cAMP synthesis in the rat oocyte

cAMP synthesis by the rat oocyte and cumulus-oocyte complex was studied using direct labeling techniques. Cumulus-oocyte complexes synthesized cAMP in response to luteinizing hormone, follicle-stimulating hormone, cholera toxin, and forskolin. However, naked oocytes prepared from cumulus-oocyte complexes by mechanically removing the cumulus cells synthesized cAMP only in response to forskolin and follicle-stimulating hormone; cholera toxin and luteinizing hormone did not stimulate cAMP synthesis. Cholera toxin could augment the response of the oocytes to FSH, indicating an intact, though atypical, adenylate cyclase system. Forskolin was found to inhibit the onset of oocyte maturation in both cumulus-oocyte complexes and naked oocytes. The implications of these findings for the relationship between cAMP synthesis and oocyte maturation in the rat are discussed.     

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

Forskolin (0-100 microM), a reversible stimulator of the catalytic subunit of adenylate cyclase, induced dose-response increases in the % germinal vesicle (GV) (ID50 = 2.68 microM, where ID50 is the dose of forskolin which maintained the meiotic arrest at the germinal vesicle stage, determined cytogenetically, of 50% cultured oocytes), and the cAMP content (determined by RIA) of cumulus-enclosed oocytes and cumulus masses. A significant positive correlation was established between the amount of cAMP within the cumulus mass and that in the corresponding enclosed oocyte (r = 0.78). In contrast, neither the % GV nor the cAMP content of cumulus-free oocytes was affected by the drug. The arresting action of forskolin upon cumulus-enclosed oocytes was dependent upon the presence of adherent cumulus cells, and was transient and fully reversible. The gradual decrease in the % GV of cumulus-enclosed oocytes cultured from 4 to 12 h in 25 microM-forskolin (84, 54 and 22% GV at 4, 8 and 12 h, respectively) was accompanied by a drastic fall in intra-oocyte cAMP at 8 h (41.2 +/- 2.5 and 1.1 +/- 0.6 fmol/oocyte at 4 and 8 h, respectively), while the cumulus cell cAMP content remained constant (135.3 +/- 14.7 and 145.9 +/- 28.7 fmol/cumulus at 4 and 8 h, respectively). Moreover, heterologous metabolic coupling, assessed by determination of the fraction of radiolabelled uridine marker that was transferred from the cumulus mass to the oocyte, significantly decreased. These results show that cumulus cell cAMP is transferred to the rat oocyte where it appears to play a pivotal role in the regulation of meiosis and that the rat oolemma does not appear to possess active catalytic subunits of adenylate cyclase in an amount adequate to stimulate measurably cAMP synthesis.     

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.     

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.     

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)     

Stimulation and inhibition of rat oocyte meiosis by forskolin

The adenylate cyclase activator forskolin was used to study the role of cAMP for oocyte meiosis and follicular steroid secretion. Follicular and cumulus cAMP production was stimulated dose-dependently by forskolin, as was the follicular secretion of progesterone, testosterone and estradiol. Forskolin induced meiosis in follicle-enclosed oocytes with a maximal effect at 1 microM, with lower and higher concentrations being less effective. The spontaneous resumption of meiosis in isolated cumulus-enclosed oocytes was dose-dependently retarded by forskolin. Meiosis of cumulus-free oocytes was also retarded but only slightly. These data support the earlier hypothesis that a limited increase in follicular cAMP levels triggers meiosis, whereas sustained levels of cAMP in the oocyte itself prevent meiosis.     

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.     

