Mitogen-Activated Protein Kinase Mediates Luteinizing Hormone-Induced Breakdown of Communication and Oocyte Maturation in Rat Ovarian Follic
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内分泌学杂志 2005年第3期
Department of Biological Regulation (S.S.-A., D.G., N.D.), The Weizmann Institute of Science, Rehovot 76100, Israel; and Department of Neurobiology (E.C.), Institute of Life Sciences, Hebrew University, Jerusalem 91904, Israel
Address all correspondence and requests for reprints to: Nava Dekel, Ph.D., Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel. E-mail: nava.dekel@weizmann.ac.il.
Abstract
Resumption of meiosis, induced by LH, is preceded by the breakdown of gap junctional communication, which terminates the supply of cAMP from the somatic cells of the ovarian follicle to the oocyte. It has recently been shown that LH-induced reinitiation of meiosis is mediated by MAPK; however, the underlying molecular mechanism involved in the action of this enzyme remains unknown. We hypothesized that activation of MAPK interrupts junctional communication within the ovarian follicle, leading, in turn, to oocyte maturation. To test this hypothesis, we blocked the activation of MAPK by UO126, which specifically inhibits the MAPK signaling pathway. We analyzed junctional communication using three complementary methods: 1) patch-clamp analysis, which determined changes in the electrical coupling between two adjacent granulosa cells; 2) the scrape-loading technique, which monitored the spread of dyes through a granulosa cell layer; and 3) a metabolic coupling assay, which evaluated the transfer of radiolabeled uridine from the cumulus cells to the oocyte. We show, herein, that the somatic follicle cells, rather than the oocyte, activate MAPK immediately after their exposure to LH. Moreover, inhibition of LH-induced MAPK activation not only prevents oocyte maturation but also blocks the reduction in junctional communication. In addition, the appearance of the two phosphorylated forms of the gap junction protein, connexin 43, in response to LH, was avoided by UO126. We concluded that MAPK mediates LH-induced oocyte maturation by interrupting cell-to-cell communication within the ovarian follicle, possibly through phosphorylation of connexin 43.
Introduction
MEIOSIS IN MAMMALIAN oocytes is arrested at the diplotene stage of the first prophase. It is reinitiated after the onset of puberty, in response to the preovulatory surge of LH. Resumption of meiosis in mammals can also occur spontaneously in vitro in oocytes liberated from their follicular environment (reviewed in Ref.1). Oocytes incubated within the intact ovarian follicle remain meiotically arrested but can be induced to resume meiosis in vitro by LH (2), epidermal growth factor (EGF) (3), or GnRH (4).
Within the ovarian follicle, the immediate neighbors of the oocyte are the cumulus cells, which represent a subpopulation of the granulosa somatic compartment. The cumulus cells communicate with the oocyte by gap junctions. An extensive network of junctional communication is also established between the cumulus and the granulose cells, as well as within each cellular compartment (reviewed in Ref.5). Gap junctions are composed of proteins from the connexin gene family, the most abundant of which in the ovary is connexin 43 (Cx43) (6, 7). Cx43 is a multiphosphorylated protein that becomes hyperphosphorylated in response to LH (8). Sequence analysis of Cx43 revealed that this protein can serve as a substrate for different kinases, such as the cAMP-dependent protein kinase A (PKA), protein kinase C (PKC), glycogen synthase kinase 3, and MAPK (reviewed in Ref.9).
A major role of junctional communication within the ovarian follicle is to supply nutrients from the somatic cells, which support oocyte growth (10, 11). In addition, gap junctions mediate the transfer of cAMP from the granulosa/cumulus cells to the oocyte (reviewed in Refs.12 and 13). cAMP serves as the regulatory signal that maintains the fully grown oocyte in meiotic arrest (14, 15). Reinitiation of meiosis, which occurs in response to the preovulatory surge of LH, takes place after the interruption of cell-to-cell communication within the ovarian follicle (16, 17, 18). Breakdown of communication arrests the supply of cAMP from the somatic cells to the oocyte, resulting in a decrease in the intraoocyte concentration of this cyclic nucleotide (reviewed in Ref.12).
Meiotically arrested oocytes contain diffuse chromosomes surrounded by an intact nuclear structure known as the germinal vesicle (GV). Upon reinitiation of meiosis, the chromosomes condense, and the GV breaks down (GVB). The first meiotic division progresses through metaphase I; its completion is manifested by the formation of the first polar body. The oocytes are then arrested again at metaphase II until fertilization (reviewed in Ref.1).
Maturation promoting factor (MPF), a heterodimer composed of the regulatory cyclin B1 and the catalytic p34cdc2 kinase, is a pivotal regulator of meiosis reinitiation (reviewed in Ref.19). The two members of the MAPK family, P44mapk and P42mapk, known as the ERKs 1 and 2, respectively, are also activated in oocytes that resume meiosis. The upstream regulator of these members of the MAPK family is the MAPK kinase, MEK, which phosphorylates them on both a serine and a threonine residue (reviewed in Ref.20).
In general, phosphorylation and activation of MEK is catalyzed by Raf1 kinase (reviewed in Ref.20). However, MEK, being in the oocyte, is regulated by Mos (21). A recent study showed that, at least in the rat, MPF activation is a prerequisite for mos translation and, in turn, for MAPK activation (22, 23). Consequently, the kinetics of activation of the MAPK signal transduction pathway in rodent oocytes is delayed (23). This late activation of the Mos/MAPK signaling pathway is consistent with its function as the cytostatic factor responsible for the second meiotic arrest (24). In somatic cells, MAPK plays a role in regulating various other cellular processes such as proliferation, differentiation, morphology, learning, apoptosis, and carcinogenesis (reviewed in Refs.20 and 25). It has been reported that MAPK is activated in ovarian granulosa cells in response to GnRH (26, 27), LH (28), and FSH (29). More recent studies have shown that LH and FSH-stimulated MAPK activation is mediated by PKA (28, 30, 31).
The obvious assumption that the effect of LH on the ovarian gap junctions is mediated by PKA was based on the commonly accepted notion that LH uses cAMP as its second messenger (32). Later reports that LH also activates PKC (33, 34) raised the possibility that this kinase is involved in the LH-induced breakdown of communication as well (8). Because both PKA and PKC activate MAPK, it may be possible that MAPK is a downstream component in the signal transduction pathway, stimulated by LH in the ovarian follicle.
It has been recently demonstrated that MAPK mediates LH-induced maturation of mouse follicle-enclosed oocytes (35). Nevertheless, the molecular events that participate in this process remain unresolved, leaving this issue open to further investigation. Our experiments explored the mechanism involved in MAPK mediation of LH-induced resumption of meiosis. More specifically, our study examined the effect of MAPK on junctional communication within the ovarian follicle and on the phosphorylation of the ovarian gap junction protein, Cx43. Importantly, our findings revealed that interruption of communication within the ovarian follicle is dependent upon an active MAPK. Furthermore, they suggest that this response apparently involves the phosphorylation of Cx43.
Materials and Methods
Reagents and antibodies
Leibovitz’s L-15 tissue culture medium and fetal bovine serum were purchased from Biological Industries (Kibbutz Beit Hemeek, Israel). Antibiotics were purchased from Bio-Lab, Ltd. (Jerusalem, Israel); U0126 and U0124 were from Calbiochem (San Diego, CA); phenylmethylsulfonylfluoride (PMSF), leupeptin, aprotinin, and dithiothreitol (DTT) were from Sigma (St. Louis, MO). Monoclonal anti-Cx43 antibodies were purchased from Transduction Laboratories (Lexington, KY). Monoclonal mouse anti-P-MAPK antibodies were kindly provided by Prof. Rony Seger, The Weizmann Institute of Science (Rehovot, Israel). Polyclonal rabbit antitubulin antibodies were purchased from Sigma. Polyclonal rabbit anti-MAPK antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Goat antimouse peroxidase-conjugated antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).
Animals
Sexually immature 23-d-old female Wistar rats were injected with 8I U pregnant mare’s serum gonadotropin (PMSG; Chrono-gest Intervet, The Netherlands). The rats were killed by cervical dislocation 48 h later. The ovaries were removed, and the large antral follicles were separated. These experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, National Academy of Science, Bethesda, MD).
