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Division of Reproductive Biology (S.P., M.H., M.F., M.C.), Department of Obstetrics and Gynecology, Stanford, University School of Medicine, Stanford, California 94305; and Department of Medical Sciences (S.P., L.P.), University of Milan, Lab of Experimental Endocrinology, Istituto di Ricovero e Cura a Carattere Scientifico, Istituto Auxologico Italiano, Milano 20095, Italy
Address all correspondence and requests for reprints to: Marco Conti, M.D., Professor, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California at San Francisco, 513 Parnassus, San Francisco, California 94143-0556. E-mail: ContiM{at}obgyn.ucsf.edu.
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ABSTRACT |
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protease inhibitor-1 (TAPI-1), suggesting the involvement of EGF-like growth factor shedding. LH- but not amphiregulin-induced oocyte maturation and EGFR phosphorylation were sensitive to protein synthesis inhibition. When granulosa cells were cultured with a combination of neutralizing antibodies against amphiregulin, epiregulin, and betacellulin, EGFR phosphorylation and MAPK activation were inhibited. In cultured follicles, LH-induced MAPK activation was partially inhibited by AG1478 and GM6001, indicating that this pathway is regulated in part by the EGF network but also involves additional pathways. Thus, complex mechanisms are involved in the rapid amplification and propagation of the LH signal within preovulatory follicles and include the early activation of the EGF network. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In recent years, members of the epidermal growth factor (EGF)-like growth factor family have emerged as likely mediators of LH action in the follicle. Specifically, amphiregulin (AREG), epiregulin (EREG), and betacellulin (BTC) are rapidly induced by LH or its analog human chorionic gonadotropin (hCG) (5, 6, 7) and are thought to function in an autocrine and paracrine manner to propagate LH signals throughout the preovulatory follicle. In vitro, these growth factors promote many events that are stimulated by LH, including cumulus expansion and oocyte meiotic resumption (5, 6). The growth factor effects require EGF receptor (EGFR, also called ErbB1) activity, because EGFR tyrosine kinase inhibitors block both processes (5, 6), as well as several other gonadotropin-dependent functions of granulosa cells (8, 9, 10, 11). Recently, we reported that disruption of the EGF signaling network in mice impairs LH-induced ovulation, demonstrating the importance of this pathway to LH action also in vivo (12). LH-induced EGFR phosphorylation was significantly reduced in POFs from Areg–/– Egfrwa2/wa2 double-mutant mice. This decrease in LH-induced EGFR phosphorylation was associated with impaired oocyte meiotic resumption in hCG-primed Areg–/– Egfrwa2/wa2 mouse ovaries in vivo.
The EGF-like growth factors are produced as transmembrane precursors that are cleaved at the cell surface by members of the matrix metalloprotease (MMP) or a disintegrin and metalloprotease (ADAM) families (13, 14). The mature growth factors bind to dimers of EGFR family members, resulting in activation of intrinsic receptor tyrosine kinase activity and autophosphorylation of specific tyrosine residues in the cytoplasmic domain. Target proteins are subsequently phosphorylated and/or recruited for the activation of downstream signaling cascades, including the MAPK pathway, to elicit distinct biological effects.
GPCRs also can activate the MAPK cascade via Gβ
-c-Src (15) or effectors such as phosphatidylinositide-3-kinase or PKC (16, 17). In addition, GPCR-induced transactivation of the EGF signaling network has been linked to activation of the MAPK cascade (18, 19). Early studies described an intracellular mechanism for GPCR-mediated EGFR transactivation, involving the intracellular mediators Src, PKC, or Ca2+ (20, 21, 22, 23, 24, 25, 26). However, later findings have also documented a ligand-dependent mechanism of EGFR transactivation. In brief, agonist-stimulated GPCR signaling has been shown to induce metalloprotease-dependent shedding of EGF-like growth factors and EGFR activation (27, 28). This mechanism of GPCR-induced EGFR transactivation has been described as the triple-membrane-passing signal (27).
