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Endocrinology Vol. 147, No. 5 2280-2286
Copyright © 2006 by The Endocrine Society

Disruption of Gap Junctional Communication within the Ovarian Follicle Induces Oocyte Maturation

Sagit Sela-Abramovich1, Iris Edry1, Dalia Galiani, Nava Nevo and Nava Dekel

Department of Biological Regulation, The Weizmann Institute of Science, Rehovot 76100, 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{at}weizmann.ac.il.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Meiotically arrested mammalian oocytes are stimulated to resume meiosis by LH. This response, which can be reversed by elevation of intraoocyte cAMP levels, is associated with interruption of gap junctional communication (GJC) within the ovarian follicle. In the present study, we examined the hypothesis that disruption of GJC within the ovarian follicle is sufficient for induction of oocyte maturation. For this purpose, we incubated rat follicle-enclosed oocytes with carbenoxolone (CBX), a known blocker of gap junctions. We found that this selective disruptor of GJC promoted maturation of almost all the follicle-enclosed oocytes after 5 h of incubation; this response was also obtained by a transient (2 h) exposure to this agent. CBX-induced oocyte maturation was accompanied by a substantial decrease in intraoocyte concentrations of cAMP that was not associated with elevated activity of type 3A phosphodiesterase (PDE3A). The effect of CBX on reinitiation of meiosis was blocked by isobutylmethylxanthine, a phosphodiesterase inhibitor. Unlike LH, CBX did not activate MAPK in the follicular cells, and inhibition of the MAPK signaling pathway by means of UO126 did not prevent the resumption of meiosis. Injection of CBX into the ovarian bursa of intact animals stimulated maturation in 30% of the oocytes, whereas no maturation was observed in the contralateral ovary injected with PBS. We conclude that, because experimentally induced breakdown of communication within the ovarian follicle is associated with a drop in intraoocyte cAMP concentrations and results in resumption of meiosis, this could be the physiological mechanism employed by LH to stimulate oocyte maturation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MEIOSIS IN OOCYTES is initiated during embryonic life and is arrested at around the time of birth. Resumption of meiosis in mammalian oocytes is stimulated by LH; however, it can also occur spontaneously in oocytes liberated from their follicular environment. Reinitiation of meiosis is morphologically identified by the germinal vesicle breakdown (GVB) that is concomitant with chromosome condensation. The oocyte then proceeds to the first metaphase and, without an intervening interphase, enters the second metaphase and remains arrested at this stage until fertilization. The sequence of events initiated by GVB and completed at the second metaphase leads to the production of a mature fertilizable egg. Therefore this process is defined as oocyte maturation (reviewed in Ref.1).

Mammalian oocytes communicate with the surrounding cumulus cells via gap junctions (2, 3, 4, 5, 6). An extensive gap junctional communication (GJC) network is also present between the cumulus and the granulosa cells, as well as within each of these cellular compartments (reviewed in Ref.7). The protein products of the connexin gene family serve as the building blocks for the gap junctions. Connexin 43, which is most abundant in the ovary (8, 9), is a multiphosphorylated protein, the phosphorylation status of which can determine gap junction conductance and permeability (10). Ovarian connexin 43 is phosphorylated in response to LH (11). This effect is mediated by members of the MAPK family (P44mapk and P42mapk) and results in disruption of cell-to-cell communication in rat ovarian follicles (12).

To date, it is well established that relatively high levels of cAMP within the oocyte are essential to keep the meiotic cycle on hold, whereas a drop in intraoocyte concentrations of this nucleotide enables resumption of meiosis. Nevertheless, the source of the inhibitory cAMP is still a matter of controversy. One theory suggests that the oocyte generates the inhibitory cAMP on its own (13, 14, 15), whereas another claims that meiotic arrest is dependent on the cumulus/granulosa cells for the supply of this inhibitor (16, 17).

According to the first theory, self-production of cAMP by the oocyte takes place in response to a ligand continuously generated by the granulosa cells, that in turn activates an oocyte membrane-bound Gs protein which stimulates oocyte adenylyl cyclase (14, 15). In this case, the spontaneous maturation that occurs upon the release of the oocyte from the ovarian follicle may represent the termination of their exposure to the granulosa-derived Gs-activating ligand.

