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Gamete Biology |
Reproductive Medicine Unit, Department of Obstetrics and Gynaecology, University of Adelaide, The Queen Elizabeth Hospital, 5011, Adelaide, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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40% of initial levels. These results, together with our previous studies, demonstrate that treatments that maintain or elevate cAMP levels in cumulus cells, oocytes, or both result in prolonged oocyte-cumulus cell communication and delayed meiotic resumption.
cumulus cells, cyclic adenosine monophosphate, meiosis, oocyte development, phosphodiesterases
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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An intricate network of gap junction transmembrane channels facilitates direct communication between the follicular cells and the oocyte [3]. Gap junctions connect the cumulus cells to each other, and the innermost layer of cumulus cells surrounding the oocyte extend cytoplasmic processes through the zona pellucida, forming gap junctions with the oolemma [4–6]. The gap junctional channel is made up of two symmetrical units called connexons, each consisting of a hexamer of proteins from the connexin family [7]. Connexons are cylindrical organelles that form hemichannels in the cell's plasma membrane and dock with a connexon in an adjacent cell's membrane, forming the intercellular hydrophilic gap junction channel. Gap junction channels metabolically couple the oocyte and the follicular cells to each other, allowing intercellular communication and transfer of low molecular weight (<1000 Mr) substrates such as ions, nucleotides, amino acids, metabolites, and regulatory molecules between the cells that are important for oocyte growth [4, 8, 9].
Many different connexins are expressed in the ovarian follicle, and often, different connexin types are expressed in the same cell type ([10, 11]; for a review of connexins in developing follicles, see [12]). Evidence that gap junctions play essential roles in folliculogenesis and oogenesis has come from the characterization of connexin-knockout mice. Mice deficient in connexin 37 (Cx37), normally found on the oocyte surface beneath the zona pellucida, display arrested folliculogenesis and oocyte growth [13], and oocytes do not achieve meiotic competence [14]. In contrast, antral follicles in ovaries from mice deficient in Cx43 (normally expressed in granulosa cells) fail to properly develop granulosa cell layers, which is detrimental for the oocyte [15]. Gap junctional communication (GJC) allows granulosa cells to provide the oocyte with small molecules that permit oocyte growth, control maturation, and, consequently, to play important roles in oocyte maturation and subsequent embryo development. For this reason, this form of intercellular communication constitutes an important component of cytoplasmic maturation of the oocyte and acquisition of developmental competence.
Gap junctional communication is also likely to influence oocyte meiotic maturation. In vivo and in intact explanted follicles in vitro, oocytes are maintained at the immature germinal vesicle (GV) stage of meiosis, but spontaneously resume meiosis and progress through to the metaphase II stage following artificial isolation from the follicle. This suggests that follicular factors are responsible for the maintenance of meiotic arrest. Several studies have suggested that a loss of GJC between the oocyte and its surrounding cumulus cells in vivo could trigger the resumption of oocyte meiosis due to a cessation in the transfer of meiotic inhibitory substances, such as cAMP, purines, and other putative regulatory molecules from the follicular cells to the oocyte [16–19]. There is abundant evidence that the second-messenger cAMP plays a role in the regulation of oocyte maturation (for a review, see [20, 21]). It has been previously shown in bovine [22, 23] and rodent [24, 25] oocytes that oocyte maturation can be differentially regulated by inhibitors of specific phosphodiesterases (PDEs) as a consequence of subtype compartmentalization of PDEs within the somatic and germ cell compartments of the ovarian follicle. We have shown that subtype 3- and 4-specific PDE inhibitors differentially elevate intracellular cAMP levels in the bovine oocyte and cumulus cells, respectively [22].