Culture of follicles
Isolated intact rat ovarian follicles were separated and grown in suspension in L-15 tissue culture medium containing 5% fetal bovine serum (both purchased from Biological Industries) in 25-ml flasks and gassed with 50% O2-50% N2. Incubations were carried out at 37°C in an oscillating water bath in the presence or absence of either 1 μg/ml ovine LH (National Institutes of Health LH S-24) or 200 ng/ml EGF, with or without 10 μM UO126 or its inactive derivative, UO124, which was added 1 h before adding the hormone. UO126 is a very potent inhibitor of MEK, the direct upstream regulator of MAPK. Even though it shows little, if any, effect on the kinase activities of PKC, Abl, Raf, MEKK, ERK, JNK, MKK-3, MKK-4/SEK, MKK-6, Cdk2, or Cdk4 (36), it is commonly used for specifically inhibiting the MAPK signaling pathway. At the end of the incubation period, the follicles were incised, and the cumulus-oocyte complexes (COCs) were recovered. The oocytes were monitored microscopically using differential interference contrast (DIC) optics for reinitiation of meiosis as indicated by the disappearance of the GV.
Protein extraction and Western blot analysis
For Cx43 analysis, the ovarian follicles were lysed in homogenization buffer (20 mM Tris, pH 7.4; 2 mM EDTA; 5 mM EGTA; 0.25 mM sucrose; 1 mM DTT) supplemented with fresh 2 mM PMSF, 10 μg/ml leupeptin, 10 μg/ml pepstatin, and 50 mM ?-glycerophosphate. Cell membranes were then sedimented by ultracentrifugation and further dissolved in homogenization buffer. The concentration of the proteins was determined by the Bradford assay. Samples (30 μg each), which were dissolved in protein sample buffer (2% ?-mercaptoethanol; 2% sodium dodecyl sulfate (SDS); 50 mM Tris; HCL, pH 6.8; 10% glycerol; and 0.01% bromophenol blue), were boiled and loaded onto 12.5% SDS-PAGE. For better resolution of the different phosphorylation forms of Cx43, the bisacrylamide in the monomer mixture was reduced from 8% to 0.12%. After electrophoretic separation, the proteins were transferred to a nitrocellulose membrane, washed for 1 h with a blocking solution (5% milk, 0.05% Tween in PBS), and then incubated with anti-Cx43 monoclonal antibodies (1:250) for 2 h. The membrane was then washed several times and incubated with antimouse horseradish peroxidase-conjugated antibodies (1:1000). Chemiluminescent signals were generated by incubation with the enhanced chemiluminescence reagent (Amersham, Buckinghamshire, UK).
For determination of the extent of MAPK activation, the cells were lysed in buffer H containing 50 mM ?-glycerophosphate, 1.5 mM EGTA, 1 mM EDTA, 1 mM Na-orthovanadate, 1 mM benzamidine, 1 mM PMSF, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 2 μg/ml pepstatin, and 1 mM DTT. The lysates were then centrifuged for 10 min, followed by a transfer of the supernatants to new tubes and determination of protein concentration. Next, equal amounts of protein were subjected to Western blot analysis. Detection of the immunoreactive band was done, as described above, and the samples were loaded onto 10% acrylamide gel. After electrophoretic separation, the proteins were transferred to a nitrocellulose membrane. Two anti-MAPK antibodies were used for the Western blot analysis: one antibody immunoreacted with the phosphorylated (active) MAPK (pMAPK), whereas the second immunoreacted with both the active and inactive MAPK (total MAPK). The relative amount of the pMAPK in each sample represents the extent of MAPK activation.
Coupling assay
To evaluate the metabolic coupling within the COCs, we incubated isolated intact ovarian follicles with the indicated agents. The follicles were then incised; the recovered COCs were further incubated for 1 h in medium containing radiolabeled (20 μCi) uridine, followed by several extensive washes. Each group was then divided into two subgroups: 1) one subgroup remained intact; and 2) in the other subgroup, the oocytes were freed mechanically from the surrounding cumulus cells. Cumulus-free oocytes and COCs were solubilized in 10% SDS, and their radioactivity was counted in the scintillation liquid. The extent of coupling was calculated as the ratio between the radioactivity counted in the oocyte and that in the cumulus cells, minus the oocyte. This equation takes into account the size of the cumulus. A group of cumulus-free oocytes was incubated with radiolabeled uridine; its radioactivity was counted to confirm the negligible uptake of uridine into the oocytes.
Cultured primary granulosa cells
Granulosa cells were recovered from the ovaries of the above-mentioned female rats. The cells were plated onto serum-coated wells (equivalent to two ovaries per six wells) in 24-multiwell plates (16 mm; Nunc, Copenhagen, Denmark) containing 0.5 ml L-15 medium. Cultures were incubated at 37 C in a humidified incubator for the indicated times.
Scrape-loading
The cultured primary granulosa cells were incubated with or without 3 μg/ml LH, in the presence or absence of 10 μM UO126 for 10 min. After incubation, the plates were washed, and PBS, containing a mixture of 0.7 mg/ml Lucifer yellow (LY) and 5 mg/ml rhodamine dextran (Rh), was added as described in (37). Next, the plates were mechanically scratched with a sharp scalpel and incubated for 3 min. After incubation, the cells were washed several times and fixed with 3% paraformaldehyde. The cells were viewed by fluorescent microscopy.
Patch-clamp analysis
Simultaneous, double whole-cell patch recordings were performed under visual control using infrared DIC optics. All recordings were performed with the current clamp configuration, using AxoClamp2B amplifiers (Axon Instruments, Foster City, CA). The recording pipettes were filled with intracellular solution containing the following (in mM): 4 NaCl, 10–3 CaCl2, 140 K-gluconate, 10–2 EGTA, 4 Mg-ATP, and 10 HEPES (pH 7.2). The voltage and current traces were digitized and stored on the computer, using a National Instrument PCI-MIO-10X DAQ card and LabView software (National Instruments, Austin, TX).
Analysis of electrical coupling.
To compare the level of electrical coupling between different pairs of cells, we calculated the coupling coefficient (CC) from the voltage responses of pre- and postjunctional cells to prolonged (150–300 msec) negative current pulses. Accordingly, CC is defined as the ratio between the voltage responses of the post- and the prejunctional cells (equations 1 and 2). To calculate the actual resistance between the two cells, we employed a method previously described by (38). Briefly, each cell was treated as an isopotential cell, consisting of a resistor (Ri, where i is the cell’s index) and a conductor (Ci), in parallel. Each cell was then coupled to a second cell, via a third resistor (Rc), which models the electrical coupling. Thus,
where V1 and V2 are the voltage responses of cell 1 and cell 2, respectively. Note that the coupling resistor can be asymmetrical. From the circuit diagram, we derived the following equations:
From equations 1–4, we derived:
Statistical analysis
The number of times that each individual experiment was repeated is indicated in the figure legend. Data points are presented as the mean ± SD. Statistical significance was determined by using the ANOVA to assess the differences between multiple experimental groups.
Results
LH stimulates MAPK activation in the ovarian follicle
The activation of MAPK by LH has, thus far, been demonstrated in several granulosa cell lines (28, 30, 31). Our initial experiment was designed to confirm these findings in the ovarian follicle. As shown in Fig. 1A, pMAPK could not be detected in ovarian follicles incubated without LH, whereas their incubation with LH resulted in the activation of the two MAPK isoforms, p44 and p42. This response was observed as soon as 10 min after exposure to the hormone.
FIG. 1. A, Timing of LH-induced MAPK activation in the ovarian follicle. Intact ovarian follicles were incubated with or without LH (1 μg/ml) for the indicated times. After incubation, the follicles were homogenized, and samples of 20 μg of the protein extract were subjected to Western blot analysis with anti-P-MAPK and anti-total-MAPK antibodies. The results of one representative of a total of at least five independent experiments are presented. B, MAPK activation in follicle-enclosed oocytes incubated with LH. Intact ovarian follicles were incubated with 1 μg/ml LH. After the indicated times of incubation, the oocytes were released from the follicles, freed mechanically from the surrounding cumulus cells, and lysed. Samples of 50 oocytes each were subjected to Western blot analysis with anti-P-MAPK and anti-total-MAPK antibodies. The results of one of two independent experiments with similar results are presented.