The second theory proposes that cAMP produced in the surrounding granulosa cells is transferred into the oocyte via gap junctions, thereby maintaining meiotic arrest (16, 17). This hypothesis further suggests that LH-induced oocyte maturation occurs subsequently to interruption of GJC that has been shown to occur in the ovarian follicle in response to this gonadotropin (6, 12, 16, 18). According to this hypothesis, the release of the oocyte from the ovarian follicle, which results in its spontaneous maturation, actually mimics the effect of LH, in that it terminates the supply of the inhibitory cAMP from the granulosa cells to the oocyte. This theory is strongly supported by the recent demonstration of cAMP transport from granulosa cells into the oocyte through gap junctions (19). That same study further revealed that this transfer of cAMP is subjected to negative regulation by gonadotropins (19).

Our present study represents an attempt to challenge the second theory. Experiments were designed to mimic the effect of LH selectively on GJC in the ovarian follicles, as well as to examine the possible consequences of this response on the oocyte. Our results suggest that GJC indeed serves as a key mediator of meiotic arrest in mammalian oocytes, by virtue of providing physiological channels that conduct the supply of the inhibitory cAMP from the somatic follicular cell mass into the oocyte.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and antibodies
Leibovitz’s L-15 tissue culture medium and fetal bovine serum (FBS) were purchased from Biological Industries (Kibbutz Beit Hemeek, Israel). Antibiotics were purchased from Bio-Lab Ltd. (Jerusalem, Israel), U0126 was purchased from Calbiochem (Darmstadt, Germany), pregnant mare’s serum gonadotropin (PMSG) was purchased from Chrono-gest Intervet (Boxmeer, The Netherlands), ovine LH (o-LH-26) was purchased from NHPP (Harbor-UCLA Medical Center, Torrance, CA), carbenoxolone (CBX) was purchased from Sigma (St. Louis, MO), and isobutylmethylxanthine (IBMX) was also purchased from Sigma. L-15 tissue culture medium and FBS both were purchased from Biological Industries. Phenylmethylsulfonyl fluoride (PMSF), leupeptin, aprotinin, and dithiothreitol were purchased from Sigma. Monoclonal mouse anti-phosphorylated MAPK (pMAPK) antibodies were kindly provided by Prof. Rony Seger (Weizmann Institute of Science, Rehovot, Israel). 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).

Ovarian follicles culture
Follicle-enclosed oocytes (FEOs) were recovered from sexually immature PMSG-primed 25-d-old female Wistar rats. The rats were killed by cervical dislocation 48 h after PMSG administration, their ovaries were removed, and the large antral follicles were separated. The isolated intact follicles were incubated in suspension in L-15 tissue culture medium containing 5% FBS in 25-ml flasks, supplied with 50% O2 and 50% N2. Incubations were carried out at 37 C in an oscillating water bath in the presence or the absence of either 1.5 µg/ml of ovine LH, or 100 µM of CBX with or without 10 µM of UO126 (a specific inhibitor of MAPK kinase) or IBMX (0.2 mM) that were added 1 h before the addition of the hormone. 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 a differential interference contrast (standard WL research microscope, Carl Zeiss, Oberkochen, Germany). The GVB served as a morphological marker for reinitiation of meiosis.

All experiments were conducted in accordance with the Guides for the Care and Use of Laboratory Animals (National Research Council, National Academy of Sciences, Washington, DC).

Protein extraction and Western blot analysis
To determine the extent of MAPK activation, the ovarian follicles were lysed in RIPA buffer containing 50 mM ß-glycerophosphate, 20 mM Tris (pH 7.4), 137 mM NaCl, 10% glycerol, 0.1% SDS, 1% Triton X-100, 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, and 2 µg/ml pepstatin. The lysates were then centrifuged for 10 min, after which the supernatants were collected and their protein concentrations were determined. Equal amounts of protein (30 µg) were dissolved in protein sample buffer (2% ß-mercaptoethanol, 2% SDS, 50 mM Tris, HCl (pH 6.8), 10% glycerol, 0.01% bromophenol blue), boiled, and loaded on 10% SDS-PAGE. After electrophoretic separation, the proteins were transferred to nitrocellulose membrane.

For the Western blot analysis, two anti-MAPK antibodies were used. One antibody immunoreacts with the pMAPK (active), whereas the second immunoreacts with both the active and the inactive MAPK (total MAPK). The relative amount of pMAPK in each sample represents the extent of MAPK activation.