It is known that cAMP is also a regulator of GJC in various cell types and tissues [26]. Therefore, the present study was designed to investigate the temporal relationship between the spontaneous onset of bovine oocyte GV breakdown and the breakdown of cumulus cell-oocyte GJC. To investigate this we have developed a new assay for the measurement of cumulus cell-oocyte GJC and have studied the resultant effects of manipulating cAMP in the somatic and germ cell compartments of the in vitro-matured cumulus-oocyte complex using subtype-specific PDE inhibitors.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Bovine ovaries were obtained at an abattoir and transported to the laboratory in warm (29–32°C) saline supplemented with penicillin G (40 IU/ml; Sigma, St Louis, MO) and streptomycin sulphate (40 µg/ml; Sigma), requiring 3–4 h for collection and transport. All ovaries collected on a day were pooled and used at random. Follicle aspiration was performed with an 18-gauge needle and a 10-ml syringe. Follicular contents were aspirated from follicles 2–8 mm in diameter of unknown atresia status and sedimented in 15-ml conical tubes (Falcon, Franklin Lakes, NJ). Cumulus-oocyte complexes (COCs) with intact, compact cumulus investments were selected under a dissecting microscope and transferred to 35-mm Petri dishes (Falcon) containing H-TCM (15 mM HEPES-buffered tissue culture medium-199 [ICN Biomedicals, Costa Mesa, CA], penicillin G [100 IU/ml], streptomycin sulfate [100 µg/ml], and BSA [Fraction V, 4 mg/ml; Sigma]). COCs were washed twice in H-TCM, and then in bicarbonate-buffered TCM-199 (B-TCM) supplemented with sodium pyruvate (0.23 mM), antibiotics, and BSA (4 mg/ml; Fraction V, Sigma) before transfer to respective treatments.
Oocyte Culture
Groups of up to 10 COCs were transferred in 50 µl of B-TCM to individual wells of 48-well multidishes (Falcon). Meiotic inhibitors were added alone or in combination, these being the type 3 PDE-specific inhibitor milrinone (final concentration 100 µM; Sigma), the type 4 PDE-specific inhibitor rolipram (final concentration 100 µM; Sigma), and the adenylate cyclase stimulator forskolin (final concentration 100 µM; Sigma). Millimolar stock concentrations of the meiotic inhibitors were stored at -20°C dissolved in anhydrous dimethyl sulfoxide (DMSO; Hybrimax; D 2650, Sigma) and solutions containing inhibitor were diluted fresh for each experiment. Inhibitors and other agents, diluted in B-TCM, were then added to give a final volume of 500 µl. All COCs were cultured in B-TCM + BSA at 39°C, 96% humidity in an atmosphere of 5% CO2 in air.
Oocyte-Cumulus Cell Gap Junctional Communication Assay
To assess the level of intercellular gap junctional connection between the oocyte and its cumulus investment during in vitro maturation, gap junctional dye transfer from the cumulus cells to the oocyte was measured using the acetoxymethyl (AM) ester derivative of the fluorescent indicator calcein (calcein-AM; 3',6'-Di(O-acetyl)-2',7'-bis[N,N-bis(carboxymethyl) amino methyl]-fluorescein, tetraacetoxy methyl ester; C-3100; Molecular Probes, Eugene, OR). Calcein-AM (MW 994.87) is nonfluorescent, electrically neutral, and highly lipophilic because of the acetoxymethyl groups in the molecule and can rapidly permeate into the cytoplasm through the cell membrane [27–29]. Once inside the cell, nonspecific endogenous esterases cleave the lipophilic acetoxymethyl groups, producing calcein (MW 622.54)—a fluorescent, negatively charged molecule that is unable to leak out of cells across the plasma membrane, but is able to pass between cells connected via gap junctions. Cumulus-oocyte GJC was measured by quantitative fluorescence microscopy as the amount of calcein in the oocyte, transferred from the cumulus cells through gap junctions via passive diffusion (see below).
Individual vials (50 µg) of calcein-AM were stored desiccated at -20°C and reconstituted in DMSO at a concentration of 5 mM fresh for each experiment (calcein-AM in solution is gradually hydrolyzed over time to generate fluorescent calcein). At the concentrations of calcein-AM used, the final concentration of DMSO in any treatment well was <0.1%.