An intercellular pathway for glucose transport into mouse oocytes

Glucose is an essential nutrient for mammalian cells. Emerging evidence suggests that glucose within the oocyte regulates meiotic maturation. However, it remains controversial as to whether, and if so how, glucose enters oocytes within cumulus-oocyte complexes (COCs). We used a fluorescent glucose derivative (6-NBDG) to trace glucose transport within live mouse COCs and employed inhibitors of glucose transporters (GLUTs) and gap junction proteins to examine their distinct roles in glucose uptake by cumulus cells and the oocyte. We showed that fluorescent glucose enters both cumulus-enclosed and denuded oocytes. Treating COCs with GLUT inhibitors leads to simultaneous decreases in glucose uptake in cumulus cells and the surrounded oocyte but no effect on denuded oocytes. Pharmacological blockade of of gap junctions between the oocyte and cumulus cells significantly inhibited fluorescent glucose transport to oocytes. Moreover, we find that both in vivo hyperglycemic environment and in vitro high-glucose culture increase free glucose levels in oocytes via gap junctional channels. These findings reveal an intercellular pathway for glucose transport into oocytes: glucose is taken up by cumulus cells via the GLUT system and then transferred into the oocyte through gap junctions. This intercellular pathway may partly mediate the effects of high-glucose condition on oocyte quality.     

Regulation of oocyte maturation in the mouse: possible roles of intercellular communication, cAMP, and testosterone

Experiments were performed to determine if elevation of cumulus cell cAMP results in an increase in mouse oocyte cAMP while the heterologous gap junctions were intact. Both follicle-stimulating hormone (FSH) and cholera toxin induced a marked increase (>20-fold) in intracellular cAMP in isolated mouse cumulus cell-oocyte complexes in the presence of 3-isobutyl-1-methyl xanthine (IBMX). Concomitantly, both FSH and cholera toxin transiently inhibited resumption of meiosis of cumulus cell-enclosed but not denuded oocytes. The transient nature of the inhibitory effect produced by either FSH or cholera toxin was correlated with the cAMP level in the cumulus cell-oocyte complex. The inhibitory effect, however, was apparently not due to movement of cumulus cell cAMP to the oocyte via the functional heterologous gap junctions between cumulus cells and the oocyte. Radioimmunoassay of cAMP in oocytes free of attached cumulus cells or cumulus cell-enclosed oocytes exposed to either FSH or cholera toxin revealed that both groups of oocytes contained similar amounts of cAMP (about 0.14 fmole/oocyte). Metabolic labeling of cumulus cell-oocyte complexes with [³H]adenosine followed by incubation with either FSH or cholera toxin resulted in a marked increase in the amount of radiolabeled cAMP compared to that in unstimulated complexes. However, similar amounts of radiolabeled cAMP were found in oocytes derived from either stimulated or unstimulated complexes. Thus, we have not detected, using two methods of assay, that increasing the cAMP content of the cumulus cells results in any increase in the cAMP content of the oocyte. The apparent compartmentalization of cumulus cell cAMP elevated in response to either FSH or cholera toxin was not due to disruption of intercellular communication between the two cell types during the incubation; metabolic cooperativity was present between the two cell types and molecules of similar molecular weight and charge relative to that of cAMP were rapidly equilibrated between the two cell types. Testosterone potentiated the FSH/cholera toxin-induced transient inhibition of maturation of cumulus cell-enclosed oocytes. However, testosterone did not increase cAMP accumulation produced by either FSH or cholera toxin, decrease the rate of cAMP degradation, or promote movement of cumulus cell cAMP to the oocyte. Since cAMP elevated in response to FSH or cholera toxin appeared to be compartmentalized to cumulus cells and since neither FSH, cholera toxin, nor testosterone inhibited resumption of meiosis in denuded oocytes, it appears that the inhibitory effect promoted by FSH or cholera toxin is directly mediated by an agent other than cAMP, although cAMP generation is required for its action and that cumulus cells mediate the inhibition. These results are discussed in terms of a possible role of cAMP and steroids in regulating maturation in the mouse.     

Effects of protein kinase inhibitors on pig oocyte maturation in vitro.