Culture of granulosa cells
Granulosa cells were recovered from the ovaries of the aforementioned female rats. The cells were plated onto serum-coated 24-multiwell plates (Nunc, Copenhagen, Denmark) containing 0.5 ml of L15 medium. Cultures were incubated at 37 C in a humidified incubator for 48 h to obtain a confluent cell layer.

Scrape loading dye transfer assay
The cultured granulosa cells were incubated with or without either CBX (100 µM) or LH (10 µg/ml) for 30 min. After incubation, the plates were washed, and PBS containing a mixture of 0.7 mg/ml lucifer yellow (LY; Sigma) and 5 mg/ml rhodamin dextran (Rh; Molecular Probes, Eugene, OR) was added as previously described (20). The plates were mechanically scratched with a sharp scalpel. After an additional 5 min of incubation, the cells were washed several times with PBS and fixed with 3% paraformaldehyde. The cells were then viewed by laser scanning confocal imaging system (Bio-Rad; radiance 2000/AGR-3) connected to Nikon T200 microscope (Melville, NY).

Intrabursal injection
To study the effect of CBX on oocyte maturation in vivo, intrabursal injection was performed on sexually immature PMSG-primed Wistar female rats (21). Rats were lightly anesthetized, and one of their ovaries was exteriorized through a small lumbosacral incision. Either CBX (100 µl of 100 mM in PBS) or PBS alone (100 µl) were injected through a 30-gauge needle threaded into the ovarian bursa via the adjoining fat pad. After injection, the ovary was returned to the abdominal cavity and skin was clipped. For positive control, LH (100 µl of 0.1 mg/ml) was administrated by intrabursal injection. After 6 h, the rats were killed by means of cervical dislocation, and the COCs were collected from the largest ovarian follicles to examine the occurrence of GVB oocytes.

Determination of cAMP levels in oocytes
Ovarian follicles cultured as described above were incubated for 2.5 h with either 1.5 µg/ml of o-LH or with 100 µM CBX. The follicles were then incised in medium containing 0.2 mM IBMX. COCs were retrieved in the presence of IBMX, and the oocytes stripped and washed in PBS before their suspension in 0.1 N HCl and flash freezing in liquid nitrogen. The measurement of cAMP content in these oocytes was conducted using a cAMP (low pH) immunoassay kit (R&D Systems, Minneapolis, MN).

Phosphodiesterase (PDE) activity assay
COCs were collected from treated follicles and flash frozen in liquid nitrogen, before homogenization in isotonic buffer containing 10 mM sodium phosphate buffer, pH 7.2 (prepared from Na2HPO4 and NaH2PO4), 50 mM NaF, 150 mM NaCl, 2 mM EDTA, 5 mM ß-mercaptoethanol, 30 mM sodium pyrophosphate, 3 mM benzamidine, 5 µg/ml leupeptin, 20 µg/ml pepstatin, 2 mM PMSF, and 1 mM microcystin (all from Sigma). The homogenates were centrifuged at 4 C for 30 min at 14,000 rpm to obtain a soluble fraction. PDE activity was assayed using 1 µM cAMP (Sigma) as substrate, according to the method described by Thompson et al. (22). Samples were assayed at 34 C at a final volume of 200 µl of a solution consisting of 40 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 5 mM ß-mercaptoethanol, 1 mg/ml BSA, and 1 µM cAMP (all from Sigma) and 25 nM [3H]cAMP (Amersham Biosciences, Piscataway, NJ) (~0.1 x 106 cpm/tube, 20 Ci/mmol). To determine the contribution of oocyte-specific PDE (PDE3A) to the overall PDE activity of COCs, which is also comprised from the activity of PDE4 expressed in the cumulus/granulosa cells, the specific PDE4 inhibitor rolipram (10 µM) (Sigma) was added to the incubation mixture.

Statistical analysis
The number of repetitions of each individual experiment is indicated in the figures. Statistical significance was determined by the ANOVA method, used to assess differences between multiple experimental groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CBX interrupts GJC between granulosa cells
To confirm the effect of CBX on GJC between granulosa cells, the scrape-loading dye transfer assay was employed (Fig. 1Go). This assay takes into consideration the fact that LY and Rh do not penetrate intact cells (20). However, both these dyes can enter the cytoplasm of injured cells. Because LY has a molecular weight of less than 1 kDa, it flows transcellularly through gap junctions. On the other hand, Rh, which is a larger molecule (10 kDa), remains within the injured cells. As seen in Fig. 1Go, the spread of LY to neighboring cells, clearly observed under control conditions, is substantially reduced in the presence of either LH or CBX. As expected, under all culture conditions, Rh staining was restricted to the injured cells.


