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Gamete Biology |
Research Centre for Reproductive Health, 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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cumulus cells, cyclic adenosine monophosphate, embryo, meiosis, phosphodiestrases
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Delaying the process of spontaneous in vitro meiotic maturation may permit a more complete oocyte cytoplasmic maturation to occur, granting the oocyte a greater capacity for fertilization and subsequent embryonic development [7]. The rationale behind this approach of delaying or temporarily inhibiting nuclear maturation is to allow for continued mRNA and protein accumulation within the ooplasm. Spontaneous oocyte maturation has been prevented using various agents such as inhibitors of protein synthesis or phosphorylation (e.g., cycloheximide and 6-dimethylaminopurine [8]), or inhibitors of specific cyclin-dependent kinases [9] (e.g., butyrolactone I [10–12] and roscovitine [13–15]), after which the inhibitor is removed and maturation is allowed to continue. Whereas in general these studies show that manipulation of spontaneous meiotic progression adversely affects oocyte developmental potential, an actual improvement in oocyte quality, as reflected in improved embryo development following fertilization, may be achievable using this approach [12].
It is well known that the second-messenger cAMP plays a role in the regulation of mammalian oocyte maturation— particularly in that of rodent oocytes (for reviews see [16, 17]). In stark contrast to that observed with rodent and primate oocytes, treatment of oocytes from most ungulate species (including bovine oocytes) with chemicals that maintain high levels of cAMP exert only a transitory, inhibitory effect on GVBD [18–24]. Despite this, Funahashi et al. [25] and Luciano et al. [26, 27] demonstrated that treatment of porcine and bovine oocytes with cAMP-elevating agents dibutyryl-cAMP and invasive adenylate cyclase, respectively, during IVM resulted in increased rates of zygote cleavage and development to the blastocyst stage.
In recent studies using bovine [23, 24, 28], rodent [29– 31], macaque [32], and human [33] oocytes, we and others have shown that oocyte maturation is differentially regulated by isoenzyme-specific phosphodiesterase (PDE) inhibitors, as a consequence of PDE isoenzyme compartmentalization within germ cells (which contain the type 3, but not the type 4 PDE) and somatic cells (which contain the type 4, but not the type 3 PDE) of the ovarian follicle [29]. PDE inhibitors, specific for either the type 3 (milrinone) or type 4 (rolipram) PDEs, prevent cAMP degradation, resulting in intracellular cAMP accumulation in the bovine oocyte or cumulus cells, respectively [23]. As such, under conditions in which cAMP synthesis is stimulated, milrinone (but not rolipram) increases intraoocyte, but not cumulus cell (CC) cAMP, whereas rolipram (but not milrinone) increases granulosa/CC cAMP levels, but not oocyte cAMP levels [23]. Most recently, we have demonstrated that treatment of oocytes with PDE inhibitors maintains or upregulates (or both) CC-oocyte gap junctional communication (GJC), which is associated with or may actually cause a delay in meiotic resumption [24]. We and others believe that increased GJC between the oocyte and the CCs in addition to prevention of precocious nuclear maturation, may allow for improved oocyte cytoplasmic maturation and therefore increased capacity for subsequent embryo development. In line with this idea, Nogueira et al. [34] recently demonstrated that rodent oocytes arrested with a type 3 PDE inhibitor had significantly improved fertilization rates, and comparable rates of preimplantation and production of live offspring, compared with those oocytes collected following IVM (although GJC levels were not examined).
An intricate network of gap junction transmembrane channels facilitate direct communication between follicular cells and the oocyte [35], subsequently allowing the surrounding cumulus and granulosa cells to provide the oocyte with small molecules (such as ions, nucleotides, metabolites, amino acids, purines, and cAMP) that enhance oocyte growth and the control of maturation. It follows that GJC would therefore constitute an important component of cytoplasmic maturation of the oocyte, and therefore also an important component in the acquisition of developmental competence. Prolonging this form of communication between the CCs and the oocyte during IVM—through the addition of cAMP-altering agents such as isoenzyme-specific PDE inhibitors—may improve oocyte developmental capacity and prove to be a beneficial component of oocyte IVM culture systems.
