Role of cyclic adenosine monophosphate (cAMP) in vitro on bovine oocyte maturation

This experiment attempted to determine the effect of cAMP on maturation of bovine oocytes in chemically-defined, serum-free medium. Cumulus-oocyte complexes were incubated in modified DME/Ham F-12 medium containing dbcAMP at 0 (control), 10(-6), 10(-4) and 10(-2) M. After 18 and 24 hours of culture, the percentage of oocyte maturation between 0 (control) and 10(-2) M dbcAMP-treated groups were significant. Some oocytes were cultured with dbcAMP (10(-2) M) for 6, 12 and 24 hours followed by incubation in control medium to test the reversibility of inhibition or of any harmful effect of dbcAMP. The inhibitory effect of 10(-2) M dbcAMP on bovine oocyte maturation was reversed by transferring cumulus-oocyte complexes to the control medium. In addition, forskolin (0.12 and 0.24 mM) was effective (P < 0.01) in preventing the resumption of meiosis. The cAMP content of oocytes cultured with forskolin was not increased, although cumulus cells responded to forskolin with an increase in cAMP content. These results indicate that elevated levels of cAMP in the culture medium are important in regulating resumption of meiosis of bovine oocytes in vitro.     

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.     

Effect of dibutyryl cAMP on the cAMP content, meiotic progression, and developmental potential of in vitro matured pre-pubertal and adult pig oocytes

Pre-pubertal pig oocytes display reduced developmental competence compared with adult oocytes following in vitro maturation (IVM). Exposure to dibutyryl cyclic adenosine monophosphate (dbcAMP) for the first 20 hr IVM improves development of pre-pubertal oocytes, suggesting that their cAMP content may be inadequate. This study examined the effect of 1 mM dbcAMP treatment for the first 22 hr of IVM on the cAMP content, meiotic progression, and embryo development of pre-pubertal and adult oocytes. In control groups, a two-fold increase in cAMP was observed in adult oocytes after 22 hr IVM, with no change in pre-pubertal oocyte cAMP content. At 22 hr IVM, dbcAMP treatment resulted in two- and five-fold increases in pre-pubertal and adult oocyte cAMP, respectively. After 22 hr control IVM, a greater proportion of pre-pubertal oocytes occupied metaphase I (MI) compared with adult oocytes (69% vs. 49%). dbcAMP treatment reduced the proportion of pre-pubertal and adult oocytes in MI stage at 22 hr. Despite dbcAMP treatment, the proportion of pre-pubertal oocytes in the MI stage at 22 hr remained higher than that of adult oocytes. In control groups, adult oocytes displayed a greater ability to form blastocysts compared with pre-pubertal oocytes following either parthenogenetic activation (59% vs. 25%) or in vitro fertilization (IVF) (47% vs. 19%). dbcAMP treatment increased subsequent blastocyst formation rates of pre-pubertal oocytes, whereas blastocyst formation rates of adult oocytes remained unchanged. Our results suggest that the reduced developmental capacity of pre-pubertal oocytes may be a consequence of their reduced ability to accumulate cAMP during IVM.     

Effects of dibutyryl cyclic AMP on oocyte maturation and ovulation in the perfused rabbit ovary

The involvement of cyclic AMP (cAMP) in mammalian oocyte maturation was assessed using cultures of rabbit cumulus-oocyte complexes and in-vitro perfused rabbit ovaries. Rabbit cumulus-oocyte complexes were cultured in Brackett's medium with or without dibutyryl cyclic AMP [Bu)2cAMP) at 10(-3), 10(-4) or 10(-5) M for 4-12 h. At 4 h spontaneous meiotic maturation was significantly inhibited by (Bu)2cAMP (P less than 0.025). With prolonged incubation, spontaneous maturation progressed despite exposure to (Bu)2cAMP. When ovaries were continuously perfused in vitro for 12 h with (Bu)2cAMP (10(-3) M) or medium alone, (Bu)2cAMP stimulated ovarian progesterone production, but did not affect ovulation or maturation of follicular oocytes. When ovaries were perfused in vitro with or without (Bu)2cAMP (10(-3), 10(-4) or 10(-5) M) for the first 2 h and then transferred to medium without (Bu)2cAMP for an additional 10 h, ovulation did not occur, but transient exposure to (Bu)2cAMP stimulated a dose-related increase in maturation of follicular oocytes. Degeneration of follicle-enclosed oocytes and cumulus-oocyte complexes was not affected by exposure to (Bu)2cAMP. These results suggest that transient, but not continuous, elevation of cAMP after the gonadotrophin surge may be required for the initiation of oocyte maturation.     

