection, the differences were diminished to 2.4- and 3.8-fold, respectively. In contrast to the striking differences in the expression of Star and Cyp11al at the time of hCG treatment, the levels of Cyp19a1 mRNA in MGCs were moderately higher (7.2-fold) than in COCs (Fig. 2F). At 4 h after hCG administration, the difference between COCs and MGCs in Cyp19a1 mRNA levels decreased to 4.9-fold, whereas at 8 h it increased to 13-fold, indicating that a more marked change of Cyp19a1 expression occurs in COCs compared with MGCs at these timepoints.

Kinetics of the Expression of Transcripts Encoding Key Steroidogenic Enzymes In Vitro in MGCs and COCs

A time-dependent induction of Star and Cyp11a1 mRNA levels by LH was observed in MGCs in vitro (Fig. 3A). A significant increase in the level of Star mRNA was detected at 4 h after LH treatment, and the highest level was detected at 48 h. Significant induction of Cyp11a1 mRNA was observed at 24–48 h after LH treatment, with slower kinetics compared with Star. In contrast to the elevation of Star and Cyp11a1 mRNA levels, a significant decrease in the levels of Cyp19a1 mRNA was observed in MGCs at all timepoints after LH treatment, with the lowest level detected at 48 h (Fig. 3B).


Figure 75060703
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  		FIG. 3. Dynamic changes in the expression of transcripts encoding key steroidogenic enzymes in MGCs (A, B) and COCs (C, D) in vitro. The steady-state levels of Star (A, C), Cyp11a1 (A, C), and Cyp19a1 (B, D) mRNA in each individual sample were detected simultaneously by real-time RT-PCR. Each experiment was repeated independently three times. The mRNA level of each gene was normalized to the level of Rpl19. Data are presented as the ratio of normalized mRNA levels in LH-treated or 8-Br-cAMP-treated groups to those in control (time 0) groups with no treatment. *P < 0.05 compared to control (time 0) group.

As few if any LH receptors are expressed in COCs, COCs do not respond to LH treatment. However, they do respond to membrane-permeable cAMP analogs or FSH treatment, and, hence, these reagents are commonly used in studies involving COCs [48, 50, 51]. Therefore, in the following studies, FSH and the cAMP analog, 8-Br-cAMP, were used to treat COCs. As shown in Figure 3C, the expression of Star mRNA was time dependently promoted by 8-Br-cAMP. Significant upregulation of Star mRNA was detected at 4 h, with a marked increase at 48 h. The kinetics for the elevation of Cyp11a1 mRNA were relatively slower, with a significant increase observed at 24–48 h after 8-Br-cAMP treatment. Interestingly, the magnitude of the elevation of mRNA levels for both genes in COCs (Fig. 3C) was significantly greater than that in MGCs (Fig. 3A). In contrast to the gradual increase in Star and Cyp11a1 mRNA expression in both MGCs and COCs, a biphasic regulation of Cyp19a1 mRNA expression by 8-Br-cAMP was observed in COCs in vitro (Fig. 3D). A transient increase in Cyp19a1 mRNA was observed between 4 h and 8 h after 8-Br-cAMP treatment, followed by a rapid decrease at 24–48 h. At 24 h and 48 h after 8-Br-cAMP treatment, the level of Cyp19a1 mRNA in COCs declined to barely detectable levels.

Differential Role of MAPK in Regulating Steroidogenesis in MGCs and COCs

To investigate whether the MAPK-dependent pathway participates in regulation of granulosa cell steroidogenesis, the effect of a specific inhibitor of MAPK activation, U0126, on the synthesis of E2 and P4 was studied in cultured MGCs and COCs. In MGCs (Fig. 4A), U0126 significantly inhibited both basal and LH-induced or 8-Br-cAMP-induced P4 production. U0126 also significantly inhibited basal, FSH-induced and 8-Br-cAMP-induced P4 production in cultured COCs (Fig. 4C).


Figure 75060704
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  		FIG. 4. Effects of inhibition of MAPK activity on the production of P4 (A, C) and E2 (B, D) by MGCs (A, B) and COCs (C, D) in vitro. Each experiment was repeated independently three times. Data are presented as the ratio of E2 and P4 levels in ligand- and/or U0126-treated groups to that of the control group with only DMSO treatment. *P < 0.05 compared to non-U0126-treated groups.