Normal oocyte maturation depends on signal transmission between granulosa cells and the oocyte. We have analysed the effects of inhibiting (I) cyclic AMP-dependent protein kinase (protein kinase A, PK-A), (II) Ca2+/phospholipid-dependent protein kinase (protein kinase C, PK-C) and (III) calmodulin (CaM) on pig oocyte maturation in vitro, protein synthesis and phosphorylation. The inhibition of PK-A using a specific inhibitor H8, decreased the maturation rate (rate of germinal vesicle breakdown, GVBD) of cumulus-enclosed pig oocytes in a dose-dependent manner by approximately 12%, reaching a plateau at 100 microM. The inhibition of PK-C with H7, an inhibitor with some side-effects on PK-A, decreased the maturation rate of cumulus-enclosed oocytes in a dose-dependent manner to a maximum of 20% at a concentration of 100 microM. The calmodulin antagonist W7 up to a concentration of 200 microM had no effects on maturation of cumulus-enclosed pig oocytes. None of the inhibitors (H7, H8 and W7) altered the patterns of protein synthesis of either pig oocytes and cumulus cells after maturation in vitro. Oocyte phosphoprotein patterns were, however, clearly changed by W7. Cumulus cell protein phosphorylation patterns were changed by all 3 agents. Since inhibition of cyclic AMP and Ca2+ phospholipid pathways by PK-A and PK-C blocking chemicals affected only a limited proportion of oocytes (12 and 20%, respectively) and inhibition of Ca2+ binding to CaM was without effect on oocyte maturation, we conclude that these pathways modulate rather than regulate oocyte maturation in the pig.     

Apoptosis in cumulus cells, but not in oocytes, may influence bovine embryonic developmental competence

Aim of our study was to clarify if the occurrence of apoptosis in oocytes and cumulus cells is correlated to bovine oocyte developmental competence. The cumulus-oocyte complexes (COCs) were selected according to cumulus status: G1 with more than five layers of compact cumulus cells, G2 with one to five layers of compact cumulus cells and G3 with expanded cumulus cells. The degree of apoptosis in cumulus cells and oocytes measured by caspase staining and TUNEL assay before and after maturation, and 24 h post-insemination was compared to the cleavage, blastocyst formation and hatching rates of each group. Highest cleavage, blastocyst and hatching rates were found in cumulus-oocyte complexes with more than five layers of compact cumulus cells, but no apoptosis was detected in immature or in vitro matured oocytes, regardless of the cumulus status. Many cumulus cells contained active caspases before maturation, but caspase activity declined dramatically after maturation. TUNEL positive cells were rarely observed in each cumulus-oocyte complex upon oocyte recovery, but a huge increase of them was seen after in vitro maturation. Significantly more TUNEL and caspase positive cells were found in G2 cumulus-oocyte complexes. Our results suggest that: (i) oocyte apoptosis does not account for the inferior oocyte quality of G2 and G3; (ii) apoptosis occurs in cumulus cells regardless of the number and compactness of cumulus cells; and (iii) the degree of apoptosis in the compact cumulus-oocyte complexes (G1 and G2) is negatively correlated to the developmental competence of oocyte.     

cAMP in ovine oocytes: localization of synthesis and its action on protein synthesis, phosphorylation, and meiosis

In the first series of experiments, the source of cAMP in the sheep oocyte was studied. Cholera toxin was shown to be a potent stimulator of cAMP in isolated sheep oocytes, demonstrating the presence of adenyl cyclase. There was no evidence for transmission of cAMP from stimulated myocardial cell monolayers to cumulus-enclosed oocytes even though the existence of a concentration gradient of cAMP and of intercellular communication were demonstrated. However, gonadotrophin-stimulated follicle shells were able to induce a rise in the cAMP content of denuded or cumulus-enclosed oocytes in the same dish, independently of cell contact. Further experiments were designed to study the effects of a cholera toxin-stimulated rise in cAMP on the maturation of oocytes. When applied to cumulus-oocyte complexes, cholera toxin did not block germinal vesicle breakdown (GVBD), nor the accompanying changes in protein synthesis and phosphorylation, although there was evidence for a delaying effect. There were, however, indications that the toxin was inducing abnormalities that became gross when the concentration was raised to 1 microgram/ml. This high concentration of cholera toxin was able to block the maturation of oocytes in intact, gonadotrophin-treated follicles, although once again abnormalities were evident. We conclude that the role of cAMP in the maturation of the sheep oocyte is different from that proposed in the mouse.     