Figure 1
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  		FIG. 1. CBX blocks GJC between granulosa cells. Granulosa cells incubated with or without either LH (10 µg/ml) or CBX (100 µM) were subjected to the scrape loading assay as described in Materials and Methods. The results of one of two independent experiments with similar results, including at least two scrapes per each treatment, are presented. The pictures represent merge of the LY (green) and Rh (orange) fluorescent images.

 
CBX induces FEO maturation without cumulus expansion
Having confirmed that CBX interrupts GJC in the ovarian follicle cells, the effect of this gap junctions blocker on the meiotic status of the FEOs was further analyzed. As shown in Fig. 2AGo, an overnight incubation of ovarian follicles with CBX stimulated almost 90% of the oocytes to resume meiosis. This effect was inhibited by IBMX. Moreover, oocytes recovered from follicles that were transiently incubated with CBX for 2 h and further incubated in CBX-free medium for 3 h resumed meiosis, whereas a transient exposure that is shorter than 2 h resulted in a partial response (Fig. 2BGo).


Figure 2
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  		FIG. 2. CBX induces FEO maturation without cumulus expansion. A, Ovarian follicles were incubated overnight with CBX (100 µM) in the presence or absence of IBMX (0.2 mM). At the end of incubation, the COCs were recovered and the oocytes were monitored for maturation. Statistically significant difference vs. CBX incubation (P < 0.05) is indicated by an asterisk (*). B, Ovarian follicles were transiently incubated with CBX for the indicated times and then transferred to CBX-free medium for a total incubation time of 5 h. At the end of incubation, the COCs were recovered and the oocytes were monitored for maturation. The mean ± SD of the percentage of GVB oocytes obtained from three independent experiments are presented. Statistically significant differences vs. control (P < 0.05) are indicated by an asterisk (*). C, COCs were recovered after an overnight incubation of ovarian follicles with or without either LH (1.5 µg/ml) or CBX (100 µM), and microscopically analyzed for their expansion. The results of one of three independent experiments with similar results are presented.

 
LH-stimulated oocyte maturation is accompanied by the expansion of the cumulus mass, and the secretion of a mucoid extracellular material (17). We found that, unlike the response to LH, no expansion of the cumulus could be observed in the presence of CBX. In fact, the appearance of cumuli recovered from follicles incubated with CBX was indistinguishable from that of the control group (Fig. 2CGo).

CBX decreases cAMP concentrations in FEOs with no elevation in PDE3A activity
As noted above, resumption of meiosis is associated with a decrease in intraoocyte concentrations of cAMP. It was previously suggested that the inhibitory cAMP is synthesized in the granulosa cells and transferred in turn to the oocyte via gap junctions (reviewed in Ref.23). More recent studies claimed that the oocyte generates the inhibitory cAMP on its own (14, 15).

If the intraoocyte cAMP is indeed supplied by the granulosa cells, closure of GJC will result in a decrease in cAMP concentrations within the oocyte. To test this assumption, the concentrations of cAMP in FEOs incubated with either LH or CBX were determined. As illustrated in Fig. 3AGo, FEOs incubated under control conditions contain an average of 1.05 fmol cAMP. Administration of either LH or CBX to the incubation medium resulted in a substantial decrease (60%) in intraoocyte cAMP concentrations. To verify that this decline in cAMP concentration was not due to increased degradation of cAMP, we examined the levels of activity of the oocyte-specific phosphodiesterase, PDE3A, in FEOs exposed to CBX. As seen in Fig. 3BGo, CBX did not up-regulate the activity of PDE3A.


Figure 3
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  		FIG. 3. cAMP concentrations and PDE3A activity in FEO incubated with CBX. A, Ovarian follicles were incubated with or without either CBX (100 µM) or LH (1.5 µg/ml) for two and a half h. Oocytes were isolated in the presence of IBMX (2 mM) and analyzed for cAMP concentration as described in Materials and Methods. Statistically significant difference vs. control (P < 0.05) is indicated by an asterisk (*). B, Ovarian follicles were incubated with or without CBX (100 µM) for the indicated times and analyzed for PDE3 activity as described in Materials and Methods. Data are expressed as mean number of FEOs ± SEM.