COCs were cultured in B-TCM + BSA (4 mg/ml) with or without either PDE inhibitor (100 µM) for 0, 1, 2, 3, 4, 6, 8, 16, or 20 h, after which they were transferred to a solution of 1 µM calcein-AM freshly made up in a modified phenol red-free and protein-free B-TCM (CAM-BTCM) + polyvinyl alcohol (PVA; 0.3 mg/ml; Sigma) ± PDE inhibitor for 15 min. Phenol red and protein were excluded from the standard oocyte culture media to avoid interference with fluorescence measurement due to nonspecific cleavage of the AM group from calcein-AM. COCs were cultured with the dye for 15 min and were then transferred to calcein-AM-free media with or without the various treatments and cultured for a further 25 min to allow for dye exchange between the cumulus cells and the oocyte. Unincorporated dye was then removed by three washes in calcein-AM-free CAM-BTCM with or without the various treatments. Prior to fluorescence analysis, COCs were completely denuded of their surrounding cumulus cells using vigorous pipetting so that only dye confined within the denuded oocyte after transport via gap junctions was measured. Carbenoxolone (CBX; 3ß-hydroxy-11-oxoolean-12-en-30-oic acid 3-hemisuccinate; Sigma), a known gap junction blocker [30], was used to confirm intraoocyte fluorescence was dependant on and due to conducting gap junctions between the cumulus cells and the oocyte (Fig. 1).
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Within 30 min of denuding, the intraoocyte fluorescence emission of calcein in pulsed oocytes was measured using a fluorophotometric-inverted microscope (Leica, Wetzlar, Germany). Denuded Oocytes (DOs) in the experimental field of view were analyzed singularly and independently from neighboring oocytes. Fluorescence readings of DOs in each replicate experiment are represented as relative fluorescence intensity compared to the t = 0 control DO reading (%).
Assessment of Oocyte Meiosis
Immediately following fluorophotometry, denuded oocytes were transferred to a fixing solution (3:1 ethanol:acetic acid) for >24 h before staining with 1% Orcein (Sigma) to assess for meiotic progression. Fixed oocytes were mounted on slides and compressed beneath a coverslip supported by petroleum jelly and retained with glue. Oocytes were examined with a phase contrast microscope at 400x and classified as being at either GV stage, or as having undergone GV breakdown (GVBD) (meiotic stages diakinesis to metaphase II).
Statistical Analysis
Differences in the proportion of oocytes that had undergone GVBD from the immature GV stage were examined using chi-square analysis. Differences in the levels of GJC between the oocyte and the surrounding cumulus cells, as indicated by measurement of intraoocyte fluorescence intensity, over time and in response to treatment, were assessed using two-way ANOVA analysis (SigmaStat version 2.0 computer software; SPSS Inc., Chicago, IL) and a Student t-test to determine individual differences between treatments at each time point. Probabilities of < 0.05 were considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To demonstrate transfer of fluorescent calcein through the gap junctions connecting the oocyte with its surrounding cumulus cells (as opposed to calcein-AM entry in a nonspecific manner across the oolemma), COCs were treated with increasing doses (0, 1, 3, 9, 27, 81, 243 µM) of the documented gap junction blocker CBX for the first 3 h of culture. After a culture period of 2 h, COCs were pulsed with calcein-AM (15 min), cultured for 25 min to allow dye transfer, denuded, and the intraoocyte fluorescence was measured at approximately 3 h. Calcein dye transfer from the cumulus cells to the oocyte decreased significantly with increasing doses of CBX (Fig. 1, line graph). Treatment of COCs with 27 µM CBX was the most effective dose, blocking 81% of the cumulus-oocyte gap junctions at t = 3 h of culture. Calcein-AM did not spontaneously degrade to fluorescent calcein in the culture media over time, and the contribution of background fluorescence to intraoocyte fluorescence was negligible and was subtracted from the latter. The level of fluorescence measured in untreated oocytes cultured for 17 h fell to 18% of that measured in untreated oocytes at 3 h (Fig. 1, bar graph), and this was comparable (P > 0.05) to 27 µM CBX-treated oocytes at 3 h of culture. This demonstrates that >80% of intraoocyte calcein was transferred from the cumulus cells via gap junctions, whereas <20% was either taken up by the oocyte across the oolemma as calcein-AM, or was representative of a basal level of communication between the oocyte and the cumulus cells. It may also be possible that a portion of this dye was being transferred not through fully mature gap junctions, but through connexin hemichannels. Undocked hemichannels exist in oocytes and cumulus cells, and it has recently been shown in other nonovarian cells, but not yet in mammalian oocytes, that hemichannels can also conduct the passage of small regulatory molecules between neighboring cells [31].