Our next goal was to examine MAPK in the oocytes that reside within the isolated intact follicles, incubated with LH. As shown in Fig. 1B, both p42 and p44 of MAPK were activated in the oocytes as well. However, whereas activation of MAPK in the follicles was detected immediately upon their exposure to LH, this response in the oocytes was not observed until 8 h after their incubation.
LH-induced resumption of meiosis is mediated by MAPK
The downstream components of the LH-stimulated signal transduction pathway, which lead to oocyte maturation, are still unknown. MAPK, the LH-induced, activation of which is demonstrated here, mediates the induction of mouse oocyte maturation (35). To examine the possible involvement of this kinase in the rat, we impeded its action by using the MEK inhibitor UO126. The U0124 derivative, which does not inhibit MEK, served as a negative control. EGF has also been shown to promote maturation in follicle-enclosed oocytes (3). Because EGF action is known to activate MAPK (39), the effect of UO126 on EGF-induced oocyte maturation was also analyzed.
We initially analyzed the dose of UO126 that effectively inhibited LH-induced MAPK activation in the ovarian follicle (Fig. 2A). This same concentration was further employed to examine its effect on LH-stimulated oocyte maturation. As seen in Fig. 2B, 99% of the control oocytes remained meiotically arrested, as indicated by the presence of GV, whereas LH and EGF induced GVB in 88% and 90% of the oocytes, respectively. The addition of U0126 blocked the effect of LH, as well as that of EGF. U0124 failed to affect LH-induced oocyte maturation.
FIG. 2. A, UO126 inhibits LH-induced MAPK activation in the ovarian follicle. Ovarian follicles were incubated with or without LH (1 μg/ml) in the presence or absence of 10 μM UO126. After 10 min of incubation, the COCs were recovered and lysed. Samples of protein extract were subjected to Western blot analysis with anti-P-MAPK and anti-total-MAPK antibodies. The results of one representative of three independent experiments are presented. B, U0126 inhibits both LH- and EGF-induced oocyte maturation. Ovarian follicles were incubated with either LH (1 μg/ml) or EGF (200 ng/ml) in the presence or absence of either 10 μM UO126 or UO124. After an overnight incubation, the COCs were recovered, and the oocytes were microscopically analyzed for maturation, as described in Materials and Methods. The means ± SD of the percentage of GVB oocytes obtained in five independent experiments, in which 20 oocytes were analyzed for each experimental treatment, are presented. *, Statistical significance of P < 0.05 (ANOVA analysis).
The breakdown of communication in the ovarian follicle is mediated by MAPK
We previously showed that LH reduces intercellular communication within the ovarian follicle and that this response is followed by resumption of meiosis (18). In attempting to decipher the mechanism by which MAPK activation induces reinitiation of meiosis, we examined the possible involvement of MAPK in the LH-induced breakdown of communication. For this purpose, we employed cultured primary granulosa cells. These cells activate MAPK immediately after their exposure to LH (Fig. 3A), which is similar to the previously demonstrated response of the intact ovarian follicles. An evaluation of the transfer of current between two adjacent granulosa cells, by patch-clamp analysis, revealed a reduction in the gap-junction conductance upon LH application. Injecting the current into one cell causes ions to pass through the gap-junctions and to charge the membrane of the coupled cell. A reduction in the conductive properties of this gap junction results in less current to the adjacent cell. Thus, the voltage response of the coupled cell decreases, whereas an increase in response takes place in the injected cell. This corresponds to a decrease in the CC. Indeed, LH caused an increase in the amplitude of the voltage response in the injected cell (Fig. 3B) and a decrease in response in the coupled cell (Fig. 3B), resulting in a decrease in the CC (Fig. 3B). From these parameters, we calculated (see Materials and Methods) a 100% increase in the resistance of the gap-junctions, with a 50% change in the conductive power of the gap-junctions resulting from the application of LH. However, this effect was prevented in the presence of U0126, whereby UO124 did not affect LH-induced uncoupling (the U0124 data are pooled with that of the control). The kinetics of the increase in coupling resistance in response to LH is shown in Fig. 3C, where an initial elevation in the resistance can be observed within seconds, with a maximal increase at 3 min after applying LH. The response of the granulosa cells to LH was not observed in the presence of UO126.
FIG. 3. A, LH-induced MAPK activation in cultured granulosa cells. Granulosa cells were seeded on serum-coated culture plates and incubated with or without LH (1 μg/ml) for 10 min. The cells were harvested, and samples of their proteins were extracted and subjected to Western blot analysis with anti-P-MAPK and anti-total-MAPK antibodies. The results of one representative of a total of three independent experiments are presented. B, LH-induced uncoupling is blocked by U0126. Simultaneous, double whole-cell patch recordings were performed under visual control conditions using infrared DIC optics, as described in Materials and Methods. The effect of LH is presented as the percentage of change compared with the control. Light gray represents the experiments in which 1–3 μg/ml of LH were added to cells cultured with or without U0124 (10 μM). Dark gray represents the results from the experiments in which LH was added to cells incubated with U0126 (10 μM). The means ± SD of the coupling obtained for each treatment in triplicate with three independent experiments are presented. *, Statistical significance of P < 0.05 (ANOVA analysis). C, Coupling resistance as a function of time. The monitoring of coupling resistance before and after administrating LH (1–3 μg/ml, arrow shows time of LH application) with (solid circles) and without (empty circles) 10 μM of U0126. The average resistance before the addition of LH was defined as 100%. The results are presented as the percentage of changes compared with this control. One representative result of a total of individual measurements performed in nine independent experiments is presented.
To confirm the above-mentioned results, we used scrape-loading as a complementary method to assess cell-to-cell communication. This technique is based on the fact that LY and Rh cannot penetrate intact cells. Scratching the cells allows these dyes to penetrate into the cells. However, because LY has a molecular mass smaller than 1 kDa, it can be further transferred between the cells through gap junctions, whereas Rh, which has a higher molecular mass, will stain only the wounded cells. As shown in Fig. 4, under all culture conditions, Rh staining was restricted to the wounded cells, as expected. However, compared with the control, the spread of LY to neighboring cells in the presence of LH was markedly reduced. The addition of UO126 restored the spread of dye, bringing it back to the control level.
FIG. 4. LH-induced breakdown of communication in granulosa cells is inhibited by UO126. Granulosa cells were incubated with or without 3 μg/ml LH in the presence or absence of 10 μM UO126 for 10 min. After incubation, the cells were assayed for intercellular communication by a low-molecular-mass (LY) dye transfer after scrape-loading, as described in Materials and Methods. The high-molecular-mass (Rh) served as a negative control. The results of one representative of two independent experiments, including at least two scrapes per treatment each, are presented. The pictures represent the merge of the LY (green) and Rh (orange).
Metabolic coupling in the COCs was analyzed to examine the possible involvement of MAPK in LH-induced uncoupling between the oocyte and the cumulus cells. As shown in Fig. 5A, incubation of the ovarian follicles with LH reduced the extent of coupling almost to zero; this effect was effectively reversed by UO126.
FIG. 5. A, LH-induced breakdown of communication in the COC is inhibited by UO126. Isolated intact follicles were incubated with or without 1 μg/ml LH. After an overnight incubation, the follicles were incised, and the COCs were analyzed for metabolic coupling, as described in Materials and Methods. The coupling value, determined under control conditions, was defined as 100%. The means ± SD of the results obtained in five independent experiments are presented. Each group receiving the different experimental treatments in every individual experiment included 80 follicles. *, Statistical significance of P < 0.05 (ANOVA analysis), compared with the group of COCs recovered from follicles incubated with LH. B, Radiolabeled uridine in GV and GVB oocytes. Isolated intact follicles were incubated with or without LH (1 μg/ml). After 24 h, the follicles were incised, and the COCs were recovered. The COCs were then incubated in medium containing radiolabeled uridine. After 1 h, the oocytes were freed mechanically from the surrounding cumulus cells. Next, oocytes containing GV were separated from the GVB oocytes. Another group of GV oocytes, recovered from follicles incubated in the absence of LH, was analyzed. The oocytes of all groups were solubilized in 10% SDS, and their radioactivity was counted. The radioactivity detected in the GV group of oocytes, recovered from follicles incubated in the absence of LH, was defined as 100%. The means ± SD of duplicates of 10 oocytes, each in three independent experiments, are presented. *, Statistical significance of P < 0.05 (ANOVA analysis).