Involvement of cAMP-dependent protein kinase and protein phosphorylation in regulation of mouse oocyte maturation

We report the results of experiments which support the hypothesis that, in mouse oocytes, a decrease in intraoocyte cyclic AMP (cAMP) initiates meiotic maturation; oocytes microinjected with cyclic nucleotide phosphodiesterase (PDE) underwent germinal vesicle breakdown (GVBD) in the presence of 3-isobutyl-1-methylxanthine (IBMX), which inhibited GVBD both in oocytes not injected with PDE and in oocytes injected with heat-inactivated PDE. Cyclic AMP-dependent protein kinase (PK) has been proposed to mediate maintenance of meiotic arrest by cAMP. In support of this hypothesis is the observation that 2'-deoxy cAMP, which does not activate PK, did not maintain meiotic arrest as did cAMP; this result was obtained both by microinjection of these compounds and by incubating oocytes in the presence of their membrane-permeable N6-monobutyryl derivatives. Furthermore, microinjection into oocytes of the heat-stable inhibitor of PK, PKI, induced GVBD in the presence of either dibutyryl cAMP (dbcAMP) or IBMX. Meiotic arrest was maintained in the absence of dbcAMP or IBMX, however, by microinjected catalytic subunit of PK, but not by catalytic subunit coinjected with PKI. In addition, specific changes in oocyte phosphoproteins that preceded resumption of meiosis were induced, in the presence of dbcAMP, by microinjected PKI; these changes were also tightly coupled with commitment of oocytes to resume meiosis. These results are discussed in terms of our model for regulation of meiotic arrest and maturation.     

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.     

beta-adrenoceptor antagonists reinitiate meiotic maturation in Clarias batrachus oocytes

Effects of beta-adrenoceptor antagonists propranolol and alprenolol in the oocyte maturation of the catfish (Clarias batrachus) were investigated under in vitro. Cyclic AMP (cAMP) levels were also measured in the control, propranolol and phosphodiesterase (PDE) inhibitor treated oocytes. When full-grown folliculated oocytes were cultured in vitro in the presence of different concentrations of propranolol or alprenolol, both the substances induced germinal vesicle breakdown (GVBD) in a dose-dependent manner. The maturational effect of alprenolol at the concentration of 1.0 mM was similar to that of the 1.5 mM dose of propranolol inducing more than 88% GVBD. In the time course study, when the oocytes were treated with 1.5 mM propranolol or with 1.0 mM alprenolol for various times, both the antagonists induced more than 80% GVBD after 4 h of incubations and this induction gradually increased with the increased duration of treatments. On the other hand, 1.5 mM propranolol treatment caused a significant decrease in oocyte cAMP which was maintained upto the duration of the study (36 h). When the oocytes were first stimulated by 1.5 mM propranolol or 1.0 mM alprenolol for 4 h and then treated with various doses of cAMP or PDE inhibitors (IBMX and theophylline), all these substances effectively blocked beta-adrenoceptor antagonist-induced GVBD. Both these PDE inhibitors promoted the accumulation of cAMP in the oocytes. These results provide the first example of an existence of a cAMP-mediated mechanism of action of beta-adrenoceptor antagonists in the induction of oocyte maturation in fish.     

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.     

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)     

β-Adrenoceptor antagonists reinitiate meiotic maturation in Clarias batrachus oocytes

Effects of β-adrenoceptor antagonists propranolol and alprenolol in the oocyte maturation of the catfish (Clarias batrachus) were investigated under in vitro. Cyclic AMP (cAMP) levels were also measured in the control, propranolol and phosphodiesterase (PDE) inhibitor treated oocytes. When full-grown folliculated oocytes were cultured in vitro in the presence of different concentrations of propranolol or alprenolol, both the substances induced germinal vesicle breakdown (GVBD) in a dose-dependent manner. The maturational effect of alprenolol at the concentration of 1.0 mM was similar to that of the 1.5 mM dose of propranolol inducing more than 88% GVBD. In the time course study, when the oocytes were treated with 1.5 mM propranolol or with 1.0 mM alprenolol for various times, both the antagonists induced more than 80% GVBD after 4 h of incubations and this induction gradually increased with the increased duration of treatments. On the other hand, 1.5 mM propranolol treatment caused a significant decrease in oocyte cAMP which was maintained upto the duration of the study (36 h). When the oocytes were first stimulated by 1.5 mM propranolol or 1.0 mM alprenolol for 4 h and then treated with various doses of cAMP or PDE inhibitors (IBMX and theophylline), all these substances effectively blocked β-adrenoceptor antagonist-induced GVBD. Both these PDE inhibitors promoted the accumulation of cAMP in the oocytes. These results provide the first example of an existence of a cAMP-mediated mechanism of action of β-adrenoceptor antagonists in the induction of oocyte maturation in fish.     