To determine whether intraoocyte fluorescent calcein leaks out of the denuded oocyte over time, control COCs were pulsed with calcein-AM at t = 2 h, denuded, and fluorescence intensity was repeatedly measured at t = 3 h +5, +10, +20, and +30 min. Intraoocyte fluorescence did not change from 0 to 30 min (i.e., 30 min = 3.5 h; Fig. 1, bar graph) following denuding (measurements at 3 h +5, +10, and +20 min not shown), hence allowing the measurement of numerous oocytes in many different treatment groups per time point.
Oocyte-Cumulus Cell Gap Junctional Communication during In Vitro Maturation
During the course of normal oocyte maturation in vitro, GJC between the oocyte and the cumulus cells fell sharply and progressively from 1 h of culture until 9 h (Fig. 2), after which gap junctional communication was more or less absent (at t = 9, 21% of the initial level measured at t = 1 compared with 13% after 16 and 20 h of culture; P > 0.05). This level is comparable to that measured in oocytes treated with 27 µM CBX—the dose that maximally inhibits GJC between the cumulus cells and the oocyte (see Fig. 1).
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Effect of Type 3 and Type 4 PDE Inhibitors on Cumulus Cell-Oocyte Gap Junctional Communication and Meiotic Maturation During Early In Vitro Maturation
Treatment of COCs with the type 3 PDE inhibitor milrinone was able to significantly attenuate (P < 0.05; two-way ANOVA) the loss of GJC that occurs during in vitro maturation. During the first 3 h of culture, treatment of COCs with milrinone did not influence the drop in cumulus cell-oocyte GJC levels, but GJC loss was significantly (P < 0.05) attenuated at 4 h (control, 49%; milrinone, 81% GJC) and 5 h (control, 42%; milrinone, 71% GJC) of culture. From 7 h of culture onward, GJC levels were statistically similar to those of controls (Fig. 3A). Treatment with the type 4 PDE inhibitor rolipram attenuated the loss of GJC at 3 h of culture only (Fig. 4A).
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We have previously demonstrated that milrinone delays bovine oocyte meiotic maturation, as manifested by a delayed MI-MII transition [22]. The results of the current study show that milrinone also delays the resumption of meiosis. The onset of GVBD in control-treated COCs occurred at t = 5 h of culture (17% GVBD), whereas milrinone treatment delayed GVBD onset until 7 h of culture (28% GVBD; Fig. 3B). The time of GVBD onset in control and milrinone treatment groups correlates with a cumulus cell-oocyte GJC level of 42% and 45% of the initial t = 1 h level (Fig. 3A), respectively. Milrinone treatment significantly inhibited GVBD at t = 7 h compared to controls, arresting 2.9 times the number of oocytes at the immature GV stage compared to that of controls (controls, 25% GV; MR, 72% GV; Fig. 3B), and maintained 20% of oocytes at the GV stage even after 20 h of culture [22].
Rolipram treatment did not affect the time of GVBD onset compared to that of controls, however, at 7 h this treatment delayed GVBD and progression to MI compared to control (control, 19% GV, 81% MI; milrinone, 50% GV, 50% MI). Again, GVBD onset generally commenced when GJC had fallen to 40% of initial t = 1 h levels.