Because the exact timing of EGFR transactivation in relationship to oocyte reentry into the cell cycle has not been established, we sought to investigate the dynamics of LH regulation of the EGF network in cultured mouse POFs. Here, we show that LH induces EGFR phosphorylation within 30 min of stimulation. Based on this observation, we hypothesized that the early activation of EGFR induced by LH may involve shedding of a preexisting or rapidly formed pool of EGF-like growth factors. We provide evidence that the early LH-induced EGFR transactivation is likely mediated by cAMP and PKA signaling, involves metalloprotease activity, and impacts the MAPK cascade. This rapid activation of the EGF network serves as a means for amplifying the initial LH signal, and propagating LH effects throughout the POF.
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).
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). Expression of both genes was maximal after 2 h stimulation and decreased at 4 h. This pattern of LH-induced Areg and Ereg expression in POFs is consistent with the results of earlier studies showing that LH/hCG induces the rapid and transient expression of Areg and Ereg mRNAs in wild-type mouse ovaries (5) and rat follicles (6) within 3 h of stimulation. Interestingly, the early induction of Areg and Ereg expression and EGFR activation by LH precedes the onset of oocyte meiotic resumption by at least 1 h (Fig. 1C).
Protein Synthesis Is Required for LH-Induced Oocyte Maturation
If de novo EGF-like protein synthesis were required for LH transactivation of EGFR and oocyte maturation, then inhibition of protein synthesis should block the LH- but not AREG-mediated effects. POFs were incubated in the presence of either protein synthesis inhibitor puromycin or cycloheximide before stimulation by either rLH or AREG. After 4 h, oocytes were examined for germinal vesicle breakdown (GVBD), a hallmark of oocyte meiotic resumption. In this follicle culture model, rLH or AREG stimulation resulted in approximately 80–90% GVBD (Fig. 2A). In agreement with previous reports, puromycin concentrations that decrease amino acid incorporation by more than 90% (see Materials and Methods) significantly inhibited rLH-induced GVBD in cultured follicles (
70% inhibition), indicating that protein synthesis is necessary for the LH effect. Conversely, inhibition of AREG-induced GVBD was minimal (Fig. 2A). Treatment with a different translation inhibitor, cycloheximide, alone promoted oocyte meiotic resumption (data not shown), confounding interpretation of the experimental results. The effect of this latter inhibitor was not further investigated.
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). Puromycin had no significant effect on AREG-stimulated EGFR phosphorylation even at high doses. These data are consistent with the view that LH-induced synthesis of AREG (and/or additional EGF-like growth factors) and EGFR activation are required for oocyte meiotic resumption.
LH-Induced MAPK1/3 Activation Is Dependent in Part on EGFR Activity
One of the pathways downstream of the EGFR activation implicated in oocyte meiotic resumption is the MAPK signaling cascade. Therefore, the time course of MAPK1/3 (also called ERK1/2) phosphorylation was examined in cultured follicles by Western blot analyses with phospho-ERK1/2-specific antibodies. In POFs, an increase in MAPK phosphorylation was induced by rLH within 30 min, and phosphorylation was maximal after 2 h of culture (Fig. 3A). Increased MAPK phosphorylation was also detected in cumulus-oocyte complexes (COCs) isolated from POFs 30 min after LH stimulation (data not shown), consistent with previous findings showing MAPK activation in cumulus cells isolated 30 min after hCG stimulation in mice, with maximal activation 2–4 h after hCG (32).
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). After 4 h hCG stimulation, signal for phosphorylated MAPK remained elevated in cumulus cells but appeared reduced in mural granulosa cells.
To understand the mechanisms underlying the rapid propagation of the LH signal within ovarian POFs, the involvement of LH-induced EGFR activation in the early activation of MAPK was investigated. When POFs were preincubated with the EGFR tyrosine kinase inhibitor AG1478, rLH-induced EGFR phosphorylation was significantly inhibited after 30 min (Fig. 4, A and E), demonstrating the effectiveness of this concentration of AG1478 (0.5 µM) in blocking the LH-induced EGFR tyrosine kinase activity. Next, activation of the downstream MAPK signaling cascade was examined. As shown in Fig. 3A, rLH stimulated an increase in MAPK1/3 phosphorylation in cultured POFs within 30 min (Fig. 4B). However, pretreatment of POFs with AG1478 resulted in approximately 60% inhibition of LH-induced MAPK1/3 phosphorylation (Fig. 4, B and E). In addition, when MAPK phosphorylation was examined in POFs from wild-type, Areg+/+ Egfrwa2/wa2, and Areg–/– Egfrwa2/wa2 mice cultured without or with rLH for 2 h, rLH-induced MAPK activation was significantly reduced in the Areg–/– Egfrwa2/wa2 follicles but not completely prevented (Fig. 5). Taken together, these results suggest that regulation of the MAPK signaling pathway is dependent in part on EGFR activity but likely involves additional LH-regulated intracellular pathways.
