Inhibition of oocyte maturation in the mouse: Participation of cAMP,steroid hormones,and a putative maturation-inhibitory factor

The hypothesis that cumulus cells inhibit oocyte maturation by a cAMP-dependent process was tested (R. M. Schultz, R. Montgomery, P. F. Ward-Bailey, and J. J. Eppig (1983). Dev. Biol.95, 294–304.). Treatment of isolated cumulus cell-oocyte complexes with follicle-stimulating hormone (FSH) resulted in a dose-dependent increase in both cumulus cell cAMP levels and in the extent of inhibition of germinal vesicle breakdown (GVBD), the first morphological manifestation of oocyte maturation. Furthermore, it was found that concentrations of a membrane-permeable analog of cAMP, dibutyryl cAMP (dbcAMP), that were below those required for complete meiotic inhibition had a greater inhibitory effect on cumulus cell-enclosed oocytes than on denuded oocytes. Cumulus cell-enclosed and denuded oocytes matured at the same time in the absence of dbcAMP. Ablation of the gap junctions that couple cumulus cells to the oocyte abolished the maturation-inhibitory action of cumulus cells that was promoted either by FSH or low concentrations of dbcAMP. These results are consistent with the hypothesis that inhibition of oocyte maturation is mediated by a factor of granulosa/cumulus cell origin, other than cAMP, which requires cAMP for its activity and/or generation, and an intact intercellular coupling pathway between cumulus cells and the oocyte. A variety of steroid hormones potentiated the FSH-induced inhibition of maturation in cumulus cell-enclosed oocytes. In addition, steroid hormones inhibited maturation in denuded oocytes, but only when oocyte cAMP levels were elevated by cAMP analogs or forskolin. Steroids alone did not inhibit maturation of either cumulus cell-enclosed or denuded oocytes. Moreover, the steroids alone or in combination with FSH did not affect metabolic coupling between the cumulus cells and oocytes, nor did testosterone affect the forskolin-induced level of cAMP in denuded oocytes. Therefore, it is proposed that the oocyte is a site for the synergistic activity of steroid hormones with a cAMP-dependent process in inhibiting maturation. Results of these studies are discussed in terms of the roles of intercellular communication, cAMP, a putative maturation-inhibiting factor, and steroid hormones in the inhibition of maturation of mouse oocytes.     

Induction of maturation in cumulus cell-enclosed mouse oocytes by follicle-stimulating hormone and epidermal growth factor: evidence for a positive stimulus of somatic cell origin

The efficacy of follicle-stimulating hormone (FSH), epidermal growth factor (EGF), and dibutyryl cGMP (dbcGMP) as inducers of germinal vesicle breakdown (GVBD) in cumulus cell-enclosed mouse oocytes was examined when meiotic arrest was maintained in vitro with purines, dibutyryl cAMP (dbcAMP), or the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). When FSH was added to hypoxanthine (HX)-containing medium, the effect on oocyte maturation was at first inhibitory and later stimulatory. EGF stimulated GVBD at all time points tested. FSH and EGF also induced GVBD when oocytes were arrested with dbcAMP, IBMX, or guanosine. Dibutyryl cGMP stimulated GVBD when meiotic arrest was maintained with HX, but not when oocytes were meiotically arrested with guanosine, and was inhibitory in dbcAMP-supplemented medium. FSH and dbcGMP produced a transient delay of oocyte maturation in control medium, but the FSH effect was much more pronounced. EGF had no effect on maturation kinetics. The actions of FSH and EGF required the presence of cumulus cells. Both agents significantly stimulated cAMP production in oocyte-cumulus cell complexes. A brief exposure of complexes to a high concentration of dbcAMP induced GVBD, suggesting that FSH and EGF may act via a cAMP-dependent process. The frequency of FSH- and EGF-induced GVBD in cumulus cell-enclosed oocytes was significantly higher than the frequency of GVBD when oocytes were cultured while denuded of cumulus cells. of maturation is apparently not mediated solely by oocyte-cumulus cell uncoupling and termination of the transfer of an inhibitory meiotic signal from cumulus cells to the oocyte. The data suggest the generation of a positive signal within cumulus cells in response to hormone treatment that acts upon the oocyte to stimulate GVBD in the continued presence of inhibitory factors.     