Effect of Forskolin With and Without Type 3 and Type 4 PDE Inhibitors on Cumulus Cell-Oocyte Gap Junctional Communication and Meiotic Maturation During Early In Vitro Maturation
Treatment of COCs with forskolin prolonged the maintenance of GJC and enhanced the effect of the type 3, but not the type 4 PDE inhibitor. Forskolin prevented the loss of GJC during the first 4 h of culture, after which levels started to decline but remained greater (P < 0.05) than that of control oocytes at every time point measured until 9 h of culture (Fig. 5A). At 9 h, levels of GJC in forskolin-treated COCs declined to a level that was not significantly different from that observed in control oocytes. After only 2 h of culture, treatment of COCs with forskolin combined with the type 3 PDE inhibitor actually increased the level of cumulus cell-oocyte GJC above that of the initial t = 1 h level (36% greater; Fig. 5A). At this time, milrinone-plus-forskolin (MR+FK) treatment of COCs increased GJC to a level 1.7-fold greater than that observed in control oocytes, and 1.4-fold greater than that in COCs treated with forskolin alone (Fig. 5A). At 3 h of culture, levels of GJC induced by MR+FK treatment began to fall, but they remained above or statistically similar (P > 0.05) to the initial t = 1 h level of GJC observed in control COCs up until 5 h of culture before beginning to decline. At 5 h of culture, GJC levels in MR+FK treated COCs were 2.2-fold greater than that in control COCs. Between 5 and 7 h of culture there was a dramatic loss of GJC in MR+FK treated COCs that continued to 9 h, however, GJC levels at this time remained statistically higher than those observed in control and FK-alone treated COCs. Treatment of COCs with the type 4 PDE inhibitor rolipram in combination with forskolin induced higher levels of GJC than that of forskolin alone, however, this increase was not significantly different (Fig. 6A). Compared to that in MR+FK treated oocytes, the GJC level induced by rolipram plus forskolin was more variable both in terms of that measured in individual oocytes in each treatment group, as well as over time in culture.
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We have previously shown that the combination of forskolin with specific PDE inhibitors markedly delays bovine oocyte meiotic maturation as a consequence of dramatic increases in cAMP in either the COC as a whole, or within the oocyte itself [22]. Results from the current study support this, with the inhibitory effect of forskolin being enhanced in the presence of both the type 3 and 4 PDE inhibitors (Figs. 5B and 6B). The onset of GVBD in control-treated COCs occurred at t = 5 h (36% GVBD), whereas treatment of COCs with forskolin alone delayed GVBD until 7 h of culture (30% GVBD; Table 1) and significantly retarded the progression of meiosis to the MI stage at t = 7 h (20% MI) compared to controls, in which 93% of oocytes had reached the MI stage. Despite its significant inhibitory effect on the resumption of oocyte nuclear maturation, forskolin only transiently prevented GVBD; by 20 h of culture no oocytes were arrested at the immature GV stage (data not shown). This result demonstrates the different efficacy of the type 3 PDE inhibitor milrinone in inhibiting GVBD in which 20% of oocytes are maintained at the GV stage even after extended cultures of up to 36 h [22].
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Combined MR+FK treatment of COCs delayed the onset of GVBD until 9 h (25% GVBD), maintaining 75% of the oocytes at the immature GV stage, compared to 0% and 43% in control and forskolin-alone treated COCs, respectively. Unlike control and forskolin-alone treated COCs, combined MR+FK treatment still maintained 25% of oocytes at the immature GV stage after 20 h of culture and significantly delayed the proportion of oocytes reaching MII stage at this time (control, 100%; forskolin, 25%; MR+FK, 13%). Treatment of COCs with rolipram combined with forskolin (RP+FK) also delayed the onset of GVBD (16% GVBD at 9 h; Fig. 6B) compared to that of control (19% GVBD at 5 h) and forskolin-alone (17% GVBD at 7 h; Table 1). RP+FK delayed GVBD onset to 9 h of culture, after which these oocytes progressed through to MI and MII, however, the population of oocytes maintained at the immature GV stage at t = 17 h was equivalent to that maintained by MR+FK treatment [22]. Again, in all treatment groups, the onset of GVBD generally occurred when GJC had fallen to approximately 40% of the initial level.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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40% of that initially measured at t = 1 h of culture, supporting the idea that GVBD occurs post-GJC breakdown (Table 2).