 
CBX-induced FEO maturation is independent of MAPK activation in granulosa cells
Previous studies in both mouse and rat have shown that activation of MAPK in the ovarian follicle mediates LH-induced resumption of meiosis (12, 24). Moreover, MAPK is involved in the LH-induced disruption of cellular communication within the ovarian follicle (12). We assumed that interruption of GJC by CBX in the ovarian follicle is independent of MAPK activation. In support of this assumption, we show that MAPK is not activated for at least 2 h of incubation with CBX (Fig. 4AGo). However, a transient (2 h) exposure of ovarian follicles to CBX requires additional incubation in CBX-free medium, if meiosis is to resume (Fig. 2BGo). We also examined whether ovarian follicles preincubated for 2 h with CBX would induce MAPK activation after their extended incubation in CBX-free medium. As shown in Fig. 4AGo, no activation of MAPK could be detected under these conditions as well. Complementary evidence that CBX-induced resumption of FEO maturation is independent of MAPK was provided by the use of UO126, a known inhibitor of the MAPK signaling pathway. UO126 was found to have no effect on CBX-induced FEO maturation, supporting the previous results (Fig. 4BGo).


Figure 4
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  		FIG. 4. CBX induces FEO maturation independently of MAPK activation. A, One group of ovarian follicles was incubated with or without either CBX (100 µM) or LH (1.5 µg/ml) for the indicated time points. Another group of ovarian follicles was initially incubated with CBX for 2 h then washed and further incubated in either CBX-free medium or LH for an additional 15 min. At the end of incubation, the follicles were subjected to Western blot analysis using either anti-pMAPK or anti-total-MAPK. The results of one of three independent experiments with similar results are presented. B, Ovarian follicles were incubated overnight with 100 µM CBX in the presence or absence of 10 µM UO126. At the end of incubation, the COCs were recovered and the oocytes were monitored for maturation. The mean ± SD of the percentage of GVB oocytes obtained from two independent experiments are presented. C, MAPK activation in FEO incubated in the presence of CBX. Ovarian follicles were incubated with or without CBX (100 µM). After the indicated times, the oocytes were released, freed mechanically from the surrounding cumulus cells, and lysed. Samples of 60 oocytes each were subjected to Western blot analysis using either anti-pMAPK or anti-total-MAPK. The results of one of two independent experiments with similar results are presented.

 
We previously demonstrated that spontaneously maturing oocytes, as well as FEOs stimulated by LH, activate their MAPK 8 h after reinitiation of meiosis (12, 25). We found that FEOs incubated with CBX also activate their MAPK at this same later time point (Fig. 4CGo).

Intrabursal injection of CBX results in resumption of meiosis
Complementary in vivo evidence that interruption of GJC is sufficient for resumption of meiosis was provided by the injection of CBX into the ovarian bursa of intact animals. Six hours later, the ovaries were removed and the COCs were collected and examined for evidence of oocyte maturation. In each experiment, the contralateral ovary that was either injected by PBS or left untreated served as negative control. LH injected into the bursa served as a positive control.

As seen in Fig. 5Go, intrabursal injection of CBX induced oocyte maturation in 30% of the oocytes, whereas no mature oocytes were observed in the contralateral ovary. Injection of LH into the bursa resulted in a greater degree (60%) of oocyte maturation. Unlike injection of CBX, oocytes in the contralateral ovary of LH-treated rats were also stimulated to resume meiosis.