Kinetics of meiotic progression, M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase) activities during in vitro maturation of porcine and bovine oocytes: species specific differences in the length of the meiotic stages

The kinetics of nuclear maturation, M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase) activities during in vitro maturation of porcine and bovine oocytes were examined. A further objective was to determine the duration of the meiotic stages during the maturation process. Porcine and bovine cumulus-oocyte complexes (COCs) were incubated in TCM 199 supplemented with 20% (v/v) heat inactivated fetal calf serum (FCS), 0.05microg/ml gentamycin, 0.02mg/ml insulin, 2.5microg/ml FSH and 5microg/ml LH. COCs were removed from the culture media in hourly intervals starting immediately after recovery from the follicle until 24 (bovine) or 48h (porcine) of culture. Oocytes were either fixed to evaluate the maturation status or the activity of MPF, assessed by its histone H1 kinase activity, and MAP kinase were determined by a radioactive assay simultaneously. In oocytes of both species, the MPF activity oscillated during the culture period with two maxima corresponding with the two metaphases: between 27-32 and after 46h (porcine) and between 6-9 and after 22h (bovine). There was a temporary decline in activity after 33-38 (porcine) and after 19h (bovine), which corresponded with anaphase I and telophase I. MAP kinase activity increased during the whole culture period and reached maximum levels after 47 (porcine) and after 22h (bovine). In porcine oocytes, the MAP kinase was activated before GVBD and MPF activation. In bovine oocytes, MPF and MAP kinase were activated at approximately the same time as the GVBD (8-9h of incubation). In average porcine, oocytes remain 23.4h in the germinal vesicle (GV) stage (13h in GV I, 5.7h in GV II, 3.2h in GV III and 1.5h in GV IV), 0.9h in diakinese, 9.6h in the metaphase I, 2.8h in anaphase I and 1.9h in telophase I of the first meiotic division. In bovine oocytes, the temporal distribution of the meiotic stages were 8.5h for the GV stage, 1.2h for diakinese, 8.3h for metaphase I, 1.6h for anaphase I and 1.9h for telophase I. These results indicate that the duration of the meiotic stages differs between the species and that MAP kinase is activated before MPF and GVBD in porcine oocytes.     

A follicular fluid component prevents gonadotropin reversal of cyclic adenosine monophosphate-dependent meiotic arrest in murine oocytes

The effects of the putative maturation inhibitor in porcine follicular fluid on gonadotropinstimulated reversal of cyclic adenosine monophosphate (cAMP)-maintained meiotic arrest in mouse oocytes in vitro were assessed in this study. When cumulus cell-enclosed oocytes were cultured in a suboptimal inhibitory concentration of dibutyryl cAMP (dbcAMP), the effect of follicle-stimulating hormone (FSH) on oocyte maturation was initially inhibitory at 3 hr, but stimulatory at 6 hr. Supplementation of the medium with an ultrafiltrate of porcine follicuiar fluid (PM10-filtrate) completely suppressed FSH-promoted reversal of inhibition at 6 hr. Charcoal extraction eliminated this effect of the PM10-filtrate. FSH reversed the inhibition of maturation of cumulus cell-enclosed oocytes maintained by a high concentration of dbcAMP and suboptimal concentrations of the phosphodiesterase inhibitor, 3-isobutyl-1-methyl xanthine (IBMX), during a 21–22-hr culture period. However, the effect of a completely inhibitory concentration of IBMX was not reversed by gonadotropin. A component of serum was also found to inhibit FSH reversal of dbcAMP-maintained meiotic arrest, and this activity was removed by charcoal extraction. In addition, when oocytes were cultured in medium containing a suboptimal concentration of dbcAMP plus a low molecular weight fraction (< 1,000) of porcine follicular fluid, porcine serum, or fetal bovine serum, a synergistic inhibition of maturation was observed. Experiments with highly purified gonadotropins revealed that reversal of dbcAMP-maintained meiotic arrest occurred only in response to FSH; neither highly purified luteinizing hormone nor human chorionic gonadotropin could mimic this action of FSH. Also, this effect was mediated by the cumulus cells, since FSH could not reverse dbcAMP-maintained meiotic arrest in denuded oocytes. Furthermore, elevating cAMP levels in denuded oocytes augmented, rather than reversed, the inhibitory action of dbcAMP on oocyte maturation. These data therefore suggest that dbcAMP- or IBMX-maintained meiotic arrest in vitro is reversed by an FSH-stimulated, cAMP-dependent process mediated by the cumulus cells and demonstrate that a factor present both in follicular fluid and serum prevents this action of the gonadotropin.     