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, C and E). Similarly, preincubation of POFs in the presence of TAPI-1, a metalloprotease inhibitor with some specificity for TNF converting enzyme/ADAM17, also blocked rLH-induced EGFR phosphorylation (data not shown). Consistent with their effects on EGFR activation, GM6001 and TAPI-1 inhibited LH-induced MAPK1/3 phosphorylation by about 50% (Fig. 4, D and E, and data not shown). In addition, GM6001 and TAPI-1 each inhibited LH-induced oocyte meiotic resumption in cultured follicles by at least 66%; in contrast, GM6001 failed to inhibit oocyte maturation induced by EREG (data not shown). Thus, metalloprotease activity involved in EGF-like growth factor shedding is required for EGFR activation and signaling.
LH-Mediated EGFR Transactivation Involves cAMP and PKA Signaling
To determine whether EGFR transactivation induced by LH is mediated by cAMP signaling, POFs were stimulated with forskolin (Fsk), a direct activator of adenylyl cyclase. Fsk promoted an increase in EGFR phosphorylation to levels comparable to those observed with rLH after 30 min (Fig. 6A). In addition, Fsk-induced EGFR phosphorylation was completely inhibited by both AG1478 and GM6001, suggesting that the cAMP cascade lies upstream of both matrix metalloprotease activity and EGFR transactivation.
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). Thus, LH activation of cAMP signaling is not impaired by these inhibitors under the conditions used, and CREB is not a downstream target of the EGFR signaling pathway.
The involvement of PKA downstream of cAMP in LH-induced EGFR transactivation was examined by incubation with the PKA inhibitor H89. When POFs were exposed to H89, rLH-stimulated EGFR phosphorylation was significantly inhibited (Fig. 6C), indicating a role for PKA in LH-stimulated EGFR transactivation.
EGF-Like Growth Factors Implicated in Early LH Signaling
In an independent strategy to define the involvement of EGF-like growth factors in the rapid LH-induced EGFR transactivation and activation of downstream signaling, POFs were cultured in the presence of neutralizing antibodies against AREG, EREG, and BTC for 30 min before rLH stimulation. A combination of the three antibodies was used based on the likely redundant functions of these growth factors (12). As a control, POFs were cultured in the presence of mouse IgG1. In this experimental paradigm, no effect of the neutralizing antibodies on GVBD was observed after 4 h, possibly due to an inefficient ability of the antibodies to concentrate in the follicle (data not shown). To circumvent this problem, the effects of the three neutralizing antibodies on LH-induced EGFR phosphorylation was examined in cultured granulosa cells. After 30 min preincubation with the antibodies, cells were stimulated with vehicle or Fsk for 30 min. Fsk stimulated EGFR phosphorylation, and this effect was completely inhibited by the neutralizing antibodies but not by the nonspecific IgG1 antibody (Fig. 7A). Fsk-induced MAPK1/3 phosphorylation was also significantly inhibited in granulosa cells preincubated with the combination of AREG, EREG, and BTC neutralizing antibodies (Fig. 7B). Taken together, the results suggest that one or more of the EGF-like growth factors are involved in the early EGFR phosphorylation and MAPK activation that occur downstream of LH and cAMP signaling in vivo.
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DISCUSSION |
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Using the in vitro POF model, we establish that LH induces the rapid expression and activation of components of the EGF network in follicular cells. LH or Fsk induced EGFR phosphorylation as early as 30 min after stimulation. Although small compared with the large increase in EGFR phosphorylation that occurs after 2–4 h exposure to LH, this early increase is significant. This transactivation of EGFR occurs at the same time that small but significant increases in Areg and Ereg mRNAs are detected by RT-PCR under the same culture conditions. Previous studies have also shown Fsk stimulation of Areg mRNA in COCs (33) and Areg and Ereg mRNAs in human granulosa cells (34). We also show that LH- and Fsk-induced EGFR transactivation is sensitive to metalloprotease inhibitors, suggesting the involvement of cAMP signaling and a requirement for shed EGF-like growth factors. Indeed, Fsk-induced EGFR phosphorylation was prevented in cultured granulosa cells by neutralizing antibodies against the AREG, EREG, and BTC growth factors. Given the rapid phosphorylation of one of the major EGFR autophosphorylation sites (Y1068), these results argue for a ligand-dependent rather than an intracellular-dependent mechanism of EGFR transactivation. Although the intracellular mediator Src has been reported to phosphorylate the EGFR autophosphorylation sites in addition to distinct residues (35), LH-induced phosphorylation of the Y1068 residue is blocked when EGF-like growth factor processing or availability is inhibited.