In contrast to the inhibition of P4 synthesis, U0126 significantly enhanced the synthesis of E2 in both control and LH-treated or 8-Br-cAMP-treated MGCs (Fig. 4B). U0126 also significantly promoted the production of E2 in control, 8-Br-cAMP-treated, and FSH-treated COCs (Fig. 4D).

Effect of MAPK Inhibitor on the Expression of Transcripts Encoding Key Steroidogenic Enzymes in MGCs and COCs

As differential expression of the three steroidogenic genes studied in granulosa cells was greatly pronounced at 48 h of in vitro culture, and differential regulation of E2 and P4 synthesis by a MAPK-dependent pathway also was observed at this timepoint, the question arises as to whether or not the MAPK-dependent pathway also simultaneously participates in the differential regulation of steroidogenic gene expression. To test this possibility, we first investigated the effect of U0126 on the expression of Star, Cyp11a1, and Cyp19a1 mRNA in MGCs and COCs at 48 h of in vitro culture. As shown in Figure 5, A and B, U0126 dramatically inhibited LH-induced and 8-Br-cAMP-induced elevation of Star and Cyp11a1 mRNA levels in MGCs. Similarly, U0126 also significantly attenuated FSH-induced and 8-Br-cAMP-induced Star and Cyp11a1 mRNA expression in COCs (Fig. 5, D and E). In both cultured MGCs and COCs, U0126 significantly decreased basal levels of Star and Cyp11a1 mRNA. In contrast to the inhibition of Star and Cyp11a1 mRNA levels, U0126 treatment dramatically elevated the expression of Cyp19a1 mRNA in both MGCs and COCs (Fig. 5, C and F). As shown in Figure 5C, U0126 blocked LH-induced and 8-Br-cAMP-induced reduction of Cyp19a1 mRNA levels in MGCs, and the levels of Cyp19a1 mRNA in U0126-treated groups were even higher than the control groups without any treatment. Similarly, in COCs U0126 also blocked FSH-induced and 8-Br-cAMP-induced reduction of Cyp19a1 mRNA levels (Fig. 5F). In both MGCs and COCs, U0126 also increased basal levels of Cyp19a1 mRNA in controls.


Figure 75060705
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  		FIG. 5. Effects of U0126 on the expression of transcripts encoding key steroidogenic enzymes in MGCs (A–C) and COCs (D–F) after 48 h of culture in vitro. The steady-state levels of Star (A, D), Cyp11a1 (B, E), and Cyp19a1 (C, F) mRNA in each individual sample were detected simultaneously by real-time RT-PCR. Each experiment was repeated independently three times. The mRNA level of each gene was normalized to the level of Rpl19. Data are presented as the ratio of normalized mRNA levels in ligand- and/or U0126-treated groups to that of control group with only DMSO treatment. *P < 0.05 compared to the non-U0126-treated group.

Because the earliest changes of steroidogenic gene expression in granulosa cells occurred at 4 h both in vivo and in vitro, it is of interest to know whether this early response of granulosa cells also is dependent on the activation of MAPK. To this end, we studied the effect of U0126 on LH- or cAMP-induced expression of Star and Cyp19a1 in granulosa cells cultured for 4 h. As the expression of Cyp11a1 did not change at this timepoint, we did not examine its expression in response to U0126 treatment. As shown in Figure 6, A and C, LH and 8-Br-cAMP significantly elevated the expression of Star in MGCs and COCs. This stimulatory effect of LH and cAMP was markedly attenuated by U0126. Interestingly, in COCs, 8-Br-cAMP promoted the expression of Cyp19a1, and treatment of U0126 significantly augmented this stimulatory effect (Fig. 6D). LH and U0126 did not significantly affect the expression of Cyp19a1 in MGCs after 4 h of treatment (Fig. 6C).