The releasing action of calcium upon cyclic AMP-dependent meiotic arrest in hamster oocytes

The effect of increasing cytoplasmic calcium on cyclic adenosine monophosphate (cAMP)-dependent meiotic arrest (%GV where GV is germinal vesicle) in hamster oocytes was investigated. The hypotheses tested were that calcium is required for the spontaneous maturation of hamster oocytes, elevation of calcium in the oocyte-cumulus complex can antagonize cAMP-dependent meiotic arrest, and the intraoocyte level of cAMP remains unchanged, but heterologous metabolic coupling decreases, concomitant with calcium-stimulation of germinal vesicle breakdown (GVBD). Levels of cAMP were elevated by culturing cells in the presence of dibutyryl cAMP (dbcAMP), isobutylmethylxanthine (IBMX) or forskolin and intracellular levels of calcium were manipulated by altering the CaCl2 concentration in the medium and/or by utilizing EGTA or A23187. Intracellular cAMP was determined by RIA, functional metabolic coupling was assessed by determination of the fraction of radiolabeled uridine marker transferred from the cumulus mass to the oocyte, and meiotic stage was determined cytogenetically. Compared with the proportion of oocytes that underwent meiotic maturation in control medium containing 1.53 mM CaCl2, that of cumulus-free (denuded) oocytes was unaffected by culture in the absence of added CaCl2, while that of cumulus-enclosed (intact) oocytes was significantly decreased (%GV = 59.5 +/- 4.8 and 4.2 +/- 0.9 in 0 and 1.53 mM CaCl2, respectively, P less than 0.001, where GV is germinal vesicle). EGTA prevented, in a dose-dependent manner, the spontaneous maturation of denuded oocytes that occurred in 0 mM CaCl2 (ID50 = 0.05 mM, where ID50 is the dose of EGTA that inhibited GVBD in 50% cultured oocytes). In contrast, compared with the control, less than 1 mM EGTA failed to increase the %GV of intact oocytes, although 5 mM EGTA significantly increased meiotic arrest. The %GVBD of oocytes cultured in medium containing 0 mM CaCl2 was dose-dependent on A23187 for both intact oocytes (ID50 = 3.0 microM) and for denuded oocytes cultured in the presence of 0.5 mM EGTA (ID50 = 2.7 microM). Elevated extracellular calcium significantly antagonized dbcAMP-maintained meiotic arrest in both types of oocyte and the %GV was significantly correlated with the pH of the medium [(r) = -0.78 and -0.60 for intact and denuded oocytes, respectively, P less than 0.001 in both cases]. Both CaCl2 and A23187 induced dose-dependent antagonistic effects on forskolin-maintained meiotic arrest in intact oocytes but neither antagonism was accompanied by significant dose-dependent decreases in either the intraoocyte content of cAMP or the extent of heterologous metabolic coupling.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of forskolin on maintenance of meiotic arrest and stimulation of cumulus expansion, progesterone and cyclic AMP production by pig oocyte-cumulus complexes

Forskolin induced biphasic responses of cumulus progesterone secretion (determined by RIA) and cumulus mass expansion, with maximal increases occurring at 6.25 microns, and subsequent dose-dependent declines observed up to 10 microns-forskolin. The diterpene induced dose-dependent responses in the % germinal vesicle (GV) of cumulus-enclosed and denuded oocytes (0.23 and 4.84 microns maintained 50% GV, respectively), it increased the cAMP content of cumulus masses, cumulus-enclosed oocytes and denuded oocytes, and increased heterologous metabolic coupling (determined by measuring transfer of radiolabelled uridine marker from the cumulus mass to the oocyte). A significant correlation was established between the amount of cAMP within the cumulus mass and that in the corresponding oocyte (r = 0.58). Above 10 microns-forskolin, the cAMP content of cumulus-enclosed oocytes was significantly greater than that of denuded oocytes (100 microns-forskolin: 0.118 +/- 0.082 and 0.006 +/- 0.001 pmol/oocyte respectively; P less than 0.001, paired t test), and the enhanced arresting action of forskolin upon cumulus-enclosed oocytes was correlated with an increase in intra-oocyte cAMP. Maintenance of meiotic arrest and stimulation of oocyte-cumulus cAMP were reversible. During 48 h of culture, the arresting action of forskolin (50 microns) was maintained on denuded and cumulus-enclosed oocytes but heterologous metabolic coupling significantly declined. The cAMP content of the cumulus mass and corresponding oocyte significantly declined, while that of the denuded oocyte remained unchanged. The cAMP content of arrested cumulus-enclosed oocytes cultured for 48 h in 50 microns-forskolin was significantly greater than that of maturing oocytes cultured for 24 h in 50 microns-forskolin and then for 24 h in control medium. These results show that (1) forskolin stimulates progesterone secretion and expansion of pig cumuli, but at high doses the drug inhibits these functions while cumulus cAMP remains elevated; (2) when heterologous metabolic coupling is maintained, cumulus cAMP may be transferred to the oocyte; (3) the pig oocyte can synthesize cAMP; and (4) forskolin-maintenance of meiotic arrest of pig oocytes is correlated with elevated intra-oocyte cAMP but a 'factor' other than cAMP is also involved in maintenance of meiotic arrest.     