For reasons unknown, some follicle-enclosed oocytes incubated with LH did not reinitiate meiosis. We separated the immature (GV) from the mature (GVB) oocytes and counted their radiolabeled uridine content. Radioactivity was also monitored in a control group of GV oocytes, recovered from follicles that were incubated without LH. As shown in Fig. 5B, the amount of radioactivity detected in both groups of GV oocytes is 3-fold higher than that in the oocytes showing GVB. These results indicate that meiotic arrest is associated with intact communication within the ovarian follicle, supporting the notion that uncoupling between the oocyte and the granulosa cells is necessary for reinitiation of meiosis.
MAPK is involved in LH-induced Cx43 phosphorylation
It was previously shown that LH induces phosphorylation of Cx43 in the ovarian follicles after 10 min of exposure to the hormone (8). To examine the possible LH-induced MAPK-mediated Cx43 phosphorylation, we exposed intact follicles, incubated with LH, to UO126, after which we examined the pattern of CX43 phosphorylation. As shown in Fig. 6, LH stimulated the appearance of two additional phosphorylated forms of Cx43. The addition of UO126 (but not UO124) to these follicles prevented the appearance of those two isoforms. This result implies that Cx43 may serve as a substrate for MAPK phosphorylation.
FIG. 6. LH-induced Cx43 phosphorylation is affected by inhibition of the MAPK signaling pathway. Intact ovarian follicles were incubated with 10 μM of either UO126 or UO124 for 1 h, followed by the addition of 1 μg/ml LH. After 20 min of incubation, the follicles were homogenized, and 30 μg from each sample was subjected to Western blot analysis using either anti-Cx43 antibodies or antitubulin antibodies. One representative of a total of six independent experiments is presented. The additional bends that appear with LH and disappear in the presence of UO126 are denoted by arrows.
Discussion
Our study presents evidence that activation of MAPK in the somatic cells of rat ovarian follicles mediates LH-induced oocyte maturation. We further show, for the first time, that the mechanism by which MAPK mediates the LH-stimulated resumption of meiosis involves down-regulation of junctional communication. This response is apparently elicited at the level of Cx43 phosphorylation.
Despite the recent demonstration that oocytes express LH receptors (40), a direct response of the female gamete to this gonadotropin has not yet been reported. Alternatively, the notion that the response of the somatic follicle cells to LH eventually leads to oocyte maturation is widely accepted. Accordingly, we show here that LH activates MAPK in the ovarian follicle cells within minutes, whereas no activation of this kinase was observed in the oocyte before GVB. The maturing oocytes in our experiments exhibited the pMAPK; however, this event takes place only 8 h after exposure of the follicle to LH and apparently does not represent a direct response of the oocyte to the gonadotropin. This late elevation in MAPK activity within the oocyte ostensibly represents a downstream event along the cascade, initiated by MPF activation and followed by mos mRNA polyadenylation and its translation (41). The late activation of MAPK, in a manner that is independent of LH, also occurs in oocytes that mature spontaneously in vitro upon their separation from the ovarian follicle (23). As expected, inhibition of MAPK activation in these isolated oocytes does not interfere with GVB (42). Reinitiation of meiosis, which is independent of MAPK activation in the oocyte, was also demonstrated in mos-null mice. Despite their failure to activate MAPK, the oocytes of these mice resume meiosis in vivo in response to LH (43, 44). Because MAPK activation in the granulosa cells is independent of Mos, their capacity to activate this enzyme in the mos knockout mouse model is not impaired. Under these conditions, LH activates the MAPK signaling pathway in the granulosa cells via Raf1 kinase; its effect on the reinitiation of meiosis in these mice is therefore not inhibited.
The mechanism underlying the signal transduction pathway, which is initiated upon the binding of LH to its receptors on the granulose cells leading to GVB, is still unclear. Based on the commonly accepted notion that LH uses cAMP as its second messenger (32), the obvious assumption was that PKA mediates the effect of LH on the ovarian follicle. Interestingly, later reports demonstrated that LH also activates PKC (34), which makes this kinase an additional candidate for serving this function. It has recently been shown that both PKA and PKC are involved in MAPK activation, suggesting that MAPK acts downstream from PKA and/or PKC in mediating LH action (reviewed in Ref.45). We further confirmed this by demonstrating that LH activates MAPK in isolated granulosa cells within the intact ovarian follicle.
It has recently been reported that MAPK activation in the cumulus cells is essential for resumption of meiosis in the mouse (35). However, the identity and the hierarchy of the downstream events in this signal transduction pathway remain largely unknown. Previous studies have shown that LH decreases intercellular communication between the oocyte and the cumulus cells (16, 17, 18). It has also been reported that LH induces uncoupling of Cx43 gap junction channels in TM3 Leydig cells (46). Taking this information into account, we hypothesized that MAPK may be involved in the LH-induced breakdown of communication and its subsequent resumption of meiosis. To explore this possibility, we employed three complementary methods, each of which uses a specific marker of a different molecular size. The transfer of ions, assessed by the patch-clamp analysis, as well as the analysis of dye transfer, examined by the scrape-loading experiments, confirmed that LH induces the breakdown of junctional communication between the granulosa cells. The patch-clamp analysis, used in our present study, not only confirms the previous findings but also provides a very fine kinetic analysis of the response of granulosa cells to LH. Using this method, we have shown, for the first time, that LH uncouples the granulosa within seconds. This, as well as the complementary scrape-loading assay, done in the presence of UO126, unequivocally revealed that the LH-induced uncoupling between the granulosa cells is mediated by MAPK. The metabolic coupling assay, which quantifies the level of communication within the COC, revealed that inhibition of MAPK activation prevents LH-induced breakdown of communication between the oocyte and the surrounding cumulus cells, suggesting that MAPK is involved in this response as well.
As mentioned before, EGF promotes maturation of follicle-enclosed oocytes (3). Recently, it has been suggested that LH induces the activation of the ovarian granulosa EGF receptors through enhanced expression of certain EGF family members such as amphiregulin, epiregulin, and ?-cellulin (47). However, because the expression of these EGF family member mRNAs could not be demonstrated earlier than 1–3 h after exposure to LH, their mediatory role in stimulating oocyte maturation is somewhat questionable. The well-characterized mechanism, which involves an immediate release of EGF storage by metalloproteinase upon stimulation of G protein-coupled receptors (48), if relevant for the ovary, could overcome this puzzle. If there is further support for the physiological relevance of EGF receptors in the induction of oocyte maturation, our findings confirm that this action is mediated by MAPK (39).
We have previously shown that exposing intact follicles to LH for 10 min results in enhanced phosphorylation of Cx43 (8). We postulated that this phosphorylation might result in some conformational changes in the protein and the consequent closure of the channel. The notion that phosphorylation events occurring on the C-terminal portion of the protein regulate the gating of gap junction channels has been extensively studied (reviewed in Ref.49). Along this line, previous studies have reported that EGF stimulates a rapid disruption of gap junctional communication and enhances phosphorylation of Cx43 (50) on ser255, ser279, and ser282, which are sites of MAPK action (51). Moreover, mutants of Cx43 at these specific sites failed to disrupt junctional communication in response to EGF (52). The changes in the LH-induced Cx43 phosphorylation pattern, in the presence of UO126, imply the possible involvement of MAPK in Cx43 phosphorylation within the ovarian follicle. Further investigations are needed to identify the exact sites of LH-stimulated Cx43 phosphorylation.
In summary, in this study, we proposed a mechanism by which MAPK mediates LH-induced oocyte maturation in the rat ovarian follicle. Our model suggests that MAPK, which is activated in the somatic follicular compartment in response to LH, phosphorylates Cx43. This phosphorylation of Cx43 may result in uncoupling among the follicular cells, which arrests the supply of the inhibitory cAMP and subsequently leads to resumption of meiosis (Fig. 7).