Therefore, the present study was designed to investigate the effect of isoenzyme-specific PDE inhibitors during IVM on the developmental competence of bovine oocytes following in vitro fertilization (IVF) and embryo culture. Using a recently developed assay to quantitate levels of communication between the CCs and the oocyte [24] we have examined the effects of manipulating cAMP in the somatic and germ cell compartments with isoenzyme-specific PDE inhibitors on the in vitro-matured cumulus-oocyte complex (COC) and related this to changes in oocyte cytoplasmic quality.
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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 sulfate (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). COCs with intact, compact cumulus vestments were selected under a dissecting microscope and transferred to 35-mm Petri dishes (Falcon) containing follicular fluid. COCs were washed twice in bicarbonate-buffered TCM-199 with Earl salts (B-TCM; ICN Biotech, Irvine, CA) supplemented with sodium pyruvate (0.23 mM; Sigma), antibiotics, and fatty acid-free BSA (FAF-BSA, 4 mg/ml; ICP Bio, New Zealand) before they were transferred to respective treatments.
Oocyte Culture
Groups of up to 10 COCs destined for the CC-oocyte GJC assay were transferred in 50 µl of B-TCM to individual wells of 48-well multidishes (Falcon), while groups of up to 50 COCs were transferred in 50 µl of B-TCM to Nunc (Roskilde, Denmark) 4-well dishes for the meiotic assessment experiments. COCs destined for both the GJC assay and for assessment of progression through meiosis were treated with combinations of the type 3 PDE specific inhibitor milrinone (100 µM; Sigma), the type 4 PDE specific inhibitor rolipram (100 µM; Sigma), recombinant human FSH (0.1 IU/ml; Organon, The Netherlands), or recombinant human hCG (0.1 IU/ml; Serono, Sydney, Australia). Interaction of the PDE inhibitors with gonadotropins were examined first, because FSH in particular, is a standard and essential additive to IVM media used for routine embryo in vitro production, and second, because PDE inhibitors have low efficacy in increasing COC cAMP in the absence of adenylate cyclase stimulators. 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 gonadotropins diluted in B-TCM were then added to the culture well to give a final volume of 500 µl. All COCs were cultured in B-TCM + FAF-BSA at 38.5°C, 96% humidity in an atmosphere of 5% CO2 in air.
Oocyte-CC GJC Assay
To assess the level of intercellular gap-junctional connection between the oocyte and its cumulus vestment during IVM, gap-junctional transfer of the fluorescent dye calcein from the CCs to the oocyte was measured as previously described [24] and briefly outlined below. Cumulus-oocyte GJC was measured by quantitative fluorescence microscopy as the amount of calcein in the oocyte.
COCs were cultured in B-TCM + FAF-BSA (4 mg/ml) in the presence or absence of FSH, with or without either PDE inhibitor (100 µM) for 0, 1, 2, 3, 4, 6, 8, or 9 h, after which COCs were transferred to a solution of 1 µM 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) freshly prepared in a modified phenol red- and BSA-free B-TCM (CAM-BTCM) supplemented with polyvinyl alcohol (0.3 mg/ ml; Sigma) ± PDE inhibitor or FSH. COCs were cultured with the dye for 15 min, then unincorporated dye was removed by three washes in calcein-AM-free CAM-BTCM with or without the various treatments. COCs were then transferred to calcein-AM-free media ± the various treatments and cultured for a further 25 min to allow for dye exchange between the CCs and the oocyte. Prior to fluorescence microphotometry, COCs were completely denuded of their surrounding CCs using vigorous pipetting so that only dye confined within the denuded oocyte (DO) after transport via gap junctions was measured.
Within 30 min of denuding, the intraoocyte fluorescence emission of calcein in pulsed oocytes was measured using a fluorophotometric-inverted microscope (Leica, Wetzlar, Germany). 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 = 1 h control DO reading (%).
Assessment of Oocyte Meiosis
Immediately following fluorophotometry, or at designated time points of standard IVM, denuded oocytes were transferred to a fixing solution (3:1 ethanol:acetic acid) for >24 h before staining with 1% Orcein (Sigma) to assess 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, diakinesis I, metaphase I (MI), anaphase I, telophase I, or MII stages. For graph simplicity, oocytes at diakinesis I were pooled with and classified as metaphase I, whereas those at anaphase I and telophase I were pooled with and classified as MII stage.