When POFs were cultured in the presence of a protein synthesis inhibitor, LH-induced but not AREG-induced EGFR phosphorylation was inhibited. In a similar fashion, LH-induced but not AREG-induced oocyte maturation was prevented. A likely explanation of this finding is that LH action requires de novo synthesis of EGF-like growth factors. That EGFR phosphorylation is indispensable for LH-stimulated oocyte maturation is indicated by pharmacological inhibition with the different inhibitors used in this study and by the analyses of oocyte maturation in follicles with genetic disruptions in the EGF network (12). In both cases, a decrease or absence of EGFR phosphorylation was associated with a failure of LH to induce oocyte maturation. That LH still produces a robust signal under these conditions is indicated by the effect of LH on CREB phosphorylation and MAPK activation. Based on the above findings, we propose that LH signaling branches to activation of the EGF network through complex mechanisms during the ovulatory process. Initially, transactivation occurs either through very rapidly synthesized growth factors or mobilization of a small preformed pool of EGF-like growth factors that may not depend on de novo synthesis. This pool could not be detected by fluorescence-activated cell sorting analysis of granulosa cells before exposure to LH (5). This initial small activation is then greatly amplified and stabilized by the de novo synthesis of EGF-like growth factors as we have previously described.
With this study, we also show that LH-induced EGFR transactivation impacts the MAPK cascade, consistent with the previous observation that AREG induces MAPK phosphorylation in COCs (33). LH-induced MAPK activation is diminished but not abolished when EGFR phosphorylation is blocked either by an EGFR tyrosine kinase inhibitor or inhibitors of EGF-like growth factor shedding or action. The finding that substantial MAPK activation is observed in Areg–/– Egfrwa2/wa2 follicles is consistent with the pharmacological data. The incomplete inhibition of MAPK activity in the absence of EGFR phosphorylation suggests that additional pathways contribute to the LH-mediated activation of MAPK in the POF. These may be intracellular pathways that may be directly downstream of cAMP. It is possible that additional mechanisms are also operating to regulate MAPK activity at times earlier than 30 min LH stimulation. Regardless of these mechanisms, both the pharmacological and genetic manipulations define an experimental paradigm whereby oocyte maturation does not occur when cAMP accumulation is induced by LH (12) and when MAPK activation is still substantial in the follicle. This finding suggests that neither cAMP nor MAPK activation by itself is sufficient to signal oocyte maturation unless the EGF network is activated. This observation is consistent with reports showing that MAPK is necessary but not sufficient to induce oocyte maturation. MAPK kinase inhibitors prevent LH-induced oocyte maturation in follicles or FSH-induced maturation in COCs (32, 36, 37); however, activation of MAPK in cumulus cells by recombinant growth and differentiation factor 9 alone or in combination with FSH in the presence of hypoxanthine fails to promote oocyte maturation in COCs (38). Thus, synergism between several pathways yet to be identified is likely required.