FIG. 7. A proposed model for LH-induced oocyte maturation. Upon LH administration, MAPK in the somatic follicular cells is activated and phosphorylates Cx43. Phosphorylation of Cx43 uncouples the follicle cells and leads to the resumption of meiosis.
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Address all correspondence and requests for reprints to: Nava Dekel, Ph.D., Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel. E-mail: nava.dekel@weizmann.ac.il.
Abstract
Resumption of meiosis, induced by LH, is preceded by the breakdown of gap junctional communication, which terminates the supply of cAMP from the somatic cells of the ovarian follicle to the oocyte. It has recently been shown that LH-induced reinitiation of meiosis is mediated by MAPK; however, the underlying molecular mechanism involved in the action of this enzyme remains unknown. We hypothesized that activation of MAPK interrupts junctional communication within the ovarian follicle, leading, in turn, to oocyte maturation. To test this hypothesis, we blocked the activation of MAPK by UO126, which specifically inhibits the MAPK signaling pathway. We analyzed junctional communication using three complementary methods: 1) patch-clamp analysis, which determined changes in the electrical coupling between two adjacent granulosa cells; 2) the scrape-loading technique, which monitored the spread of dyes through a granulosa cell layer; and 3) a metabolic coupling assay, which evaluated the transfer of radiolabeled uridine from the cumulus cells to the oocyte. We show, herein, that the somatic follicle cells, rather than the oocyte, activate MAPK immediately after their exposure to LH. Moreover, inhibition of LH-induced MAPK activation not only prevents oocyte maturation but also blocks the reduction in junctional communication. In addition, the appearance of the two phosphorylated forms of the gap junction protein, connexin 43, in response to LH, was avoided by UO126. We concluded that MAPK mediates LH-induced oocyte maturation by interrupting cell-to-cell communication within the ovarian follicle, possibly through phosphorylation of connexin 43.
Introduction
MEIOSIS IN MAMMALIAN oocytes is arrested at the diplotene stage of the first prophase. It is reinitiated after the onset of puberty, in response to the preovulatory surge of LH. Resumption of meiosis in mammals can also occur spontaneously in vitro in oocytes liberated from their follicular environment (reviewed in Ref.1). Oocytes incubated within the intact ovarian follicle remain meiotically arrested but can be induced to resume meiosis in vitro by LH (2), epidermal growth factor (EGF) (3), or GnRH (4).
Within the ovarian follicle, the immediate neighbors of the oocyte are the cumulus cells, which represent a subpopulation of the granulosa somatic compartment. The cumulus cells communicate with the oocyte by gap junctions. An extensive network of junctional communication is also established between the cumulus and the granulose cells, as well as within each cellular compartment (reviewed in Ref.5). Gap junctions are composed of proteins from the connexin gene family, the most abundant of which in the ovary is connexin 43 (Cx43) (6, 7). Cx43 is a multiphosphorylated protein that becomes hyperphosphorylated in response to LH (8). Sequence analysis of Cx43 revealed that this protein can serve as a substrate for different kinases, such as the cAMP-dependent protein kinase A (PKA), protein kinase C (PKC), glycogen synthase kinase 3, and MAPK (reviewed in Ref.9).
A major role of junctional communication within the ovarian follicle is to supply nutrients from the somatic cells, which support oocyte growth (10, 11). In addition, gap junctions mediate the transfer of cAMP from the granulosa/cumulus cells to the oocyte (reviewed in Refs.12 and 13). cAMP serves as the regulatory signal that maintains the fully grown oocyte in meiotic arrest (14, 15). Reinitiation of meiosis, which occurs in response to the preovulatory surge of LH, takes place after the interruption of cell-to-cell communication within the ovarian follicle (16, 17, 18). Breakdown of communication arrests the supply of cAMP from the somatic cells to the oocyte, resulting in a decrease in the intraoocyte concentration of this cyclic nucleotide (reviewed in Ref.12).
Meiotically arrested oocytes contain diffuse chromosomes surrounded by an intact nuclear structure known as the germinal vesicle (GV). Upon reinitiation of meiosis, the chromosomes condense, and the GV breaks down (GVB). The first meiotic division progresses through metaphase I; its completion is manifested by the formation of the first polar body. The oocytes are then arrested again at metaphase II until fertilization (reviewed in Ref.1).
Maturation promoting factor (MPF), a heterodimer composed of the regulatory cyclin B1 and the catalytic p34cdc2 kinase, is a pivotal regulator of meiosis reinitiation (reviewed in Ref.19). The two members of the MAPK family, P44mapk and P42mapk, known as the ERKs 1 and 2, respectively, are also activated in oocytes that resume meiosis. The upstream regulator of these members of the MAPK family is the MAPK kinase, MEK, which phosphorylates them on both a serine and a threonine residue (reviewed in Ref.20).
In general, phosphorylation and activation of MEK is catalyzed by Raf1 kinase (reviewed in Ref.20). However, MEK, being in the oocyte, is regulated by Mos (21). A recent study showed that, at least in the rat, MPF activation is a prerequisite for mos translation and, in turn, for MAPK activation (22, 23). Consequently, the kinetics of activation of the MAPK signal transduction pathway in rodent oocytes is delayed (23). This late activation of the Mos/MAPK signaling pathway is consistent with its function as the cytostatic factor responsible for the second meiotic arrest (24). In somatic cells, MAPK plays a role in regulating various other cellular processes such as proliferation, differentiation, morphology, learning, apoptosis, and carcinogenesis (reviewed in Refs.20 and 25). It has been reported that MAPK is activated in ovarian granulosa cells in response to GnRH (26, 27), LH (28), and FSH (29). More recent studies have shown that LH and FSH-stimulated MAPK activation is mediated by PKA (28, 30, 31).
The obvious assumption that the effect of LH on the ovarian gap junctions is mediated by PKA was based on the commonly accepted notion that LH uses cAMP as its second messenger (32). Later reports that LH also activates PKC (33, 34) raised the possibility that this kinase is involved in the LH-induced breakdown of communication as well (8). Because both PKA and PKC activate MAPK, it may be possible that MAPK is a downstream component in the signal transduction pathway, stimulated by LH in the ovarian follicle.
It has been recently demonstrated that MAPK mediates LH-induced maturation of mouse follicle-enclosed oocytes (35). Nevertheless, the molecular events that participate in this process remain unresolved, leaving this issue open to further investigation. Our experiments explored the mechanism involved in MAPK mediation of LH-induced resumption of meiosis. More specifically, our study examined the effect of MAPK on junctional communication within the ovarian follicle and on the phosphorylation of the ovarian gap junction protein, Cx43. Importantly, our findings revealed that interruption of communication within the ovarian follicle is dependent upon an active MAPK. Furthermore, they suggest that this response apparently involves the phosphorylation of Cx43.
Materials and Methods
Reagents and antibodies
Leibovitz’s L-15 tissue culture medium and fetal bovine serum were purchased from Biological Industries (Kibbutz Beit Hemeek, Israel). Antibiotics were purchased from Bio-Lab, Ltd. (Jerusalem, Israel); U0126 and U0124 were from Calbiochem (San Diego, CA); phenylmethylsulfonylfluoride (PMSF), leupeptin, aprotinin, and dithiothreitol (DTT) were from Sigma (St. Louis, MO). Monoclonal anti-Cx43 antibodies were purchased from Transduction Laboratories (Lexington, KY). Monoclonal mouse anti-P-MAPK antibodies were kindly provided by Prof. Rony Seger, The Weizmann Institute of Science (Rehovot, Israel). Polyclonal rabbit antitubulin antibodies were purchased from Sigma. Polyclonal rabbit anti-MAPK antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Goat antimouse peroxidase-conjugated antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).
Animals
Sexually immature 23-d-old female Wistar rats were injected with 8I U pregnant mare’s serum gonadotropin (PMSG; Chrono-gest Intervet, The Netherlands). The rats were killed by cervical dislocation 48 h later. The ovaries were removed, and the large antral follicles were separated. These experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, National Academy of Science, Bethesda, MD).