In Vitro Production of Embryos
Bovine oocytes were collected and prepared as described above and underwent routine embryo in vitro production procedures [36] with the exception of PDE treatments during IVM. Prior to IVM, COCs were washed in maturation medium (B-TCM supplemented with 4 mg/ml FAF-BSA, cysteamine [100 µM; 2-mercapto-ethylamine; Sigma] and 0.1 IU/ ml FSH), and then transferred to wells of a 48-well dish (approximately 25 oocytes per treatment group, per time point, per well, in each of five replicate experiments) containing the same medium with or without the type 3 PDE inhibitor milrinone or the type 4 PDE inhibitor rolipram to give a final volume of 250 µl. Media in each of the wells was overlayed with 350 µl of sterile, washed mineral oil (embryo tested, M 8410; Sigma), and dishes were incubated at 38.5°C in humidified 6% CO2 in air for 24, 26, or 28 h.
At specific IVM time intervals; namely, at either 24, 26, or 28 h, COCs were removed from the IVM wells and washed twice (Cook Bovine In Vitro Wash; Cook Australia, Eight Miles Plains, Australia), and transferred to insemination dishes containing IVF medium (Cook Bovine In Vitro Fert, Cook Australia) supplemented with penicillamine (0.2 mM; Sigma), hypotaurine (0.1 mM; Sigma), and heparin (2 mg/ml; Sigma). Treatment groups of approximately 25 COCs from each of the culture times were inseminated with freshly prepared Percoll-purified motile spermatozoa preparation at a final sperm concentration of 1 x 106 sperm per milliliter, and incubated for 22 h at 38.5°C in humidified 6% CO2 in air.
Following insemination, presumptive zygotes were removed from IVF wells, washed and denuded of the surrounding CCs, then transferred to early cleavage culture medium (Cook Bovine In Vitro Cleave; Cook Australia) and cultured (5 to 8 oocytes per 20-µl drop) in humidified 6% CO2, 7% O2, and 87% N2 at 38.5°C.
On Day 5 of development (where Day 0 = the day of insemination) the numbers of cleaving embryos was recorded and those were then transferred into blastocyst culture medium (Cook Bovine Blast Media, Cook Australia), regassed, and cultured for a further 48 h at 38.5°C. All embryos were removed from culture on Day 7 of development and were examined under a dissecting microscope, morphologically assessed, and graded. Expanded and hatched blastocysts were stained to determine cell numbers. Zonae pellucidae were removed by Tyrode treatment and embryos were then stained with Hoechst 33342 (4 µg/ml, bisbenzimide; Sigma). Embryos were then mounted on glass slides in 90% glycerol in PBS and evaluated under a fluorescence microscope (Olympus, Japan).
Statistical Analysis
Differences in the proportion of oocytes that had progressed to the various meiotic stages (GV, MI, MII) were examined using chi-square analysis. Differences in the number of blastomeres and the levels of GJC between the oocyte and the surrounding CCs, as indicated by measurement of intraoocyte fluorescence intensity, over time and in response to treatment, were assessed using two-way analysis of variance (ANOVA; SigmaStat version 2.0 computer software; SPSS Inc., Chicago, IL). The main treatment effects were determined by comparison of least square means (LSM), and individual differences between treatments at any time point were detected by Student-Newman-Keuls posthoc pairwise comparisons. Differences in the proportion of cleaved embryos that had progressed to the blastocyst stage were examined using chi-square analysis. Probabilities of <0.05 were considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Levels of GJC between the oocyte and the CCs dramatically decreased upon removal of the COC from the follicle and transfer to culture medium, and continued to drop until 9 h of culture, whereupon levels were effectively zero and did not decrease further (Fig. 1A). Addition of FSH to the culture medium of COCs had no effect on the level of GJC between the oocyte and its surrounding CCs at any time during early IVM compared with that measured in control COCs. Moreover, FSH did not augment the stimulatory effect of the type 3 PDE inhibitor milrinone on cumulus cell-oocyte GJC, as was observed with forskolin treatment in a previous study [24]. However, the main treatment effect of FSH + milrinone (two-way ANOVA; P = 0.04; LSM = 70.5), but not FSH + rolipram (P = 0.06; LSM = 71.7) was significantly increased over that of control and FSH alone (treatment main effect: LSM = 57.8 [control]; 57.7 [FSH]; standard error of LSM = 4.2). In combination with the type 4 PDE inhibitor, however, an increase in GJC was observed at t = 2 h (P < 0.05) compared with that induced by FSH alone at this time (Fig. 1A). Apart from this difference, GJC induced by all treatments followed the same trend as in control COCs, falling dramatically during early IVM, and was effectively zero after 9 h of culture.