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.     

Effects of meiosis-inhibiting agents and equine chorionic gonadotropin on nuclear maturation of canine oocytes

Experiments were conducted to determine the effects of meiosis-inhibiting-agents and gonadotropins on nuclear maturation of canine oocytes. The culture medium was TCM199 + 10 ng/ml epidermal growth factor supplemented with 25 microM beta-mercaptoethanol, 0.25 mM pyruvate, and 1.0 mM L-glutamine (Basal TCM). Initially, oocytes were cultured in Basal TCM alone or in Basal TCM + dibutylryl cyclic adenosine monophosphate (0.5, 1, 5, or 10 mM dbcAMP) for 24 hr. Dibutylryl cAMP inhibited resumption of meiosis in a dose-dependent manner; 60% of oocytes remained at the germinal vesicle (GV) stage after being cultured for 24 hr in 5 mM dbcAMP. The meiosis-inhibitory effect of dbcAMP appeared to be reversible, as the oocytes resumed meiosis and completed nuclear maturation after being cultured for an additional 48 hr in its absence. Oocytes were then cultured in Basal TCM alone or in Basal TCM + roscovitine (12.5, 25, or 50 microM) for 24 hr. Although approximately 60% of oocytes cultured in 25 microM roscovitine remained at the GV stage, this percentage was not significantly different from the 48% that also remained at the GV stage when cultured in its absence. Oocytes were cultured in Basal TCM + 25 microM roscovitine for 17 hr, exposed briefly to equine chorionic gonadotropin (eCG), and then cultured in Basal TCM for 48 hr. Short exposure of oocytes to eCG was beneficial, as it significantly increased the proportion of oocytes developing beyond germinal vesicle breakdown (P < 0.05) with approximately 20-30% of these were metaphase I (MI) oocytes. Study of the kinetics of nuclear maturation demonstrated that large numbers of oocytes remained at MI even after being cultured for 52 hr following brief exposure to eCG. This study showed that in vitro maturation of canine oocytes can be somewhat improved by short exposure of oocytes to eCG. However, further studies are still required to derive effective methods to mature canine oocytes in vitro.     

Effect of testosterone and dibutyryl c-AMP on the meiotic competence in pig oocytes of various size categories

Spontaneous meiosis resumption was investigated in small pig oocytes of various size categories. Two parameters were studied: 1) the possibility for inhibiting spontaneous meiosis resumption in small oocytes of various size categories and 2) the level of meiotic competence of small oocytes in which meiosis resumption had been temporarily blocked. It was observed that 1 mM dibutyryl cyclic adenosine monophosphate (dbcAMP) combined with 0.5 μM testosterone were able to prevent spontaneous maturation in pig oocytes of various size categories cultured in vitro for up to 10 d. Moreover, a culture of these oocytes with dbcAMP and testosterone has a beneficial effect on the integrity of the oocyte-granulosa cell complex. When oocytes were allowed to mature after long-term inhibition of maturation, the percentage of oocytes able to resume maturation was significantly increased. However, the portion of oocytes which completed maturation, reaching the metaphase II (M II) stage, did not increase. It was found that dbcAMP, in combination with testosterone, also efficiently inhibited spontaneous maturation in fully grown pig oocytes. When the inhibitory effect of dbcAMP and testosterone was reversed in fully grown oocytes, maturation was significantly accelerated.     

In vivo regulation of cyclic AMP phosphodiesterase in Xenopus oocytes. Stimulation by insulin and insulin-like growth factor 1