Culture of follicles
Isolated intact rat ovarian follicles were separated and grown in suspension in L-15 tissue culture medium containing 5% fetal bovine serum (both purchased from Biological Industries) in 25-ml flasks and gassed with 50% O2-50% N2. Incubations were carried out at 37°C in an oscillating water bath in the presence or absence of either 1 μg/ml ovine LH (National Institutes of Health LH S-24) or 200 ng/ml EGF, with or without 10 μM UO126 or its inactive derivative, UO124, which was added 1 h before adding the hormone. UO126 is a very potent inhibitor of MEK, the direct upstream regulator of MAPK. Even though it shows little, if any, effect on the kinase activities of PKC, Abl, Raf, MEKK, ERK, JNK, MKK-3, MKK-4/SEK, MKK-6, Cdk2, or Cdk4 (36), it is commonly used for specifically inhibiting the MAPK signaling pathway. At the end of the incubation period, the follicles were incised, and the cumulus-oocyte complexes (COCs) were recovered. The oocytes were monitored microscopically using differential interference contrast (DIC) optics for reinitiation of meiosis as indicated by the disappearance of the GV.
Protein extraction and Western blot analysis
For Cx43 analysis, the ovarian follicles were lysed in homogenization buffer (20 mM Tris, pH 7.4; 2 mM EDTA; 5 mM EGTA; 0.25 mM sucrose; 1 mM DTT) supplemented with fresh 2 mM PMSF, 10 μg/ml leupeptin, 10 μg/ml pepstatin, and 50 mM ?-glycerophosphate. Cell membranes were then sedimented by ultracentrifugation and further dissolved in homogenization buffer. The concentration of the proteins was determined by the Bradford assay. Samples (30 μg each), which were dissolved in protein sample buffer (2% ?-mercaptoethanol; 2% sodium dodecyl sulfate (SDS); 50 mM Tris; HCL, pH 6.8; 10% glycerol; and 0.01% bromophenol blue), were boiled and loaded onto 12.5% SDS-PAGE. For better resolution of the different phosphorylation forms of Cx43, the bisacrylamide in the monomer mixture was reduced from 8% to 0.12%. After electrophoretic separation, the proteins were transferred to a nitrocellulose membrane, washed for 1 h with a blocking solution (5% milk, 0.05% Tween in PBS), and then incubated with anti-Cx43 monoclonal antibodies (1:250) for 2 h. The membrane was then washed several times and incubated with antimouse horseradish peroxidase-conjugated antibodies (1:1000). Chemiluminescent signals were generated by incubation with the enhanced chemiluminescence reagent (Amersham, Buckinghamshire, UK).
For determination of the extent of MAPK activation, the cells were lysed in buffer H containing 50 mM ?-glycerophosphate, 1.5 mM EGTA, 1 mM EDTA, 1 mM Na-orthovanadate, 1 mM benzamidine, 1 mM PMSF, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 2 μg/ml pepstatin, and 1 mM DTT. The lysates were then centrifuged for 10 min, followed by a transfer of the supernatants to new tubes and determination of protein concentration. Next, equal amounts of protein were subjected to Western blot analysis. Detection of the immunoreactive band was done, as described above, and the samples were loaded onto 10% acrylamide gel. After electrophoretic separation, the proteins were transferred to a nitrocellulose membrane. Two anti-MAPK antibodies were used for the Western blot analysis: one antibody immunoreacted with the phosphorylated (active) MAPK (pMAPK), whereas the second immunoreacted with both the active and inactive MAPK (total MAPK). The relative amount of the pMAPK in each sample represents the extent of MAPK activation.
Coupling assay
To evaluate the metabolic coupling within the COCs, we incubated isolated intact ovarian follicles with the indicated agents. The follicles were then incised; the recovered COCs were further incubated for 1 h in medium containing radiolabeled (20 μCi) uridine, followed by several extensive washes. Each group was then divided into two subgroups: 1) one subgroup remained intact; and 2) in the other subgroup, the oocytes were freed mechanically from the surrounding cumulus cells. Cumulus-free oocytes and COCs were solubilized in 10% SDS, and their radioactivity was counted in the scintillation liquid. The extent of coupling was calculated as the ratio between the radioactivity counted in the oocyte and that in the cumulus cells, minus the oocyte. This equation takes into account the size of the cumulus. A group of cumulus-free oocytes was incubated with radiolabeled uridine; its radioactivity was counted to confirm the negligible uptake of uridine into the oocytes.
Cultured primary granulosa cells
Granulosa cells were recovered from the ovaries of the above-mentioned female rats. The cells were plated onto serum-coated wells (equivalent to two ovaries per six wells) in 24-multiwell plates (16 mm; Nunc, Copenhagen, Denmark) containing 0.5 ml L-15 medium. Cultures were incubated at 37 C in a humidified incubator for the indicated times.
Scrape-loading
The cultured primary granulosa cells were incubated with or without 3 μg/ml LH, in the presence or absence of 10 μM UO126 for 10 min. After incubation, the plates were washed, and PBS, containing a mixture of 0.7 mg/ml Lucifer yellow (LY) and 5 mg/ml rhodamine dextran (Rh), was added as described in (37). Next, the plates were mechanically scratched with a sharp scalpel and incubated for 3 min. After incubation, the cells were washed several times and fixed with 3% paraformaldehyde. The cells were viewed by fluorescent microscopy.
Patch-clamp analysis
Simultaneous, double whole-cell patch recordings were performed under visual control using infrared DIC optics. All recordings were performed with the current clamp configuration, using AxoClamp2B amplifiers (Axon Instruments, Foster City, CA). The recording pipettes were filled with intracellular solution containing the following (in mM): 4 NaCl, 10–3 CaCl2, 140 K-gluconate, 10–2 EGTA, 4 Mg-ATP, and 10 HEPES (pH 7.2). The voltage and current traces were digitized and stored on the computer, using a National Instrument PCI-MIO-10X DAQ card and LabView software (National Instruments, Austin, TX).
Analysis of electrical coupling.
To compare the level of electrical coupling between different pairs of cells, we calculated the coupling coefficient (CC) from the voltage responses of pre- and postjunctional cells to prolonged (150–300 msec) negative current pulses. Accordingly, CC is defined as the ratio between the voltage responses of the post- and the prejunctional cells (equations 1 and 2). To calculate the actual resistance between the two cells, we employed a method previously described by (38). Briefly, each cell was treated as an isopotential cell, consisting of a resistor (Ri, where i is the cell’s index) and a conductor (Ci), in parallel. Each cell was then coupled to a second cell, via a third resistor (Rc), which models the electrical coupling. Thus,
where V1 and V2 are the voltage responses of cell 1 and cell 2, respectively. Note that the coupling resistor can be asymmetrical. From the circuit diagram, we derived the following equations:
From equations 1–4, we derived:
Statistical analysis
The number of times that each individual experiment was repeated is indicated in the figure legend. Data points are presented as the mean ± SD. Statistical significance was determined by using the ANOVA to assess the differences between multiple experimental groups.
Results
LH stimulates MAPK activation in the ovarian follicle
The activation of MAPK by LH has, thus far, been demonstrated in several granulosa cell lines (28, 30, 31). Our initial experiment was designed to confirm these findings in the ovarian follicle. As shown in Fig. 1A, pMAPK could not be detected in ovarian follicles incubated without LH, whereas their incubation with LH resulted in the activation of the two MAPK isoforms, p44 and p42. This response was observed as soon as 10 min after exposure to the hormone.
FIG. 1. A, Timing of LH-induced MAPK activation in the ovarian follicle. Intact ovarian follicles were incubated with or without LH (1 μg/ml) for the indicated times. After incubation, the follicles were homogenized, and samples of 20 μg of the protein extract were subjected to Western blot analysis with anti-P-MAPK and anti-total-MAPK antibodies. The results of one representative of a total of at least five independent experiments are presented. B, MAPK activation in follicle-enclosed oocytes incubated with LH. Intact ovarian follicles were incubated with 1 μg/ml LH. After the indicated times of incubation, the oocytes were released from the follicles, freed mechanically from the surrounding cumulus cells, and lysed. Samples of 50 oocytes each were subjected to Western blot analysis with anti-P-MAPK and anti-total-MAPK antibodies. The results of one of two independent experiments with similar results are presented.