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Effect of Type 3 and Type 4 PDE Isoenzyme Inhibitors on Oocyte Meiotic Maturation in the Presence of FSH
In contrast to the slight effects of FSH + PDE inhibitor on CC-oocyte GJC, these treatment combinations completely inhibited meiotic resumption up to at least 9 h (Fig. 1B). This effect is similar to that previously observed with forskolin. We have previously shown that the inhibitory effect of forskolin on bovine oocyte meiotic maturation is markedly enhanced in the presence of either PDE inhibitor, as a consequence of dramatic increases in cAMP in either the COC as a whole, or within the oocyte itself [23, 24]. In the current study, treatment of COCs with FSH alone significantly (P < 0.05) delayed the onset of GVBD until 7 h of culture, at which stage 7% had undergone GVBD compared with 50% of control COCs (Fig. 1B). In addition, this meiotic-delaying effect of FSH was greatly enhanced in the presence of both the type 3 and type 4 PDE inhibitors, when even after 9 h of culture, 100% of COCs remained at the GV stage. This result is in contrast to that produced by treatment of COCs with milrinone and rolipram alone, when by 9 h of culture, 77% and 93%, respectively, had already undergone GVBD [24].
After 16 h, milrinone treatment alone maintained 20% of oocytes at the immature GV stage (compared with 0% in the absence of milrinone; Fig. 2A), and these oocytes did not undergo GVBD, even after 48 h of culture (FSH–/ milrinone+; Fig. 2B). Those oocytes that were not maintained at the GV stage by milrinone progressed through to the MII stage over time, but at a rate slower than control oocytes (present study and [23]). It is interesting that the meiosis-arresting effect of milrinone (20% GV) was overcome by addition of FSH to the culture media (FSH+/milrinone+; Fig. 2). During early IVM the effect of FSH + milrinone was to significantly delay GVBD, such that after 16–22 h of culture, 10% of oocytes remained at the GV stage (Figs. 2A and 3). However, by 24 h, all FSH + milrinone-treated oocytes had undergone GVBD (Fig. 3) and progressed through to the MII stage with time (90% MII after 26 h of culture; Figs. 2B and 3). In contrast to the meiosis-inducing effects of FSH, addition of hCG to the culture media had no effect on GVBD of meiotically arrested, milrinone-treated oocytes (Table 1). When hCG was combined with FSH (data not shown), the inhibitory effect of milrinone on GVBD was again overcome (16 h, 6% GV; 48 h, 0% GV).
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Effect of Type 3 and Type 4 PDE Isoenzyme Inhibitors During IVM on Embryo Development
Inclusion of milrinone in IVM medium of isolated bovine oocytes delays the onset of GVBD by 2 h, and in the combined presence of cAMP-stimulators forskolin [24] or FSH (current study), GVBD onset is delayed by at least 4 h. Consequently, oocytes were inseminated at 24, 26, or 28 h after IVM with FSH and either the type 3 PDE inhibitor milrinone or the type 4 PDE inhibitor rolipram. The overall cleavage rate was approximately 80%, and there were no significant differences between treatments across any of the time points examined (data not shown). Thirty-eight percent of control cleaved embryos that were fertilized at the 24 h time point developed to blastocysts (Fig. 4). The rate of blastocyst development from those cleaved embryos fertilized after 24 or 26 h of culture in the presence of either PDE inhibitor was not significantly increased above their respective FSH controls. However, oocytes fertilized at 28 h yielded a significant (P < 0.05) increase in blastocysts when matured with FSH + milrinone (39% increase in yield above 28 h control to 53% blastocysts) or FSH + rolipram (34% increase from control to 51% blastocysts; Fig. 4). Addition of either PDE inhibitor during IVM significantly increased blastocyst cell numbers when oocytes were inseminated at 24 h (Table 2; treatment x time interaction, P < 0.05). PDE inhibitors did not significantly increase embryo blastomere numbers when oocytes were inseminated at 26 or 28 h.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The experiments reported here show that the addition of isoenzyme-specific PDE inhibitors to bovine oocyte IVM media, which are known to increase CC and oocyte cAMP levels [23], slow the progression of oocyte meiotic maturation, and lead to increased oocyte capacity to support early embryo development. Application of an assay established to measure GJC between the oocyte and its surrounding cumulus cell vestment [24], indicates that the level of communication between the two cell types is slightly increased in response to this treatment, simultaneously corresponding to a marked delay in spontaneous meiotic maturation.