Inhibition of LH-induced and spontaneous meiotic maturation in mouse oocytes by N alpha-tosyl-L-lysine chloromethylketone

The effect of N alpha-tosyl-L-lysine chloromethylketone (TLCK), an inhibitor of trypsin-type proteases, on luteinizing hormone (LH)-induced and spontaneous meiotic maturation and follicular production of cAMP in mice was determined. When follicle-enclosed mouse oocytes were incubated with LH (1 micron/ml), they underwent the breakdown of the germinal vesicle (GVBD). TLCK (0.02-0.5 mM) inhibited LH-induced GVBD in folliculated oocytes. The concentration (0.5 mM) of TLCK that inhibited LH-induced GVBD did not significantly suppress LH-induced cAMP production by follicle cells. The effect of TLCK on spontaneous maturation in cumulus cell-enclosed and denuded oocytes was also determined. TLCK strongly inhibited spontaneous maturation in denuded oocytes only if it was added to the incubation medium for 1-3 h before oocytes were liberated from the follicular tissue. The inhibition of oocyte maturation by TLCK was significantly greater in cumulus cell-enclosed oocytes than in denuded oocytes, either with or without preincubation with TLCK. These results suggest that trypsin-type protease in oocytes participates in the process of meiotic maturation in mouse oocytes.     

Time-dependent effects of cycloheximide and alpha-amanitin on meiotic resumption and progression in bovine follicular oocytes

To identify the stage during maturation at which new protein and RNA are synthesized for meiotic resumption, follicular oocytes were cultured in TCM-199 with the protein synthesis inhibitor cycloheximide or the hnRNA synthesis inhibitor alpha-amanitin. Although the meiotic resumption of cumulus-enclosed oocytes was completely blocked by the addition of 25 microg/ml cycloheximide at 4 h after the onset of culture, 23% of oocytes cultured from 5 h post cultivation in the medium with cycloheximide underwent germinal vesicle breakdown (GVBD). By further delaying the addition of cycloheximide, the proportion of oocytes which underwent GVBD increased. Addition of the inhibitor at 8 h or more post cultivation resulted in GVBD occurring in more than 87% of oocytes, though none of them were able to proceed beyond the metaphase I stage. In contrast, the addition of 50 microg/ml alpha-amanitin from the onset of culture significantly reduced the proportion of GVBD to 75% in cumulus-enclosed oocytes, while no significant reduction in the proportions of GVBD was noted in the case of its addition from 1 h of culture onward. However, denuded oocytes were almost insensitive to any treatments with alpha-amanitin. These results indicate that protein synthesis in the oocytes and RNA synthesis in the cumulus cells soon after the onset of culture are necessary for GVBD and that continuous protein synthesis following GVBD is indispensable for progression of the meiotic division in bovine oocytes.     

Exogenous dibutyryl cAMP affects meiotic maturation via protein kinase A activation; it stimulates further embryonic development including blastocyst quality in pigs

High concentrations of cyclic AMP in germinal vesicle oocytes generally inhibit GVBD. Thus, maintaining the GV stage in growing oocytes is essential for the developmental competence of the eggs. In this study, we traced the effects of dibutyryl cyclic AMP on meiotic maturation and early embryonic development in pigs. We also investigated several blastocyst qualities, including structural integrity, mitochondrial membrane potential, and apoptosis, which are affected by dbcAMP. To determine whether increased concentrations of cAMP inhibit GVBD, we explored the meiotic patterns and during maturation of pig oocytes. When treated with dbcAMP for 22h, 91.1% of the oocytes were arrested in the GV stage compared to only 38.8% of the oocytes in the control group (P<0.05). After completion of IVM, a higher proportion of the dbcAMP-treated oocytes were in metaphase II than the untreated ones (91.3% vs. 72.8%, P<0.05). Western blot analysis showed a reduction (at 22h) and/or increase (at 44h) in MPF and MAP kinase activities in porcine oocytes treated with dbcAMP for the first 22h of IVM compared to the untreated control. We also confirmed that protein kinase A activity increased in dbcAMP-treated oocytes, indicating an elevated intracellular concentration of cAMP. After IVF, the frequency of polyspermy in the dbcAMP-treated group decreased compared to that in the control group (22.4% vs. 47.4%, P<0.05). Furthermore, blastocyst formation, the blastocyst cell number, mitochondrial membrane potential, and apoptosis were enhanced and/or reduced by dbcAMP in both IVF and SCNT embryos. We concluded that synchronizing meiotic resumption by dbcAMP treatment improved the developmental capacity and embryonic qualities of IVF and SCNT embryos by increasing the mitochondrial membrane potential and decreasing the incidence of apoptosis in preimplantation-stage porcine embryos.     