All the pharmacological or genetic manipulations that interfere with activation of the EGF network prevent LH-mediated induction of oocyte meiotic resumption. These observations reinforce our hypothesis that transactivation of the EGFR mediates the signal that induces oocyte meiotic resumption. The detailed time course of EGFR phosphorylation documents that the timing of this activation is compatible with the proposed mediatory role in downstream events. The EGFR phosphorylation observed after 30–60 min of LH stimulation precedes oocyte GVBD by at least 2 h. This time interval is identical to the time required for EGF-like growth factors to induce maturation in the POF in vitro (5). Assuming that the time required for the oocyte to process the maturation stimulus into GVBD requires 60 min, as inferred from spontaneous maturation, there is an interval of 1 h between the EGFR phosphorylation and the trigger of the signal for oocyte maturation. Some of this time may be required to propagate the signal from mural to cumulus cells and for amplification of the signal. However, the exact steps that are activated during this window of time in somatic cells and that are required to signal oocyte reentry into the cell cycle remain elusive. Connexin phosphorylation by PKA or MAPK and closure of gap junction communication between the somatic cells and the oocyte is a possible candidate step (39). This closure should lead to a decrease in the diffusion of any inhibitory signals. However, MAPK is still substantially activated when EGFR phosphorylation is prevented, and LH-mediated activation of PKA still occurs in view of the phosphorylation of CREB. Under these conditions, oocyte maturation is not induced. Pending additional experiments directly probing the junctional permeability in follicles where EGFR activation is prevented, connexin phosphorylation should still occur in view of the increase in cAMP and MAPK activation. It is possible that additional signals downstream of EGFR phosphorylation are indispensable for meiotic maturation. Indeed, EGFR activation produces signaling through a plethora of cascades that include activation of the Ras-MAPK, c-Jun N-terminal kinase (JNK), Src, phosphatidylinositide-3-kinase, phospholipase C, signal transducer and activator of transcription (STAT), and Cbl pathways, as well as regulation of cytoskeleton modification, cell adhesion, and lipid mediator production (14, 40, 41). Thus, each one or a combination of these pathways may be involved in the final signals for oocyte meiotic resumption.
In conclusion, we demonstrate the rapid transactivation of the EGF network by LH in POFs that likely serves to amplify the initial LH signal, thus propagating the LH effects within the follicle. This early transactivation of the EGF network may be a critical signal for oocyte maturation. These findings provide new insight into the mechanisms regulating oocyte meiotic resumption and have important implications for improving the in vitro maturation of oocytes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Animals
Immature (22–24 d old) female C57BL/6 mice (Charles River Laboratories, Wilmington, MA) were injected ip with 5 IU PMSG to stimulate follicle development to the preovulatory stage. After 44–48 h, animals were euthanized and ovaries excised. For immunohistochemical analyses, PMSG-primed mice were injected ip with an ovulatory dose of 5 IU hCG, and ovaries were excised at selected times after injection. Areg+/+ Egfr+/+, Areg+/+ Egfrwa2/wa2, and Areg–/– Egfrwa2/wa2 mice were generated and genotyped as previously described (12). All animal procedures were approved and followed the guidelines of the Stanford University Administrative Panel on Laboratory Animal Care.
RNA Extraction and Semiquantitative RT-PCR
Total RNA was extracted from POFs cultured in the presence of 5 IU rLH for 0, 0.5, 1, 2, or 4 h using Trizol Reagent (Invitrogen) as instructed by the manufacturer. Total RNA (100 ng) was reverse-transcribed using 1x Thermocycle buffer, 3.75 mM MgCl2, 1 mM dNTPs (Promega Corp., Madison, WI), 500 ng poly-dT (Amersham Pharmacia, Newark, NJ), and 2.5 U avian myeloblastosis virus-reverse transcriptase (Promega) at 42 C for 75 min and 95 C for 5 min. Afterward, one fourth of each RT reaction was used for PCR amplification of the genes of interest, and 0.1 µM of each primer, 0.25 µCi [32P]dCTP (PerkinElmer, Boston, MA), 0.625 U Taq polymerase in 1x Thermocycle buffer, and 2 mM MgCl2 (Promega Corp.) were used in each reaction. PCR conditions were 94 C for 2 min followed by cycles of 94 C for 30 sec, 60 C for 45 sec, and 72 C for 1 min and a final extension at 72 C for 7 min. Specific primers used were as follows: Areg forward, 5'-CACAGCGAGGATGACAAGGACC-3'; Areg reverse, 5'-AATGTCATTTCCGGTGTGGCTTGG-3'; Ereg forward, 5'-CATGGAGTCTGACTCTAGTC-3'; Ereg reverse, 5'-TCAGTGACTCACGAAGACCT-3'; and L19 (42, 43). The amplified PCR products were resolved on 5% polyacrylamide gels. The gels were dried, and radioactive bands were quantified using the Storm 840 PhosphorImager and ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA).