This study shows that in combination with FSH, either PDE inhibitor appreciably delays the onset of COC GVBD by at least 4 h compared with COCs treated with FSH alone, and that it slows the progression of meiosis through to MI and MII stages. This is reminiscent of a previous finding in which we showed that in combination with milrinone, the adenylate cyclase stimulator forskolin maintains more COCs at the GV stage and delays progression to MII, compared with treatment with either agent alone [24]. FSH stimulates intracellular cAMP production [39] and delays spontaneous bovine ([40, 41] and present study) and rodent [42, 43] oocyte meiotic maturation. To therefore determine a feasible time for fertilization, we investigated the meiotic status of FSH + milrinone-treated COCs at 16 h, between 22 and 30 h, and after 48 h of culture. This experiment was not conducted for rolipram-treated oocytes because, unlike milrinone, rolipram does not cause GV arrest [24]. By 16 h, when the vast majority of untreated or FSH-treated oocytes had reached MII, just 20% of FSH + milrinone-treated oocytes had matured. By 22 h, 67% of these oocytes had reached MII, with just 9% of oocytes remaining at the GV stage. However, by 24 h of culture, 88% had reached the MII stage; consequently, groups of oocytes were fertilized at 24, 26, and 28 h of culture.
It is interesting that addition of either PDE inhibitor during IVM significantly improved oocyte cytoplasmic maturation as measured by an increased quantity and quality of embryos produced. The PDE inhibitors increased blastocyst development rates in oocytes that had been matured for 28 h (52% blastocyst) compared with those of the control (38% blastocyst). Although control (FSH treated) oocytes were presumably meiotically aged at 28 h by 6 h, blastocyst rates from PDE inhibitor + FSH-treated oocytes inseminated at 28 h were still 10%–15% higher than from nonaged control oocytes inseminated at 24 or 26 h. Embryo quality, as assessed by blastocyst cell numbers, was also improved by oocyte PDE treatment, although at this stage it is unclear why this effect appears to manifest only in embryos from oocytes inseminated early.
The precise mechanisms by which bovine oocyte cytoplasmic maturation is improved by addition of PDE inhibitors during IVM are unclear at this stage. FSH activates CC adenylate cyclase, leading to cAMP accumulation in the CCs. CCs are the major source of oocyte cAMP, which rapidly traverses into the oocyte via gap junctions at the end of CC transzonal cytoplasmic processes. Although CCs can generate massive increases in ooplasm cAMP content [23], this content can also be rapidly degraded by the very active oocyte PDE. Hence, a most effective means of generating sustained, high ooplasmic cAMP levels is by combined treatment of COCs with a PDE inhibitor together with an adenylate cyclase stimulator, such as FSH or forskolin. In the current study, this approach (which improved oocyte developmental capacity) caused a substantial delay in meiotic resumption, but only a mild extension in CC-oocyte GJC. We [24] and Luciano et al. [27] hypothesized that maintaining high intra-COC cAMP levels would slow the very rapid loss of oocyte-CC GJC that occurs during spontaneous oocyte maturation, in turn improving oocyte cytoplasmic quality. The current results provide some circumstantial evidence to support this hypothesis. Alternatively, continued total GVBD inhibition after 9 h of IVM despite an almost complete loss of oocyte-CC communication, which contrasts with our previous result using forskolin [24], suggests the lack of a causal link between these two events. Perhaps it is more likely that the improvement in oocyte cytoplasmic quality from combined FSH + PDE inhibitor treatment can be attributed to the effects of cAMP on the autonomous oocyte cell cycle, rather than to alterations in oocyte-CC interactions. This notion is supported by a recent study that has demonstrated that temporarily inhibiting GVBD, by specifically targeting cyclin-dependent kinases using butyrolactone I, improves oocyte developmental competence [12]. Most recently, it has also been demonstrated that mouse oocyte developmental competence can be promoted during meiotic arrest using a PDE3 inhibitor [34].