Figure 75060706
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  		FIG. 6. Effect of U0126 on the expression of transcripts encoding key steroidogenic enzymes in MGCs (A, B) and COCs (C, D) after 4 h of culture in vitro. The steady-state levels of Star (A, C) and Cyp19a1 (B, D) mRNA in each individual sample were detected simultaneously by real-time RT-PCR. Each experiment was repeated independently three times. The mRNA level of each gene was normalized to the level of Rpl19. Data are presented as the ratio of normalized mRNA levels in ligand- and/or U0126-treated groups to that of control group with only DMSO treatment. Bars with different letters indicate significant difference, P < 0.05.

DISCUSSION

Data presented here using the mouse model confirmed previous findings in rat [2–7] and other mammalian species [52–56] showing that the LH surge induces the downregulation of Cyp19a1 and the upregulation of Cyp11a1 and Star in MGCs. More importantly, the data indicated similar changes in the expression of Cyp19a1, Cyp11a1, and Star transcripts in COCs after the LH surge, despite the many known characteristic differences between cumulus cells and MGCs. The differential expression of Cyp19a1, Cyp11a1, and Star transcripts in both MGCs and COCs correlated with the ultimate changes in levels of E2 and P4 in circulation.

The immediate changes in levels of E2 and P4 in serum after hCG administration preceded the changes in levels of Cyp19a1, Star, and Cyp11a1 transcripts in both MGCs and COCs, suggesting the initial changes of steroids in circulation are not dependent on the change of these transcripts in granulosa cells. This early change in E2 and P4 levels is probably caused by the immediate inhibition of CYP17A1 and augmentation of CYP11A1 activity, respectively, in theca cells by the LH surge, as reported previously [56–59]. Nevertheless, our data indicate that the differential expression of Cyp19a1, Cyp11a1, and Star transcripts in both MGCs and COCs correlates well with the subsequent changes in circulating steroids, and, hence, marks the terminal differentiation of both types of granulosa cells.

Differential expression of Cyp19a1, Cyp11a1, and Star transcripts in both MGCs and COCs also occurred in vitro after treatment with LH or 8-Br-cAMP. However, the transient increase in the level of Cyp19a1 transcript in MGCs as observed in vivo within 4 h of hCG administration was not observed in vitro. This could be caused by the loss of paracrine factors after releasing MGCs from the follicle that contributes to the transient elevation of Cyp19a1 within the follicle.

The dramatic upregulation of Cyp11a1 and Star transcripts in COCs indicates that cumulus cells acquire vigorous P4-producing capacity after the LH surge. The similar observation has been reported recently by Richards' group [34, 51]. This suggests that, in addition to the well-known phenomenon of cumulus expansion, cumulus cells also undergo another remarkable remodeling process after the LH surge, becoming more prone to producing P4. Indeed, cumulus cells of ovulated COCs have been found to produce high levels of P4 [15, 20, 60]. Because cumulus cells do not luteinize, P4 produced by cumulus cells must play a unique role that differs from MGCs. A recent study by Jamnongjit et al. [61] suggests that P4 produced by cumulus cells and MGCs may function as a paracrine factor to mediate LH-induced oocyte meiotic resumption in the mouse. However, given that mouse oocyte meiotic resumption normally starts at 2–4 h after the LH surge, which, as demonstrated here, precedes the apparent upregulation of Cyp11a1 and Star transcripts in COCs and MGCs, it is unlikely that P4 produced by cumulus cells and MGCs plays such a physiologic role. Nevertheless, P4 produced by cumulus cells may promote oocyte cytoplasmic maturation and fertilization [21–27]. It may also function as an autocrine factor to regulate the function of cumulus cells during the periovulatory period [51].