Our next goal was to examine MAPK in the oocytes that reside within the isolated intact follicles, incubated with LH. As shown in Fig. 1B, both p42 and p44 of MAPK were activated in the oocytes as well. However, whereas activation of MAPK in the follicles was detected immediately upon their exposure to LH, this response in the oocytes was not observed until 8 h after their incubation.
LH-induced resumption of meiosis is mediated by MAPK
The downstream components of the LH-stimulated signal transduction pathway, which lead to oocyte maturation, are still unknown. MAPK, the LH-induced, activation of which is demonstrated here, mediates the induction of mouse oocyte maturation (35). To examine the possible involvement of this kinase in the rat, we impeded its action by using the MEK inhibitor UO126. The U0124 derivative, which does not inhibit MEK, served as a negative control. EGF has also been shown to promote maturation in follicle-enclosed oocytes (3). Because EGF action is known to activate MAPK (39), the effect of UO126 on EGF-induced oocyte maturation was also analyzed.
We initially analyzed the dose of UO126 that effectively inhibited LH-induced MAPK activation in the ovarian follicle (Fig. 2A). This same concentration was further employed to examine its effect on LH-stimulated oocyte maturation. As seen in Fig. 2B, 99% of the control oocytes remained meiotically arrested, as indicated by the presence of GV, whereas LH and EGF induced GVB in 88% and 90% of the oocytes, respectively. The addition of U0126 blocked the effect of LH, as well as that of EGF. U0124 failed to affect LH-induced oocyte maturation.
FIG. 2. A, UO126 inhibits LH-induced MAPK activation in the ovarian follicle. Ovarian follicles were incubated with or without LH (1 μg/ml) in the presence or absence of 10 μM UO126. After 10 min of incubation, the COCs were recovered and lysed. Samples of protein extract were subjected to Western blot analysis with anti-P-MAPK and anti-total-MAPK antibodies. The results of one representative of three independent experiments are presented. B, U0126 inhibits both LH- and EGF-induced oocyte maturation. Ovarian follicles were incubated with either LH (1 μg/ml) or EGF (200 ng/ml) in the presence or absence of either 10 μM UO126 or UO124. After an overnight incubation, the COCs were recovered, and the oocytes were microscopically analyzed for maturation, as described in Materials and Methods. The means ± SD of the percentage of GVB oocytes obtained in five independent experiments, in which 20 oocytes were analyzed for each experimental treatment, are presented. *, Statistical significance of P < 0.05 (ANOVA analysis).
The breakdown of communication in the ovarian follicle is mediated by MAPK
We previously showed that LH reduces intercellular communication within the ovarian follicle and that this response is followed by resumption of meiosis (18). In attempting to decipher the mechanism by which MAPK activation induces reinitiation of meiosis, we examined the possible involvement of MAPK in the LH-induced breakdown of communication. For this purpose, we employed cultured primary granulosa cells. These cells activate MAPK immediately after their exposure to LH (Fig. 3A), which is similar to the previously demonstrated response of the intact ovarian follicles. An evaluation of the transfer of current between two adjacent granulosa cells, by patch-clamp analysis, revealed a reduction in the gap-junction conductance upon LH application. Injecting the current into one cell causes ions to pass through the gap-junctions and to charge the membrane of the coupled cell. A reduction in the conductive properties of this gap junction results in less current to the adjacent cell. Thus, the voltage response of the coupled cell decreases, whereas an increase in response takes place in the injected cell. This corresponds to a decrease in the CC. Indeed, LH caused an increase in the amplitude of the voltage response in the injected cell (Fig. 3B) and a decrease in response in the coupled cell (Fig. 3B), resulting in a decrease in the CC (Fig. 3B). From these parameters, we calculated (see Materials and Methods) a 100% increase in the resistance of the gap-junctions, with a 50% change in the conductive power of the gap-junctions resulting from the application of LH. However, this effect was prevented in the presence of U0126, whereby UO124 did not affect LH-induced uncoupling (the U0124 data are pooled with that of the control). The kinetics of the increase in coupling resistance in response to LH is shown in Fig. 3C, where an initial elevation in the resistance can be observed within seconds, with a maximal increase at 3 min after applying LH. The response of the granulosa cells to LH was not observed in the presence of UO126.
FIG. 3. A, LH-induced MAPK activation in cultured granulosa cells. Granulosa cells were seeded on serum-coated culture plates and incubated with or without LH (1 μg/ml) for 10 min. The cells were harvested, and samples of their proteins were extracted and subjected to Western blot analysis with anti-P-MAPK and anti-total-MAPK antibodies. The results of one representative of a total of three independent experiments are presented. B, LH-induced uncoupling is blocked by U0126. Simultaneous, double whole-cell patch recordings were performed under visual control conditions using infrared DIC optics, as described in Materials and Methods. The effect of LH is presented as the percentage of change compared with the control. Light gray represents the experiments in which 1–3 μg/ml of LH were added to cells cultured with or without U0124 (10 μM). Dark gray represents the results from the experiments in which LH was added to cells incubated with U0126 (10 μM). The means ± SD of the coupling obtained for each treatment in triplicate with three independent experiments are presented. *, Statistical significance of P < 0.05 (ANOVA analysis). C, Coupling resistance as a function of time. The monitoring of coupling resistance before and after administrating LH (1–3 μg/ml, arrow shows time of LH application) with (solid circles) and without (empty circles) 10 μM of U0126. The average resistance before the addition of LH was defined as 100%. The results are presented as the percentage of changes compared with this control. One representative result of a total of individual measurements performed in nine independent experiments is presented.
To confirm the above-mentioned results, we used scrape-loading as a complementary method to assess cell-to-cell communication. This technique is based on the fact that LY and Rh cannot penetrate intact cells. Scratching the cells allows these dyes to penetrate into the cells. However, because LY has a molecular mass smaller than 1 kDa, it can be further transferred between the cells through gap junctions, whereas Rh, which has a higher molecular mass, will stain only the wounded cells. As shown in Fig. 4, under all culture conditions, Rh staining was restricted to the wounded cells, as expected. However, compared with the control, the spread of LY to neighboring cells in the presence of LH was markedly reduced. The addition of UO126 restored the spread of dye, bringing it back to the control level.
FIG. 4. LH-induced breakdown of communication in granulosa cells is inhibited by UO126. Granulosa cells were incubated with or without 3 μg/ml LH in the presence or absence of 10 μM UO126 for 10 min. After incubation, the cells were assayed for intercellular communication by a low-molecular-mass (LY) dye transfer after scrape-loading, as described in Materials and Methods. The high-molecular-mass (Rh) served as a negative control. The results of one representative of two independent experiments, including at least two scrapes per treatment each, are presented. The pictures represent the merge of the LY (green) and Rh (orange).
Metabolic coupling in the COCs was analyzed to examine the possible involvement of MAPK in LH-induced uncoupling between the oocyte and the cumulus cells. As shown in Fig. 5A, incubation of the ovarian follicles with LH reduced the extent of coupling almost to zero; this effect was effectively reversed by UO126.
FIG. 5. A, LH-induced breakdown of communication in the COC is inhibited by UO126. Isolated intact follicles were incubated with or without 1 μg/ml LH. After an overnight incubation, the follicles were incised, and the COCs were analyzed for metabolic coupling, as described in Materials and Methods. The coupling value, determined under control conditions, was defined as 100%. The means ± SD of the results obtained in five independent experiments are presented. Each group receiving the different experimental treatments in every individual experiment included 80 follicles. *, Statistical significance of P < 0.05 (ANOVA analysis), compared with the group of COCs recovered from follicles incubated with LH. B, Radiolabeled uridine in GV and GVB oocytes. Isolated intact follicles were incubated with or without LH (1 μg/ml). After 24 h, the follicles were incised, and the COCs were recovered. The COCs were then incubated in medium containing radiolabeled uridine. After 1 h, the oocytes were freed mechanically from the surrounding cumulus cells. Next, oocytes containing GV were separated from the GVB oocytes. Another group of GV oocytes, recovered from follicles incubated in the absence of LH, was analyzed. The oocytes of all groups were solubilized in 10% SDS, and their radioactivity was counted. The radioactivity detected in the GV group of oocytes, recovered from follicles incubated in the absence of LH, was defined as 100%. The means ± SD of duplicates of 10 oocytes, each in three independent experiments, are presented. *, Statistical significance of P < 0.05 (ANOVA analysis).