The relative potencies of FSH and forskolin in regulating oocyte meiotic maturation and GJC are remarkably contrasting. While forskolin in combination with either PDE inhibitor delayed the onset of GVBD [24], FSH was noticeably more effective than forskolin in augmenting the inhibitory effects of milrinone and rolipram on the onset of GVBD (current study), maintaining 100% of oocytes at the immature GV stage for at least the first 9 h of culture. In contrast, FSH, in combination with either PDE inhibitor (current study), was far less effective than forskolin + PDE inhibitor [24] in prolonging oocyte-CC GJC. Likewise, invasive adenylate cyclase (iAC) was more effective than human menopausal gonadotropin at maintaining high intraoocyte cAMP levels and prolonging oocyte-CC GJC [27]. If the hypothesis that prolonged communication between the follicular cells and the oocyte during IVM improves oocyte cytoplasmic maturation and therefore developmental competence holds, then in light of the superior level of GJC observed during IVM in the presence of forskolin or iAC compared to gonadotropins, it may be favorable to substitute FSH with forskolin (or iAC) during the initial period of IVM to further enhance GJC between the cumulus cells and the oocyte.
Ungulate oocytes are remarkably less sensitive than rodent or primate oocytes to cAMP-mediated meiotic control. In bovine oocytes, agents that modulate cAMP levels typically reversibly inhibit a small proportion of oocytes at the GV stage, with the majority of oocytes undergoing GVBD with a delayed progression through MI to MII [18–22]. Indeed, milrinone, the inhibitor of the type 3 oocyte PDE, indefinitely arrests just 20% of bovine COCs at the immature GV stage ([23, 28] and present study). In contrast, much lower doses of PDE3 inhibitor are required to reversibly arrest >90% of rodent [29–31], monkey [32], and human [33] oocytes at the GV stage.
The meiosis-arresting effect of milrinone on bovine oocytes in this study was, interestingly, overcome by addition of FSH, but not by hCG. In the absence of FSH, 20%–30% of milrinone-treated oocytes remain arrested at the GV stage for up to 48 h of culture, but addition of FSH, while initially delaying GVBD, eventually induces all milrinone-treated oocytes to undergo GVBD by 24 h. This observation may be the first model described in a ruminant species for induced oocyte maturation (as opposed to spontaneous maturation, in which simple removal of oocytes from their follicle results in meiotic resumption [44]). In vivo, meiosis resumes in response to the preovulatory surge of gonadotropins—the mechanism by which this occurs is not clear; however, several studies have suggested production of a granulosa/CC-derived positive stimulus in response to the gonadotropic surge that actively promotes meiotic resumption [45–47]. In line with the results of the current study, Byskov et al. [48] demonstrated that hypoxanthine-arrested mouse COCs secrete a meiosis-activating substance following FSH, but not LH or hCG stimulation, that could override the effects of the meiotic inhibitor. Other studies have also demonstrated the induction of meiosis in rodent oocytes arrested at the GV stage by agents such as hypoxanthine, 3-isobutyl-1-methylxanthine, and dibutyryl-cAMP [49], and have implicated follicular fluid meiosis-activating sterol (FF-MAS) [48] as the causal factor. It remains to be determined whether a similar mechanism is operating in the current model of FSH-induced maturation of milrinone-arrested bovine oocytes, and whether FF-MAS is involved.
In vitro oocyte maturation is a vitally important platform technology for many reproductive technologies and has enormous potential applications in reproductive medicine. There is much to be gained from improving the efficiency of IVM for embryo production to levels approaching those for in vivo-matured oocytes. Manipulating meiotic resumption and the pace of meiotic progression, as has been achieved in this study, may be an important approach to improving the cytoplasmic quality of IVM oocytes. This and many other studies show that modulators of cAMP clearly have roles in these processes and improve subsequent embryo development. Understanding the mechanisms by which these agents regulate oocyte-CC function will aid their further applications. The cell-specific compartmentalization of PDE isoenzymes in the ovarian follicle is an intriguing model, and the PDE isoenzyme specific inhibitors are important experimental tools.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 61 8 8222 7521; robert.gilchrist{at}adelaide.edu.au
Received: 27 October 2003.
First decision: 14 November 2003.
Accepted: 27 May 2004.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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