POF Culture
POFs were microdissected from PMSG-primed mouse ovaries under a stereomicroscope, in Leibowitz’s L15 medium supplemented with 5% FBS, 100 U penicillin, and 100 µg streptomycin. At least 30 follicles per treatment group were cultured in 1 ml MEM supplemented with 10% FBS and antibiotics at 37 C in glass vials under 95% O2/5% CO2. After equilibration, POFs were cultured in the absence or presence of 5 IU rLH or 50 µM Fsk. At the end of culture, POFs were washed in DPBS and collected for protein extraction. In some experiments, POFs were preincubated in the presence of the inhibitors AG1478 (0.5 µM), galardin (20 µM), TAPI-1 (30 µM), puromycin (10 µg/ml), H89 (20 µM), or vehicle [dimethylsulfoxide (DMSO)] for 30 min before hormone stimulation.
For oocyte maturation studies, POFs were cultured in the presence of 5 IU rLH for the indicated times. POFs preincubated in the presence of puromycin were stimulated for 4 h with 5 IU rLH or 100 nM AREG. Follicles were punctured using 26.5-gauge needles (Becton Dickinson & Co., Franklin Lakes, NJ) to release COCs. Cumulus cells were gently removed using a Drummond microdispenser, and oocytes were examined for evidence of GVBD, a hallmark of oocyte meiotic resumption. COCs that were collected 4 h after culture were treated briefly with hyaluronidase (1µg/ml) to facilitate removal of cumulus cells from oocytes.
Granulosa Cell Culture
Granulosa cells were isolated from ovaries of immature mice 46 h after PMSG stimulation by needle puncture in Leibowitz’s L15 medium supplemented with 5% FBS, penicillin, and streptomycin. Cells were passed through a 40-µm cell strainer to remove oocytes, spun down, and resuspended in DMEM/F12 medium supplemented with 1% FBS, penicillin, and streptomycin. Cells were seeded at a density of about 5–6 x 106 cells per well in six-well culture plates. On the next day, cells were cultured in 1 ml serum-free DMEM/F12 containing vehicle (DMSO) or 10 µM Fsk, an activator of adenylyl cyclase. At the end of culture, cells were washed once with DPBS and then collected in RIPA buffer [50 mM Tris (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.25% deoxycholic acid, Complete, Mini, EDTA-free protease inhibitor cocktail, 1 mM EDTA, 1 mM Na-orthovanadate, 1 mM NaF, and 1 mM Na-pyrophosphate] for protein extraction. In some experiments, antimouse IgG1 or neutralizing antibodies against AREG (3 µg/ml), EREG (2.5 µg/ml), and BTC (2 µg/ml) were added to culture 30 min before treatment with Fsk.
Immunoprecipitation and Immunoblotting
Cultured POFs or granulosa cells were homogenized and lysed in ice-cold RIPA buffer containing a cocktail of EDTA-free protease inhibitors, 1 mM EDTA, 1 mM Na-orthovanadate, 1 mM NaF, and 1 mM Na-pyrophosphate. After centrifugation at 14,000 x g for 5 min at 4 C, supernatants were collected and protein concentrations assayed using the BCA protein assay kit (Pierce). A fixed amount of protein (140–190 µg) was immunoprecipitated by incubation with 4 µg anti-EGFR antibody overnight at 4 C, followed by 2.5 h incubation with rec-Protein G-Sepharose 4B beads. After centrifugation, lysates were retained for additional assays. Pellets were washed in DPBS containing protease inhibitors, and then bound EGFR proteins were released from the pellets by boiling for 5 min in 3x SDS buffer. The eluted samples were separated on 7.5% polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Membranes were blocked in Tris-buffered saline with 0.1% Tween 20 (TBST) plus 5% nonfat dry milk, washed with TBST, probed with an anti-phospho-EGFR antibody (specific for autophosphorylation site Tyr1068) diluted 1:1000 in TBST plus 5% BSA (Sigma) overnight at 4 C, and then incubated for 1 h at room temperature with an antirabbit IgG horseradish peroxidase (HRP)-conjugated antibody (Amersham) diluted in TBST plus 0.5% nonfat dry milk (Bio-Rad). After washing in TBST, membranes were incubated with ECL Plus Western blotting reagent for detection of specific signal.