We and others have previously demonstrated the activation of MAPK3/1 in MGCs and cumulus cells after administration of hCG in vivo or treatment with gonadotropins and cAMP analogs in vitro [28–31, 62, 63]. Activation of MAPK3/1 in both MGCs and cumulus cells, as well as oocyte meiotic resumption and cumulus expansion induced by gonadotropins or 8-Br-cAMP, was effectively blocked by 10 µM U0126, a specific inhibitor of MAPK3/1 activation [28–31, 62, 63]. These observations indicate that the MAPK3/1-dependent pathway in granulosa cells, presumably in cumulus cells, mediates the LH-induced early processes during ovulation (i.e., maturation of COCs). Here, using the same culture system of COCs and MGCs, we demonstrated that the same dose of U0126 (10 µM) inhibited gonadotropin-induced or 8-Br-cAMP-induced differential synthesis of E2 and P4, as well as differential expression of Cyp19a1, Cyp11a1, and Star. This suggests that the MAPK3/1-dependent pathway also participates in the LH-induced late process during ovulation (i.e., the terminal differentiation of granulosa cells). Therefore, further evidence is provided in support of the role of MAPK3/1-dependent pathway in propagating LH signaling in the preovulatory follicle. Although here we only addressed the MAPK3/1-dependent pathway, some other pathways, such as protein kinase A and MAPK14 (also commonly known as p38MAPK) pathways, may also participate in this process. As suggested by a recent study in Richards' group [51], all three pathways may be involved in regulating the function of COCs during the ovulatory process, and potential crosstalk may exist. Thus, further studies are necessary to unveil other potential pathways involving in this process and its interactions with the MAPK3/1 pathway.

The role of MAPK3/1 in the regulation of steroidogenesis in granulosa cells appears controversial in the literature. Consistent with the results presented here, studies in rats using primary cultures of granulosa cells isolated from early antral follicles demonstrated that inhibition of MAPK3/1 activation attenuated FSH-induced P4 secretion [38, 39, 41, 42] and Star expression [38, 39, 42] but enhanced E2 production and Cyp19a1 expression [38]. Similar observations were made in a study using short-term cultured human granulosa-luteal cells, where inhibition of MAPK3/1 activation blocked cAMP- and PGE2-induced P4 production [36]. However, contradictory results were shown in studies using immortalized granulosa cell lines and long-term cultured human luteinized granulosa cells, where inhibition of MAPK3/1 activity potentiated gonadotrophin-induced elevation of Star mRNA and P4 levels [43, 44]. Because different species (i.e., rodents vs. humans), as well as different culture systems (i.e., primary short-term vs. cell line or long-term culture), were used in different studies, interpretation of these discrepancies needs to be made cautiously.

Because few, if any, LH receptors are expressed in COCs, changes in COCs during the ovulatory process are likely not induced directly by LH. Studies by Conti's group and others indicated that the epidermal growth factor (EGF)-like factors (i.e., amphiregulin, epiregulin, and betacellulin) are probably the paracrine factors that propagate LH signaling to COCs [64, 65]. Most intriguingly, a very recent study by Richards' group [51] demonstrated that these EGF-like factors induce the expression of Star and Cyp11a1 and the activation of MAPK3/1 in mouse COCs. These studies suggest that EGF-like factors are probably the intermediary mediators of the LH-induced MAPK3/1 activation, as well as the subsequent key processes in COCs (i.e., oocyte meiotic resumption, cumulus expansion, and terminal differentiation [acquisition of high steroidogenic activity]).

In summary, cumulus cells undergo remarkable changes in their steroidogenic capabilities after the LH surge, as demonstrated by the dramatic upregulation of Star and Cyp11a1 and the simultaneous downregulation of Cyp19a1 transcripts. These changes are comparable to those in MGCs. This differential expression of Cyp19a1, Star, and Cyp11a1 transcripts in both cumulus cells and MGCs is correlated with ultimate changes in circulating E2 and P4 levels and is mediated by the MAPK3/1-dependent pathway.

ACKNOWLEDGMENTS

We are grateful to Suzanna Tulac and Amy Hamilton for the assistance in using the Mx4000 Multiplex Quantitative PCR system.

FOOTNOTES

3Current address: The Jackson Laboratory, 600 Main St., Bar Harbor, ME 04609. Back

4Current address: Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10, DK-8000 Aarhus C, Denmark. Back

5Current address: Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing 100083, China. Back

1Supported by National Institute of Child Health and Human Development grant HD31579 to L.C.G. and by fellowships from Diagnostic Systems Laboratories, Inc., to M.N. and M.T.O. Back

Correspondence: 2 Linda C. Giudice, Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco, 505 Parnassus Ave., M1496, Box 0132, San Francisco, CA 94143-0132. FAX: 415 476 1811; e-mail: giudice{at}obgyn.ucsf.edu

Received: 20 March 2006.

First decision: 10 April 2006.

Accepted: 16 August 2006.

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