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Treatments used in the current study attenuated the loss, or increased levels, of GJC between the cumulus cells and the oocyte. The addition of the oocyte type 3 PDE inhibitor milrinone to the culture medium attenuated the drop in GJC over time during early culture. Treatment of COCs with forskolin, a stimulator of adenylate cyclase, was most effective in delaying the loss of GJC, and this effect was augmented in the presence of a PDE inhibitor (most significantly by milrinone, the type 3 oocyte PDE inhibitor), when GJC was elevated above those levels measured at the initiation of in vitro maturation. In a previous study, we have demonstrated that these same treatments dramatically increase intraoocyte cAMP—in which a combined treatment of denuded oocytes with forskolin and milrinone (but not rolipram) caused a 2.8-fold increase in intraoocyte cAMP above that of forskolin alone (which only induced an approximate doubling in cAMP [22]), demonstrating the compartmentalization of PDE subtypes and consequent differential regulation of cAMP within the two compartments of the bovine follicle. In COCs, cAMP synthesized in the surrounding cumulus cells in response to forskolin with or without rolipram treatment was an even greater contributor to intraoocyte cAMP levels, generating an approximate 90-fold increase [22]. Agents that increase intracellular cAMP levels have been observed to increase levels of intercellular GJC in several other cell types and tissues—including those in the ovarian follicle [32–34]—as a result of increased connexin transcription rates, connexin RNA or protein levels [35, 36], altered connexin phosphorylation states [35, 37], altered connexin trafficking to and from cell membranes [38, 39], or changes in the conductance of gap junction channels [37].
Compared to COCs cultured in the presence of milrinone and forskolin, RP+FK treatment induces a massive increase in cumulus cell cAMP levels, which is subsequently transferred to the oocyte [22]. However, it was treatment with MR+FK that induced the most significant increase in cumulus cell-oocyte communication compared to forskolin alone. It is possible that cAMP levels in the two cell types may have different roles or efficacies on cumulus cell-oocyte gap junction conductivity. Alternatively, this lack of statistical significance in response to RP+FK treatment may be explained by the natural variability in the total number of cumulus cells in COCs used in these experiments. Because the cumulus cells are the major contributor to the total cAMP content of the oocyte, variability in cumulus cell numbers would vary the amount of cAMP delivered to the oocyte. Although the importance of oocyte cAMP as opposed to cumulus cell cAMP in regulating spontaneous oocyte meiotic maturation is well documented [22, 24], the relative importance of these two sources of cAMP in regulating cumulus cell-oocyte GJC remains unclear. The type 4 PDE inhibitor rolipram was able to attenuate the drop in GJC at 3 h of culture, but it had little effect on the time of onset of GVBD (Fig. 4). It is possible that cAMP induced in the cumulus compartment by rolipram may be transferred to the oocyte, thus attenuating the loss of GJC at this time. However, in the absence of an oocyte PDE3 inhibitor, up-regulation of PDE3 activity in response to the increase in intraoocyte cAMP, may reduce oocyte cAMP levels to those comparable in control oocytes, allowing GJC levels to decrease and meiosis to resume. Treatment of COCs with forskolin alone is more effective than either PDE inhibitor in delaying GVBD (Table 1), but in the absence of forskolin, milrinone is more effective than rolipram alone. In combination with forskolin however, the type 4 PDE inhibitor rolipram is more effective than milrinone in delaying GVBD, confirming that the cumulus compartment is a significant contributor to the total intraoocyte cAMP content [22], and that this external source of cAMP can significantly alter the kinetics of oocyte meiotic progression [current study].
In the current study, GVBD was consistently observed to occur after GJC had decreased to 40% of that initially measured. Hence these results show a temporal correlation, but not a causal relationship, between the breakdown of cumulus cell-oocyte GJC and the onset of GVBD. This temporal correlation is consistent with a well-established theory that the resumption of oocyte meiosis is a direct result of disrupted transfer of an inhibitory substance or substances from the cumulus cells to the oocyte [18, 40, 41]. In addition to the transfer of factors and molecules required for oocyte growth, gap junction channels between the cumulus cells and the oocyte may facilitate communication of factors that regulate oocyte meiotic maturation. Cyclic AMP, and purines such as hypoxanthine, guanosine, and adenosine are among the molecules suspected to act as meiotic inhibitors due their gap junction-mediated transfer from cumulus cells to the oocyte [42]. The precise relationship between the loss of this coupling and the resumption of oocyte meiosis has been the subject of much controversy, with various studies suggesting that gap junction disconnection precedes meiotic resumption—however, conflicting experiments suggest that the resumption of meiosis precedes the uncoupling of gap junctions (see [43] for a review).