Effects of cycloheximide on chromatin condensations and germinal vesicle breakdown (GVBD) of cumulus-enclosed and denuded oocytes in cattle

To determine if newly synthesized protein is imperative for the resumption of meiosis in bovine follicular oocytes collected from small antral follicles, cumulus-enclosed and denuded oocytes were cultured in TCM-199 both with and without various concentrations of the protein synthesis inhibitor, cycloheximide. After 11 h of culture in inhibitor-free medium, all oocytes had undergone germinal vesicle breakdown (GVBD). However, when concentrations of more than 1.0 mug/ml cycloheximide were added to the medium, the meiotic resumption of bovine oocytes was completely blocked. This inhibitory effect of cycloheximide was fully reversible after removal of the inhibitor from maturation media. Germinal vesicle breakdown following removal of cycloheximide occurred twice as fast as in the control medium. Nevertheless, when oocytes were arrested at the germinal vesicle (GV) stage by cycloheximide, a significantly higher proportion of chromatin condensation (40 to 57%) was observed in denuded oocytes than in cumulus-enclosed oocytes (11 to 22%). Thus the cycloheximide treatment could not prevent the chromatin condensation in only denuded oocytes. We conclude that protein synthesis is a prerequisite for GVBD in bovine follicular oocytes and that cumulus cells are responsible for the complementary regulation of the chromatin condensation at the GV stage, regardless of protein synthesis in the oocytes.     

Meiotic state of bovine oocytes is regulated by interactions between cAMP,cumulus, and granulosa

Bovine oocytes are arrested at the prophase of first meiotic cell cycle. Meiosis resumes in oocytes of pre-ovulatory follicles upon LH surge. However, oocytes from secondary follicles spontaneously resume meiosis in the absence of hormones if removed from the follicle and cultured in vitro. The nature of meiotic arrestor in bovine follicles is poorly understood. In this study we investigated the role of cell-cell interactions between granulosa and cumulus cells and the oocyte in mediating maintenance of meiotic arrest by cAMP. We sorted oocytes as granulosa-cumulus oocyte complexes (GCOC) if surrounded with cumulus cells attached to a large granulosa investment or cumulus oocytes complexes (COC) if surrounded with cumulus cells only and investigated the role cAMP in maintenance of meiotic arrest in these oocytes under various conditions. In hormone- and serum-free medium both GCOC and COC enclosed oocytes resumed meiosis. When [cAMP](i) was elevated with addition of invasive adenylate cyclase (iAC) GCOC enclosed oocytes were maintained in the prophase with intact germinal vesicle (GV) while COC enclosed oocytes underwent GV breakdown (GVBD). iAC elevated [cAMP](i) in both types of oocytes to the same level. If oocytes were liberated from the cumulus and granulosa cells, they re-initiated meiosis in serum and hormone free medium, but remained in the GV stage if iAC was added to the medium. Untreated GCOC and COC enclosed oocytes extruded first polar body at the same frequency in hormone-supplemented media. GCOC and COC enclosed oocytes but not denuded oocytes (DO) cultured without somatic cells acquired developmental competence if cultured in hormone-containing medium. It is concluded that maintenance of meiotic arrest is regulated by the interplay of [cAMP](i), and cumulus and granulosa cells.     