Figure 5
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  		FIG. 5. Intrabursal injection of CBX induces oocyte maturation. Either CBX (100 mM, n = 11) or LH (0.1 µg/ml, n = 5) were injected into the ovarian bursa of PMSG-primed female Wistar rats, as described in Materials and Methods. The contralateral ovary that served as a control was either untreated or injected with PBS. After 6 h, the ovaries were recovered, the ovarian follicles were punctured, and the oocytes were released and microscopically monitored for maturation. The number of rats per treatment is indicated. The mean ± SD of the percentage of GVB oocytes are presented. Statistically significant difference vs. PBS injection (P < 0.05) is indicated by an asterisk (*).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we demonstrate that interruption of GJC within the ovarian follicle that takes place either in vivo, in the intact animal, or in vitro, in organ culture, effectively promotes the resumption of meiosis. Moreover, transient exposure of the ovarian follicle to CBX, which blocks gap junctions, forces an irreversible commitment of the enclosed oocyte to reinitiate meiosis. This effect of CBX, which is accompanied by a decrease in intraoocyte cAMP concentrations, is reversed by the phosphodiesterase inhibitor IBMX. Unlike the response to LH, the resumption of meiosis that follows exposure of the ovarian follicle to CBX is independent of MAPK activation in the granulosa cells, and is not accompanied by cumulus expansion. These findings seem to deny the catalytic capacity of the oocyte to generate cAMP that is sufficient to maintain meiotic arrest. They further provide evidence strongly supporting the idea that down-regulation of the gap junction-mediated cAMP supply derived from the follicle cells is a causal factor for reinitiation of meiosis.

As mentioned in the introduction, in parallel to the idea underlined by our findings, an additional theory suggests that the oocyte itself generates the inhibitory cAMP that is sufficient for maintaining meiotic arrest. In support of this theory, expression of adenylyl cyclase by rodent oocytes (13), as well as its response to forskolin has been reported (26, 27). More recently, Mehlmann et al. (15) demonstrated that microinjection of antibodies against Gs into mouse FEOs resulted in resumption of meiosis. Along this line, a knockout mouse model of GPR3, a G protein-coupled receptor that activates Gs (28, 29, 30), is characterized by females bearing mature oocytes in their antral follicles independently of LH stimulation (14). However, the ligand that activates GPR3 remains unidentified, and the precise regulatory mechanism employed by LH to terminate its action has not yet been determined. Furthermore, the aforementioned knockout model was not oocyte-specific; hence, GPR3 was omitted from granulosa cells as well and the catalytic capacities of cAMP production in these granulosa cells has not been reported (14).

Self generation of inhibitory cAMP by the oocyte was also suggested by another study that hypothesized the existence of a positive stimulus that overcomes meiotic arrest (31). According to this study, insulin-like 3, generated by theca cells in response to the LH surge, binds the GPR8 expressed on the oolema, leading to the activation of an oocyte-inhibitory G protein (Gi) and a subsequent down-regulation of cAMP production by the oocyte. The resultant drop in intraoocyte cAMP concentrations in turn enables the resumption of meiosis. However, because theca cells express the receptor for LH from the early stages of folliculogenesis (32), a response to LH that is mediated by these cells would fail to select the large antral preovulatory follicles. Moreover, the requirement for a positive stimulus originating in follicle cells does not apply to spontaneous maturation of isolated oocytes.

It was previously shown that LH-induced oocyte maturation is associated with disruption of GJC in the ovarian follicle (5, 12, 16, 18, 33, 34). Taking this association into account, it has been suggested that the stimulatory effect of LH on reinitiation of meiosis is mediated by the closure of gap junctions (reviewed in Ref.23). Our present study revisited this concept by the use of CBX, a pharmacological agent that blocks gap junctions (19, 35, 36, 37). CBX provided us with a highly advantageous experimental tool, because it enabled us to single out one specific response of the ovarian follicle to LH, out of several others.

In a previous study, we demonstrated that in the rat, both LH-induced down-regulation of GJC and LH-induced oocyte maturation are mediated by MAPK (12). An earlier study showed that, similar to the rat, mouse oocyte maturation is also dependent on MAPK and that the activation of this signaling pathway is a prerequisite for cumulus expansion (24). By using CBX, whose activity is specifically directed at the level of the gap junction, we were able to dissect the interruption of GJC from its upstream regulatory MAPK signaling pathway. Interestingly, we show in this study that CBX enabled FEOs to resume meiosis in the absence of an active MAPK, but failed to promote cumulus expansion under these conditions. These results suggest that, unlike the dispersal of the cumulus, oocyte maturation takes place downstream to breakdown of communication. Therefore, our experimental system, in which CBX bypassed the necessity for MAPK activation in stimulating oocyte maturation, enabled us to confirm that breakdown of GJC within the ovarian follicle is a causative event for the induction of oocyte maturation. This outcome of disruption of GJC was further confirmed in vivo, by intrabursal injections. Direct application of a saturated concentration of CBX to the ovary resulted in resumption of meiosis of more than 30% of the ovarian oocyte.