Afterward, the same membranes were incubated in a solution containing 62.5 mM Tris-HCl (pH 6.8), 2% SDS, and 100 mM β-mercaptoethanol for 30 min at 50 C, washed in TBST, and reprobed for total EGFR. Membranes were blocked for 1 h at room temperature in TBST plus 1% nonfat dry milk, incubated with a polyclonal anti-EGFR antibody diluted 1:200 in TBST plus 0.2% nonfat dry milk for 2 h at room temperature, washed in TBST, and then incubated for 1 h at room temperature with an antirabbit IgG HRP-linked antibody diluted 1:5000 in TBST plus 0.2% nonfat dry milk. After washing in TBST, membranes were incubated with ECL Western blotting reagent for detection of specific signal.
MAPK (p44/42) and CREB phosphorylation levels in cultured POFs were analyzed in protein lysates retained after EGFR immunoprecipitation. Briefly, 2 or 5 µg lysate, respectively, were separated on 10% polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Membranes were blocked in TBST plus 0.2% nonfat dry milk, incubated for 2 h at room temperature with anti-phospho-p44/42 MAPK (1:1000) or anti-phospho-CREB (1 µg/ml) antibody diluted in TBST plus 0.2% nonfat dry milk, washed in TBST, and then incubated for 1 h at room temperature with antirabbit IgG-HRP (1:5000) or antimouse IgG-HRP antibodies (1:3000), respectively. Specific signals were detected using ECL reagent. Afterward, membranes were stripped of bound antibodies as described above, blocked in TBST plus 1% nonfat dry milk, and then incubated with anti-pan-ERK or anti-ERK1 (diluted 1:2500 in TBST plus 0.2% nonfat dry milk) for 2 h at room temperature or with anti-CREB (diluted 1:1000 in TBST plus 0.2% nonfat dry milk) overnight at 4 C. After washing in TBST, membranes were incubated for 1 h at room temperature with antimouse IgG-HRP (1:5000) or antirabbit IgG-HRP (1:3000), respectively. Specific signals were detected using ECL Western blotting reagent and visualized by autoradiography. For Western blots, specific bands were quantified by scanning and densitometric analyses using Scion Image software (Scion Corp., Frederick, MD).
Metabolic Labeling
Intact POFs isolated from PMSG-primed mice were washed three times in methionine-free DMEM (GIBCO), supplemented with 10% dialyzed FBS, 100 U penicillin, 100 µg streptomycin, and 2 mM L-glutamine (GIBCO). Three follicles were then preincubated for 15 min in this medium with 50 µCi/ml [35S]methionine (Amersham) to deplete intracellular pools of methionine. The follicles were incubated for an additional 30 min in the absence or presence of puromycin and then stimulated with 5 IU rLH for 4 h. After the incubations, follicles were washed twice in DPBS and homogenized on ice in 500 µl 5% (wt/vol) trichloroacetic acid solution. After centrifugation at 3000 rpm for 30 min at 4 C, the pellets were resuspended in 1 ml 5% trichloroacetic acid solution and centrifuged again at 3000 rpm for 30 min at 4 C. Afterward, the pellet was resuspended with 200 µl DPBS. The supernatant was reserved for use as a control. After vortexing and sonication of the samples to break down the cells, the amount of [35S]methionine incorporation was counted.
Immunohistochemistry
Excised ovaries were fixed in Bouin’s solution overnight at 4 C. After dehydration, the ovaries were embedded in Paraplast and sectioned at 6 µm onto Superfrost Plus slides. For immunohistochemical staining, sections were deparaffinized and rehydrated, treated with 3% H2O2 for 15 min to inactivate intrinsic peroxidase activity, and incubated with sodium citrate buffer for 20 min at 90 C for antigen retrieval. Washes were performed with automation buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.02% Tween 20]. After blocking for 20 min at room temperature with normal goat serum, sections were incubated for 1 h at room temperature with anti-phospho-p44/42 MAPK antibody diluted 1:100 in buffer containing 1% BSA. Sections were next incubated with biotinylated goat antirabbit antibody for 30 min at room temperature, followed by a 30-min incubation with the avidin/biotinylated enzyme complex (ABC). Blocking serum and antibodies were from the antirabbit Vectastain ABC kit (Vector Laboratories). Staining was performed using the diaminobenzidine chromogen. The staining reactions were stopped in distilled water, and sections were dehydrated and mounted with Permount.