For reasons unknown, some follicle-enclosed oocytes incubated with LH did not reinitiate meiosis. We separated the immature (GV) from the mature (GVB) oocytes and counted their radiolabeled uridine content. Radioactivity was also monitored in a control group of GV oocytes, recovered from follicles that were incubated without LH. As shown in Fig. 5B, the amount of radioactivity detected in both groups of GV oocytes is 3-fold higher than that in the oocytes showing GVB. These results indicate that meiotic arrest is associated with intact communication within the ovarian follicle, supporting the notion that uncoupling between the oocyte and the granulosa cells is necessary for reinitiation of meiosis.
MAPK is involved in LH-induced Cx43 phosphorylation
It was previously shown that LH induces phosphorylation of Cx43 in the ovarian follicles after 10 min of exposure to the hormone (8). To examine the possible LH-induced MAPK-mediated Cx43 phosphorylation, we exposed intact follicles, incubated with LH, to UO126, after which we examined the pattern of CX43 phosphorylation. As shown in Fig. 6, LH stimulated the appearance of two additional phosphorylated forms of Cx43. The addition of UO126 (but not UO124) to these follicles prevented the appearance of those two isoforms. This result implies that Cx43 may serve as a substrate for MAPK phosphorylation.
FIG. 6. LH-induced Cx43 phosphorylation is affected by inhibition of the MAPK signaling pathway. Intact ovarian follicles were incubated with 10 μM of either UO126 or UO124 for 1 h, followed by the addition of 1 μg/ml LH. After 20 min of incubation, the follicles were homogenized, and 30 μg from each sample was subjected to Western blot analysis using either anti-Cx43 antibodies or antitubulin antibodies. One representative of a total of six independent experiments is presented. The additional bends that appear with LH and disappear in the presence of UO126 are denoted by arrows.
Discussion
Our study presents evidence that activation of MAPK in the somatic cells of rat ovarian follicles mediates LH-induced oocyte maturation. We further show, for the first time, that the mechanism by which MAPK mediates the LH-stimulated resumption of meiosis involves down-regulation of junctional communication. This response is apparently elicited at the level of Cx43 phosphorylation.
Despite the recent demonstration that oocytes express LH receptors (40), a direct response of the female gamete to this gonadotropin has not yet been reported. Alternatively, the notion that the response of the somatic follicle cells to LH eventually leads to oocyte maturation is widely accepted. Accordingly, we show here that LH activates MAPK in the ovarian follicle cells within minutes, whereas no activation of this kinase was observed in the oocyte before GVB. The maturing oocytes in our experiments exhibited the pMAPK; however, this event takes place only 8 h after exposure of the follicle to LH and apparently does not represent a direct response of the oocyte to the gonadotropin. This late elevation in MAPK activity within the oocyte ostensibly represents a downstream event along the cascade, initiated by MPF activation and followed by mos mRNA polyadenylation and its translation (41). The late activation of MAPK, in a manner that is independent of LH, also occurs in oocytes that mature spontaneously in vitro upon their separation from the ovarian follicle (23). As expected, inhibition of MAPK activation in these isolated oocytes does not interfere with GVB (42). Reinitiation of meiosis, which is independent of MAPK activation in the oocyte, was also demonstrated in mos-null mice. Despite their failure to activate MAPK, the oocytes of these mice resume meiosis in vivo in response to LH (43, 44). Because MAPK activation in the granulosa cells is independent of Mos, their capacity to activate this enzyme in the mos knockout mouse model is not impaired. Under these conditions, LH activates the MAPK signaling pathway in the granulosa cells via Raf1 kinase; its effect on the reinitiation of meiosis in these mice is therefore not inhibited.
The mechanism underlying the signal transduction pathway, which is initiated upon the binding of LH to its receptors on the granulose cells leading to GVB, is still unclear. Based on the commonly accepted notion that LH uses cAMP as its second messenger (32), the obvious assumption was that PKA mediates the effect of LH on the ovarian follicle. Interestingly, later reports demonstrated that LH also activates PKC (34), which makes this kinase an additional candidate for serving this function. It has recently been shown that both PKA and PKC are involved in MAPK activation, suggesting that MAPK acts downstream from PKA and/or PKC in mediating LH action (reviewed in Ref.45). We further confirmed this by demonstrating that LH activates MAPK in isolated granulosa cells within the intact ovarian follicle.
It has recently been reported that MAPK activation in the cumulus cells is essential for resumption of meiosis in the mouse (35). However, the identity and the hierarchy of the downstream events in this signal transduction pathway remain largely unknown. Previous studies have shown that LH decreases intercellular communication between the oocyte and the cumulus cells (16, 17, 18). It has also been reported that LH induces uncoupling of Cx43 gap junction channels in TM3 Leydig cells (46). Taking this information into account, we hypothesized that MAPK may be involved in the LH-induced breakdown of communication and its subsequent resumption of meiosis. To explore this possibility, we employed three complementary methods, each of which uses a specific marker of a different molecular size. The transfer of ions, assessed by the patch-clamp analysis, as well as the analysis of dye transfer, examined by the scrape-loading experiments, confirmed that LH induces the breakdown of junctional communication between the granulosa cells. The patch-clamp analysis, used in our present study, not only confirms the previous findings but also provides a very fine kinetic analysis of the response of granulosa cells to LH. Using this method, we have shown, for the first time, that LH uncouples the granulosa within seconds. This, as well as the complementary scrape-loading assay, done in the presence of UO126, unequivocally revealed that the LH-induced uncoupling between the granulosa cells is mediated by MAPK. The metabolic coupling assay, which quantifies the level of communication within the COC, revealed that inhibition of MAPK activation prevents LH-induced breakdown of communication between the oocyte and the surrounding cumulus cells, suggesting that MAPK is involved in this response as well.
As mentioned before, EGF promotes maturation of follicle-enclosed oocytes (3). Recently, it has been suggested that LH induces the activation of the ovarian granulosa EGF receptors through enhanced expression of certain EGF family members such as amphiregulin, epiregulin, and ?-cellulin (47). However, because the expression of these EGF family member mRNAs could not be demonstrated earlier than 1–3 h after exposure to LH, their mediatory role in stimulating oocyte maturation is somewhat questionable. The well-characterized mechanism, which involves an immediate release of EGF storage by metalloproteinase upon stimulation of G protein-coupled receptors (48), if relevant for the ovary, could overcome this puzzle. If there is further support for the physiological relevance of EGF receptors in the induction of oocyte maturation, our findings confirm that this action is mediated by MAPK (39).
We have previously shown that exposing intact follicles to LH for 10 min results in enhanced phosphorylation of Cx43 (8). We postulated that this phosphorylation might result in some conformational changes in the protein and the consequent closure of the channel. The notion that phosphorylation events occurring on the C-terminal portion of the protein regulate the gating of gap junction channels has been extensively studied (reviewed in Ref.49). Along this line, previous studies have reported that EGF stimulates a rapid disruption of gap junctional communication and enhances phosphorylation of Cx43 (50) on ser255, ser279, and ser282, which are sites of MAPK action (51). Moreover, mutants of Cx43 at these specific sites failed to disrupt junctional communication in response to EGF (52). The changes in the LH-induced Cx43 phosphorylation pattern, in the presence of UO126, imply the possible involvement of MAPK in Cx43 phosphorylation within the ovarian follicle. Further investigations are needed to identify the exact sites of LH-stimulated Cx43 phosphorylation.
In summary, in this study, we proposed a mechanism by which MAPK mediates LH-induced oocyte maturation in the rat ovarian follicle. Our model suggests that MAPK, which is activated in the somatic follicular compartment in response to LH, phosphorylates Cx43. This phosphorylation of Cx43 may result in uncoupling among the follicular cells, which arrests the supply of the inhibitory cAMP and subsequently leads to resumption of meiosis (Fig. 7).
FIG. 7. A proposed model for LH-induced oocyte maturation. Upon LH administration, MAPK in the somatic follicular cells is activated and phosphorylates Cx43. Phosphorylation of Cx43 uncouples the follicle cells and leads to the resumption of meiosis.
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