Cyclic AMP phosphodiesterase activity was measured in vivo after microinjection of [3H]cAMP into intact Xenopus oocytes. This activity was inhibited by extracellular application of methylxanthines, and the dose-dependent inhibition of phosphodiesterase activity correlated with the abilities of isobutylmethylxanthine and theophylline to inhibit oocyte maturation induced by progesterone, with IC50 values of approximately 0.3 and 1.5 mM, respectively. Insulin stimulated in vivo phosphodiesterase activity measured after microinjection of 200 microM [3H]cAMP in a time- and dose-dependent fashion without affecting phosphodiesterase activity measured after microinjection of 2 microM [3H]cAMP. Although progesterone alone had no effect on in vivo phosphodiesterase activity, low concentrations of progesterone (0.01 microM) accelerated the time course of insulin stimulation of both phosphodiesterase activity and oocyte maturation. The EC50 for stimulation of in vivo phosphodiesterase activity by insulin correlated with the IC50 for inhibition of oocyte membrane adenylate cyclase activity measured in vitro (2 and 4 nM, respectively). Twenty-fold higher concentrations of insulin were required to stimulate oocyte maturation. In contrast, insulin-like growth factor 1 stimulated in vivo phosphodiesterase, inhibited in vitro adenylate cyclase, and induced oocyte maturation at concentrations of 0.3-1.0 nM. These results demonstrate a dual regulation of oocyte phosphodiesterase and adenylate cyclase by insulin and insulin-like growth factor 1.     

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.     

Maintenance of meiotic arrest in isolated rat oocytes by the invasive adenylate cyclase of Bordetella pertussis

Rat oocytes resume meiosis spontaneously in vitro within 3 h after their isolation from the ovarian follicles. We report here that the spontaneous maturation of isolated rat oocytes is preceded by a drop in intracellular levels of cyclic adenosine 3',5'-monophosphate (cAMP). Further experiments were carried out to examine the possible correlation between the meiotic status and cAMP levels within the oocyte. To challenge rat cumulus-free oocytes to generate cAMP, bypassing their own adenylate cyclase, a preparation of an invasive adenylate cyclase from Bordetella pertussis was used. We found a dose-dependent elevation of cAMP levels within these oocytes that corresponded to inhibition of their spontaneous maturation. Persistent inhibition of meiosis was obtained with the continuous presence of the enzymatic preparation, whereas its removal resulted in a transient inhibition associated with a drop in cAMP. We suggest that the presence of elevated cAMP levels in the oocyte is directly responsible for the maintenance of meiotic arrest.     

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.     

Cyclic AMP level and phosphodiesterase activity during 17alpha,20beta-dihydroxy-4-pregnen-3-one induction and theophylline inhibition of oocyte maturation in the catfish,Clarias batrachus

This study directly tested the hypothesis that the induction of oocyte maturation in the catfish Clarias batrachus is followed by a transient decrease in oocyte cyclic AMP (cAMP) level that is due to an increase in phosphodiesterase (PDE) activity. Further, the PDE inhibitor theophylline was used to investigate the possible role of PDE in the maturation-inducing action of 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-DP), the physiological maturation-inducing steroid of this catfish species. The results obtained from batches of oocytes taken from the same donor at the same time clearly show a close relationship between dose-dependent induction of germinal vesicle breakdown (GVBD) and PDE activity with a concomitant decrease in cAMP in the oocytes treated with different concentrations of 17alpha,20beta-DP. In contrast, theophylline prevents GVBD and inhibits PDE activity by promoting cAMP accumulation in oocytes. A time-dependent decrease in PDE activity and an increase in cAMP content with a marked inhibition of GVBD were recorded even in oocytes pre-stimulated with 1 microgram/ml 17alpha,20beta-DP for 6 h and then treated with 1 mM theophylline for various times. These results suggest that cAMP plays a key role in the regulation of oocyte maturation in C. batrachus which may be mediated by PDE activity.     

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.     

Interaction of signal cascades induced by cAMP and prolactin in bovine oocyte-cumulus complexes

Prolactin (PRL) is one of the pituitary hormones participating in the control of mammalian folliculo- and oogenesis. In the present study, the joint effect of PRL (50 ng/ml) and dibutyryl cAMP (dbcAMP, 1 mM) on oocyte maturation and the morphologic-functional state of surrounding cumulus cells was investigated in vitro. It has been shown that PRL totally suppresses the braking impact of dbcAMP on meiosis reinitiation and the completion of oocyte nuclear maturation. Furthermore, PRL partly inhibited cumulus expansion induced by dbcAMP, although it exerted the opposite effect in the control medium. In the presence of PRL, the inhibitory impact of dbcAMP on the proliferative activity of cumulus cells and on the PRL-elicited braking of destructive processes in the cells has been found. In cumulus cells, mRNA expression of PRL receptor long isoform was revealed by the RT-PCR method. The data obtained suggest an interaction of signal cascades induced by PRL and cAMP in bovine oocyte-cumulus complexes, with the coupling site of these cascades in oocytes being apparently different from that in cumulus cells.