Statistics
Data are presented as the mean ± SEM. Data were analyzed by Student’s t test or one-way ANOVA and Bonferroni’s multiple comparison test where appropriate. P < 0.05 was considered statistically significant.
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FOOTNOTES |
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The work done in the authors’ laboratory was supported by the National Research Service Award F32-HD049966 (M.H.) from the National Institute of Child Health and Human Development (NICHD) and NICHD/National Institutes of Health Grant 1R01HD052909 (M.C.) and through a cooperative agreement (U54-HD31389 to M.C.) as part of the Specialized Cooperative Centers Program in Reproduction Research. A grant from Organon (M.C.) is also acknowledged.
Disclosure Summary: S.P., M.H., M.F., and L.P. have nothing to declare. M.C. received grant support (10/06-9/07) from N.V. Organon.
First Published Online January 10, 2008
1 S.P. and M.H. contributed equally to this work. 
Abbreviations: ADAM, A disintegrin and metalloprotease; AREG, amphiregulin; BTC, betacellulin; COC, cumulus-oocyte complex; CREB, cAMP-response element-binding protein; DMSO, dimethylsulfoxide; DPBS, Dulbecco’s PBS; EGF, epidermal growth factor; EGFR, EGF receptor; EREG, epiregulin; FBS, fetal bovine serum; Fsk, forskolin; GPCR, G-protein-coupled receptor; GVBD, germinal vesicle breakdown; hCG, human chorionic gonadotropin; HRP, horseradish peroxidase; LHR, LH receptor; PKA, protein kinase A; PMSG, pregnant mare serum gonadotropin; POF, preovulatory follicle; rLH, recombinant LH; TBST, Tris-buffered saline with 0.1% Tween 20.
Received for publication May 10, 2007. Accepted for publication January 2, 2008.
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. Science 275:394–397 subunits mediate Src-dependent phosphorylation of the epidermal growth factor receptor. A scaffold for G protein-coupled receptor-mediated Ras activation. J Biol Chem 272:4637–4644. J Biol Chem 270:15591–15597This article has been cited by other articles:
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Q. Li, F. Jimenez-Krassel, J. J Ireland, and G. W Smith Gene expression profiling of bovine preovulatory follicles: gonadotropin surge and prostanoid-dependent up-regulation of genes potentially linked to the ovulatory process Reproduction, February 1, 2009; 137(2): 297 - 307. [Abstract] [Full Text] [PDF]
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 I. Ben-Ami, L. Armon, S. Freimann, D. Strassburger, R. Ron-El, and A. Amsterdam EGF-like growth factors as LH mediators in the human corpus luteum Hum. Reprod., January 1, 2009; 24(1): 176 - 184. [Abstract] [Full Text] [PDF]
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 K. Evaul and S. R. Hammes Cross-talk between G Protein-coupled and Epidermal Growth Factor Receptors Regulates Gonadotropin-mediated Steroidogenesis in Leydig Cells J. Biol. Chem., October 10, 2008; 283(41): 27525 - 27533. [Abstract] [Full Text] [PDF]
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R. P. Norris, M. Freudzon, L. M. Mehlmann, A. E. Cowan, A. M. Simon, D. L. Paul, P. D. Lampe, and L. A. Jaffe Luteinizing hormone causes MAP kinase-dependent phosphorylation and closure of connexin 43 gap junctions in mouse ovarian follicles: one of two paths to meiotic resumption Development, October 1, 2008; 135(19): 3229 - 3238. [Abstract] [Full Text] [PDF]
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) and Ereg (
) mRNAs by rLH (5 IU) was examined in cultured POFs by semiquantitative RT-PCR. Gene expression levels were normalized to Rpl19 levels at each time point and are expressed as percentage of maximal induction. Data shown are the mean ± SEM of three separate experiments. Inset shows Areg and Ereg expression levels in control (Cont) vs. 0.5-h rLH-stimulated follicles. *, P < 0.05. C, Time course of GVBD in POFs. POFs (20–30 follicles per group) were cultured in the presence of rLH (5 IU) and punctured at the indicated times to release COCs. Oocytes were denuded of cumulus cells and evaluated for evidence of GVBD, a hallmark of oocyte meiotic resumption. Data are expressed as the mean percent GVBD ± SEM of at least four separate experiments.