Possible role of 5'AMP-activated protein kinase in the metformin-mediated arrest of bovine oocytes at the germinal vesicle stage during in vitro maturation

The 5'AMP-activated protein kinase (AMPK) activation is involved in the meiotic maturation of oocytes in the ovaries of mice and pigs. However, its effects on the oocyte appear to be species-specific. We investigated the patterns of AMPK and mitogen-activated protein kinases (MAPK3/1) phosphorylation during bovine in vitro maturation (IVM) and the effects of metformin, an AMPK activator, on oocyte maturation in cumulus-oocyte complexes (COCs) and denuded bovine oocytes (DOs). In bovine COCs, PRKAA Thr172 phosphorylation decreased, whereas MAPK3/1 phosphorylation increased in both oocytes and cumulus cells during IVM. Metformin (5 and 10 mM) arrested oocytes at the GV stage in COCs but not in DOs. In COCs, this arrest was associated with the inhibition of cumulus cell expansion, an increase in PRKAA Thr172 phosphorylation, and a decrease in MAPK3/1 phosphorylation in both oocytes and cumulus cells. However, the addition of compound C (10 muM), an inhibitor of AMPK, accelerated the initiation of the GV breakdown (GVBD) process without any alteration of MAPK3/1 phosphorylation in oocytes from bovine COCs. Metformin decreased AURKA and CCNB1 protein levels in oocytes. Moreover, after 1 h of IVM, metformin decreased RPS6 phosphorylation and increased EEF2 phosphorylation, suggesting that protein synthesis rates were lower in oocytes from metformin-treated COCs. Most oocytes were arrested after the GVBD stage following the treatment of COCs with the MEK inhibitor, U0126 (100 micromoles). Thus, in bovine COCs, metformin blocks meiotic progression at the GV stage, activates PRKAA, and inhibits MAPK3/1 phosphorylation in both the oocytes and cumulus cells during IVM. Moreover, cumulus cells were essential for the effects of metformin on bovine oocyte maturation, whereas MAPK3/1 phosphorylation was not.     

Cumulus cells secrete a meiosi-inducing substance by stimulation with forskolin and dibutyric cyclic adenosine monophosphate

The role of the cumulus cells in initiating the resumption of meiosis after exposure to forskolin and dbcAMP was studied in the mouse. The resumption of meiosis was monitored by the percentage of germinal vesicle breakdown (GVBD) and polar body formation (PB). The cumulus-enclosed oocytes (CEO) and denuded oocytes (DO) were cultured with and without hypoxanthine (HX) in the culture medium. Three types of experiments were performed: (1) Effect of forskolin on spontaneous resumption of meiosis, i.e. cultures without HX, and two experiments in which HX is present throughout the culture: (2) Effect of transient exposure to forskolin or dibutyric-cyclic adenosinemonophosphate (dbcAMP) on GVBD prior to continued culture without forskolin or dbcAMP (oocyte priming). (3) Priming of CEO with forskolin for 2 hr, separation of cumulus cells and oocytes, followed by coculture of rejoined cumulus cells and oocytes, or coculture of the cumulus cells and new, unprimed DO. (1) Forskolin inhibited a spontaneous resumption of meiosis in a dose-dependent manner during the first 5 hr of culturing. After 22 hr all controls and CEO resumed meiosis, whereas only half of the DO did. (2) At least 1 hr of priming the CEO with forskolin is needed to induce GVBD and PB formation, but forskolin inhibited the resumption of meiosis when present for 24 hr. Similar results were obtained with a high concentration of dbcAMP. (3) A separation and rejoining of oocytes and cumulus cells after priming induced the resumption of meiosis in a significantly greater number of oocytes than in the control oocytes which were not primed. The GVBD of unstimulated DO also increased significantly when cocultured with cumulus cells from primed CEO. The percentage of GVBD in unprimed DO and in DO isolated from primed CEO was the same. We suggest that within 1–2 hr, forskolin and cAMP stimulate cumulus cells to produce a diffusible meiosis-inducing substance which overcomes HX-inhibition and induces oocyte maturation, including both GVBD and PB formation. The CEO must be primed for more than 2 hr before the resumption of meiosis in DO isolated from such CEO is induced. Oocyte-cumulus connections are crucial as far as initiating the production of a meiosis-inducing substance is concerned. Oocyte-cumulus connections are not needed for transferring this substance to the oocyte. © 1994 Wiley-Liss, Inc.     