Reinitiation of meiosis is commonly diagnosed by the disappearance of the germinal vesicle. This morphological marker apparently represents a distal end point along a cascade that is initiated by a decrease in intraoocyte cAMP levels, and continues with the inactivation of cAMP-dependent protein kinase A; this is followed by the elevation of maturation promoting factor activity and apparently other biochemical events that are not yet fully elucidated (reviewed in Ref.38). To precisely detect the timing of the onset of CBX-induced oocyte maturation, we transiently exposed FEOs to this pharmacological agent, monitoring the oocyte for GVB after an additional incubation period in CBX-free medium. This experiment was designed to pinpoint the time at which the oocyte becomes irreversibly committed to resume meiosis. We found that a transient exposure of 2 h to CBX followed by 3 h of incubation in a CBX-free medium induced GVB in a fraction of oocytes that was as high as that obtained after a continuous 5-h incubation with this disruptor of GJC. A transient exposure that was shorter than 2 h resulted in a partial GVB response. The scrape loading dye transfer assay performed to characterize the effect of CBX demonstrated that GJC in cultured granulosa cells was disrupted within 30 min of incubation. Taken together, these temporal relationships suggest that interruption of GJC within the ovarian follicle takes place before the onset of the intraoocyte events that give rise to GVB.

We further hypothesize that the resultant cessation of transfer of inhibitory cAMP from the follicle to the oocyte provides the ultimate trigger for reinitiation of meiosis. In support of this notion, we demonstrate in this study that the positive effect of CBX on meiosis is associated indeed with a drop in the levels of intraoocyte cAMP concentration. Along this line, a recent study revealed that CBX effectively inhibits the flow of cAMP from the cumulus cells into the oocyte (19). The decline in intraoocyte cAMP apparently represents the termination of its supply from the somatic follicular cells, which would obviously not take place in the absence of the hydrolyzing activity of PDE within the oocyte. Indeed, inhibition of PDE activity by the addition of IBMX blocked the CBX-induced oocyte maturation.

The possibility that the drop in cAMP levels reflects its decreased supply, rather than enhanced PDE-catalyzed hydrolysis, was examined by determining the levels of activity of this enzyme in oocytes exposed to CBX. Of eleven tissue-specific PDE families identified in mammals (39, 40), PDE3A was shown to be expressed in rat oocytes, whereas PDE4D and PDE4B were exclusively observed in mural granulosa and in theca cells, respectively (41). The availability of type-specific PDE inhibitors enabled us to single out the activity of type 3 PDE when assayed in COCs in the presence of the PDE4 inhibitor, rolipram. Our results indeed demonstrate that the activity of PDE3A is not enhanced in oocytes exposed to CBX.

In summary, cAMP could possibly be generated by both the oocyte as well as the granulosa cells. Nevertheless, the catalytic capacity of rat oocytes to generate cAMP is insufficient to maintain meiotic arrest (18). Therefore, it appears that the decline in the gap junction-mediated cAMP supply derived from granulosa cells is a causal factor for reinitiation of meiosis. We speculate that the effect of CBX on GJC in the ovarian follicle, which is followed by a decrease in intraoocyte cAMP concentrations, faithfully represents the mechanism employed by LH to induce the resumption of meiosis.


   Acknowledgments
 
We thank Ms. B. Morgestern for her excellent editorial assistance.


   Footnotes
 
This work was supported by the Dwek Fund for Biomedical Research. N.D. is the incumbent of the Phillip M. Klutznick Professorial Chair of Developmental Biology.

S.S., I.E., D.G., N.N., and N.D. have nothing to declare.

First Published Online January 26, 2006

1 The first two authors contributed equally to this study. Back

Abbreviations: CBX, Carbenoxolone; COC, cumulus-oocyte complex; FBS, fetal bovine serum; FEO, follicle-enclosed oocyte; GJC, gap junctional communication; GVB, germinal vesicle breakdown; IBMX, isobutylmethylxanthine; LY, lucifer yellow; PDE, phosphodiesterase; PDE3A, type 3A phosphodiesterase; pMAPK, phosphorylated MAPK; PMSF, phenylmethylsulfonyl fluoride; PMSG, pregnant mare’s serum gonadotropin; Rh, rhodamin dextran.

Received August 8, 2005.

Accepted for publication January 13, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Dekel N 1995 Molecular control of meiosis. Trends Endocrinol Metab 6:165–169
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