A causal relationship (i.e., a decrease in intercellular communication induces meiotic resumption) has been suggested by some studies in rodent oocytes [33, 41, 44, 45], while other studies in human, porcine [19], ovine, and murine [43] oocytes suggest GVBD precedes the decrease in gap junctions. Hyttel [6] demonstrated a marked decrease in corona cell projections to the oolemma after 3 h of bovine oocyte in vitro culture and observed a close, but not causal, relationship between meiotic resumption and cumulus-oocyte gap junction disconnection. Some studies have suggested cumulus cell-oocyte gap junctions are maintained well after the loss of coupling within the cumulus investment itself [5, 9, 19, 33, 46], whereas Sutovsky et al. [47] observed the disappearance of gap junctions between bovine cumulus cells and the oocyte after 6 h of culture. This result is consistent with those shown in the current study, in which GJC had reduced to 29% of that initially measured and 75% of oocytes had undergone GVBD (by 7 h of culture). The increase in GJC observed in this study (due to prolonged gap junction conductance or the prevention of gap junction removal from the cumulus cell membrane/oolemma as a result of elevated intracellular cAMP) may maintain the transfer of one or more cumulus cell-derived meiosis-modulating factors to the oocyte, thereby delaying the onset of GVBD. It is possible, however, that the decrease in gap junctions is only chronologically correlated with GVBD—that is, elevated oocyte cAMP may inhibit spontaneous maturation through one mechanism, and maintain or increase cumulus cell-oocyte GJC by another completely separate system. Consistent with this suggestion, Wert and Larsen [48] showed that inhibition of gap junctional loss by treatment with a microfilament destabilizing agent did not result in a change in course of GVBD.
The spontaneous maturation of isolated bovine COCs is delayed by elevating or maintaining intraoocyte cAMP levels, or by inducing a massive increase in cAMP in the cumulus cells that surround the oocyte. While 90% to 100% of rodent oocytes are arrested at the GV stage by the type 3 PDE inhibitor milrinone [24, 49], only 20% of bovine oocytes remain indefinitely arrested [22, 23]. As a consequence, the effect of milrinone on cumulus cell-oocyte GJC observed in this study may be underestimated because the level of GJC is determined by averaging intraoocyte fluorescence intensity of the entire treatment group at each time point. To obtain a more accurate appreciation of the role of cumulus cell-oocyte GJC in the resumption of oocyte meiosis, it would be necessary to compare the level of GJC in each individual oocyte with its stage of meiosis at each time point.
In conclusion, this study has utilized a novel assay to measure changes in cumulus cell-oocyte gap junctional communication during in vitro maturation, and has exploited the compartmentalization of PDE4 and PDE3 within the bovine follicle to alter cAMP levels in the somatic and germ cell compartments, respectively. There is a dramatic loss of GJC during in vitro maturation, and this loss is attenuated in response to the PDE3 inhibitor milrinone, and to a lesser extent by the PDE4 inhibitor rolipram. This potentially may alter the capacity of an oocyte to undergo cytoplasmic maturation and to improve oocyte developmental potential. The inhibitory effect of milrinone on spontaneous meiotic resumption is either a direct result of the attenuation in GJC loss, or is a separate phenomenon attributable to the elevation of oocyte cAMP. Cumulus cell production of cAMP (as induced by forskolin and the type 4 PDE inhibitor) is a significant contributor to the total cAMP content of the oocyte, and this alters GJC accordingly. Exploitation of the differential localization of PDE subtypes within the follicle by use of specific PDE inhibitors is a powerful experimental tool to demonstrate the functions of cAMP levels in the two follicular compartments and may prove useful in the elucidation of precise mechanisms regulating mammalian oocyte maturation, with potential for improving oocyte cytoplasmic maturation during in vitro maturation, and therefore in vitro embryo production for assisted reproduction applications in laboratory animals, livestock, and humans.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: FAX: 61 08 8222 7521; robert.gilchrist{at}adelaide.edu.au
Received: 11 July 2003.
First decision: 23 July 2003.
Accepted: 17 October 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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R.E. Thomas, J.G. Thompson, D.T. Armstrong, and R.B. Gilchrist Effect of Specific Phosphodiesterase Isoenzyme Inhibitors During In Vitro Maturation of Bovine Oocytes on Meiotic and Developmental Capacity Biol Reprod, October 1, 2004; 71(4): 1142 - 1149. [Abstract] [Full Text] [PDF]
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