Effect of forskolin on the spontaneous maturation and cyclic AMP content of hamster oocyte-cumulus complexes

Forskolin, a reversible stimulator of the catalytic subunit of adenylate cyclase, has been used to determine: whether an increase in hamster cumulus cell cyclic adenosine monophosphate (cAMP) results in an elevation of intraoocyte cAMP and an accompanying increase in the maintenance of meiotic arrest (%GV where GV is germinal vesicle) when heterologous coupling is maintained, whether the hamster oolemma possesses the catalytic subunit of adenylate cyclase in an amount adequate to stimulate sufficient cAMP synthesis to maintain arrest, and whether release from meiotic arrest is accompanied by a decrease in the content of intraoocyte cAMP. Intracellular cAMP was determined by RIA, functional metabolic coupling was assessed by determination of the fraction of radiolabeled uridine marker transferred from the cumulus mass to the oocyte, and meiotic stage was determined cytogenetically. While the %GV of both cumulus-enclosed (intact) and cumulus-free (denuded) oocytes was dose-dependent upon forskolin, that of intact oocytes was much more sensitive to the drug (intact: ID50 3.4 microM; denuded: ID50 65.0 microM, where ID50 is the dose of forskolin that inhibits the maturation of 50% of cultured oocytes). Forskolin stimulated a significant, dose-dependent increase in the amount of cAMP within the cumulus mass [(r) = 0.789, P less than 0.001)], the intact oocyte [(r) = 0.715, P less than 0.001], and the denuded oocyte [(r) = 0.673, P less than 0.01)]. The cAMP content of intact oocytes was significantly greater than that of denuded oocytes above 6.25 microM forskolin (25 microM forskolin: 9.28 +/- 1.01 vs. 3.98 +/- 0.15 fmol cAMP, intact and denuded oocytes, respectively; P less than 0.001, paired t test). A highly significant positive correlation was established between the amount of cAMP in groups of cumulus masses and that in the corresponding enclosed oocytes [(r) = 0.635, P less than 0.001]. The enhanced sensitivity of meiotic arrest in intact, as compared to denuded, oocytes was due to the presence of adherent cumulus cells but was not attributable to a significant increase in the cAMP content of intact oocytes (at 6.25 microM forskolin; %GV intact = 73.0 +/- 10.7, denuded = 20.3 +/- 7.4; fmol cAMP intact = 5.02 +/- 1.50; denuded = 4.63 +/- 0.81). The arresting action of forskolin on intact oocytes was transient and fully reversible, but release from arrest was not accompanied by a decrease in either intraoocyte cAMP or heterologous metabolic coupling.(ABSTRACT TRUNCATED AT 400 WORDS)     

Manipulation of chromosome condensation by protein synthesis inhibitors and cyclic AMP during maturation of bovine oocytes

We investigated the effects of cycloheximide on bovine oocyte chromosomes during meiotic maturation in vitro. Bovine oocytes at Metaphase I (MI) of the meiotic maturation were treated with 10 mug/ml cycloheximide alone or in addition to 5 mM dibutyrylcAMP (dbcAMP) plus 1 mM isobutylmetylxantine (IBMX). A maturation period of 15 to 18 h followed by 12-h treatment with cycloheximide appeared to be most efficient to induce interphase (86% with 16 h maturation). About 60% of oocytes returned to a metaphase state 12 h after the oocytes were transferred to cycloheximide-free medium. In contrast, up to 73% of cycloheximide-treated oocytes at 17 h of maturation remained in interphase if dbcAMP plus IBMX was included in the cycloheximide-free medium. This shows that dbcAMP plus IBMX can inhibit the development of conditions in the oocytes that are required for the transition to metaphase. The chromosome decondensation induced by protein synthesis inhibition at Metaphase I is reversible. This study shows that transition to interphase in bovine oocyte depends on the stage of maturation of oocytes and is sensitive to cAMP levels.     

Combined use of dbcAMP and IBMX minimizes the damage induced by a long‐term artificial meiotic arrest in mouse germinal vesicle oocytes

Phosphodiesterase (PDE)‐mediated reduction of cyclic adenosine monophosphate (cAMP) activity can initiate germinal vesicle (GV) breakdown in mammalian oocytes. It is crucial to maintain oocytes at the GV stage for a long period to analyze meiotic resumption in vitro. Meiotic resumption can be reversibly inhibited in isolated oocytes by cAMP modulator forskolin, cAMP analog dibutyryl cAMP (dbcAMP), or PDE inhibitors, milrinone (Mil), Cilostazol (CLZ), and 3‐isobutyl‐1‐methylxanthine (IBMX). However, these chemicals negatively affect oocyte development and maturation when used independently. Here, we used ICR mice to develop a model that could maintain GV‐stage arrest with minimal toxic effects on subsequent oocyte and embryonic development. We identified optimal concentrations of forskolin, dbcAMP, Mil, CLZ, IBMX, and their combinations for inhibiting oocyte meiotic resumption. Adverse effects were assessed according to subsequent development potential, including meiotic resumption after washout, first polar body extrusion, early apoptosis, double‐strand DNA breaks, mitochondrial distribution, adenosine triphosphate levels, and embryonic development. Incubation with a combination of 50.0 μM dbcAMP and 10.0 μM IBMX efficiently inhibited meiotic resumption in GV‐stage oocytes, with low toxicity on subsequent oocyte maturation and embryonic development. This work proposes a novel method with reduced toxicity to effectively arrest and maintain mouse oocytes at the GV stage.     

Regulation of nuclear membrane assembly and maintenance during in vitro maturation of mouse oocytes: role of pyruvate and protein synthesis

Summary In the absence of a suitable energy source, mouse oocytes cultured in vitro resume, but fail to complete, meiotic maturation. However, little is known about the underlying mechanisms leading to this meiotic failure. We utilized pyruvate-deficient medium to test for the role of pyruvate throughout the meiotic maturation process. Germinal vesicle-stage (GV) oocytes underwent germinal vesicle breakdown (GVBD), but failed to form a polar body when cultured continuously in pyruvate-free medium. However, when GV oocytes were preincubated for 4 h in pyruvate-free medium containing dibutyryl cyclic adenosine monophosphate (dbcAMP) and then cultured in pyruvate-free medium, GVBD was markedly inhibited. Preincubation of GV oocytes in dbcAMP and cycloheximide, followed by culture in cycloheximide only, also inhibited GVBD. A longer preincubation period was required in the cycloheximide-dbcAMP case (12 h) than in pyruvate-free-dbcAMP medium situation (4 h). Strikingly, reassembly of the nuclear membrane without polar body formation was observed following GVBD in oocytes continuously cultured in pyruvate-free medium. The reassembled nuclear membrane increased in size with continued culture, and it surrounded partially-decondensed chromatin. Nuclear membrane reassembly also occurred in oocytes which had undergone GVBD during continuous culture in medium containing only cycloheximide. Reformation of nuclear membranes after GVBD was confirmed by electron-microscopic analyses of oocytes cultured in pyruvate-free medium or in the presence of cycloheximide. We conclude that both pyruvate and protein synthesis are required for nuclear membrane disassembly, whereas lack of pyruvate or protein synthesis is associated with interruption of the metaphase state and reassembly of the nuclear membrane. The evidence suggests that assembly and maintenance of an intact nucleus and its disintegration are all amenable to regulation by pyruvate, possibly via mechanism(s) involving protein synthesis.     

Meiotic arrest maintained by cAMP during the initiation of maturation enhances meiotic potential and developmental competence and reduces polyspermy of IVM/IVF porcine oocytes

We investigated effects of invasive adenylate cyclase (iAC), 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl cyclic AMP (dbcAMP) on porcine oocyte in vitro maturation (IVM), in vitro fertilisation (IVF) and subsequent embryonic development. Porcine oocytes were collected in Hepes-buffered NCSU-37 supplemented with or without 0.1 microg/ml iAC and 0.5 mM IBMX. IVM was performed in a modified NCSU-37 supplemented with or without 1 mM dbcAMP for 22 h and then without dbcAMP for an additional 24 h. After IVF, oocytes were cultured in vitro for 6 days. After 12 h of IVM, no difference in nuclear status was observed irrespective of supplementation with these chemicals during collection and IVM. At 22 h, most (95%) of the oocytes cultured with dbcAMP remained at the germinal vesicle (GV) stage, whereas 44.3% of the oocytes cultured without dbcAMP underwent GV breakdown. At 36 h, oocytes cultured with dbcAMP had progressed to prometaphase I or metaphase I (MI) (32.6% and 49.3%, respectively), whereas non-treated oocytes had progressed further to anaphase I, telophase I or metaphase II (MII) (13.6%, 14.3% and 38.0%, respectively). At 46 h, the rate of matured oocytes at MII was higher in oocytes cultured with dbcAMP (81%) than without dbcAMP (57%), while the proportion of oocytes arrested at MI was lower when cultured with dbcAMP (15%) than without dbcAMP (31%). The rate of monospermic fertilisation was higher when oocytes were cultured with dbcAMP (21%) than without dbcAMP (9%), with no difference in total penetration rates (58% and 52%, respectively). The blastocyst rate was higher in oocytes cultured with dbcAMP (32%) than without dbcAMP (19%). These results suggest that a change in intracellular level of cAMP during oocyte collection does not affect maturational and developmental competence of porcine oocytes and that synchronisation of meiotic maturation using dbcAMP enhances the meiotic potential of oocytes by promoting the MI to MII transition and results in high developmental competence by monospermic fertilisation.