Gonadotropin-induced murine oocyte maturation in vivo is not associated with decreased cyclic adenosine monophosphate in the oocyte-cumulus cell complex

The cyclic adenosine monophosphate (cAMP) content of intact oocyte-cumulus cell complexes at various times after the induction of oocyte maturation in mice in vivo was correlated with the time of commitment by the oocytes to undergo germinal vesicle breakdown (GVB) and metabolic coupling between the oocyte and cumulus cells. Seventy-nine percent of the oocytes either underwent GVB or were committed to do so by 2 h after injection of human chorionic gonadotropin (hCG). This occurred without a decrease in the coupling between cumulus cells and the oocyte and with increasing cAMP levels in the oocyte-cumulus cell complex. Maintenance of threshold levels of cAMP within mammalian oocytes appears essential for the maintenance of meiotic arrest, but data presented here suggest that oocyte maturation in mice is induced by gonadotropins in nonatretic follicles in vivo by some mechanism other than one which decreases the cAMP content of the intact oocyte-cumulus cell complex.     

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.     

Forskolin and mouse oocyte maturation in vitro

Oocytes isolated from mature follicles undergo spontaneous maturation when cultured in vitro. Forskolin, an adenylate cyclase stimulator, inhibited resumption of meiosis of cumulus-free mouse oocytes in vitro. Germinal vesicle breakdown (GVBD) was prevented in more than 85% of the oocytes treated by forskolin at concentrations of 20 micrograms/ml and higher. The inhibiting effect of forskolin was dose-dependent and reversible. FSH, LH, FSH plus LH, estrogen, progesterone, and estrogen plus progesterone did not reverse the block induced by forskolin in cumulus-free and cumulus-enclosed oocytes. The present results suggest that intracellular cAMP may play a role in the regulation of oocyte maturation.     

Induction of meiotic maturation of follicle-enclosed oocytes of rabbits by a transient increase followed by an abrupt decrease in cyclic AMP concentration.

The involvement of cyclic adenosine monophosphate (cAMP) in mammalian oocyte maturation was assessed using cultures of rabbit cumulus-oocyte complexes and perfused rabbit ovaries. Rabbit cumulus-oocyte complexes were cultured in Brackett's medium with or without forskolin at 10(-4), 10(-5) or 10(-6) mol l-1 for 3-6 h. At 3 or 4 h spontaneous meiotic maturation was significantly (P < 0.05) inhibited by forskolin at 10(-4) mol l-1. With prolonged incubation, spontaneous maturation progressed despite exposure to forskolin. In the second experiment ovaries were perfused for 12 h with forskolin (10(-4), 10(-5) or 10(-6) mol l-1) or medium alone. Neither ovulation nor degeneration of follicular oocytes occurred in any perfused ovary. The percentage of follicular oocytes achieving germinal vesicle breakdown was significantly (P < 0.001) increased in response to forskolin in a dose-related manner. In an additional experiment, ovaries were perfused with forskolin at 10(-4) mol l-1. A significant increase in the cAMP content in the follicle was observed within 30 min, but the ability to produce cAMP in response to forskolin decreased as the duration of perfusion was increased. Intraoocyte cAMP increased significantly within 30 min and reached its maximum 2 h after exposure to forskolin. Thereafter, cAMP levels in the oocytes decreased abruptly. This drop in intraoocyte cAMP concentration was followed by the resumption of meiosis. The alterations of intraoocyte cAMP contents following exposure to hCG in vivo paralleled those observed in the ovaries perfused with forskolin. These data suggest that a transient, but not continuous, increase in cAMP concentration after the gonadotrophin surge may be required to initiate oocyte maturation.     

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)     

Antagonistic actions of estradiol and tamoxifen upon forskolin-dependent meiotic arrest, intercellular coupling, and the cyclic AMP content of hamster oocyte-cumulus complexes

The effects of estradiol (E2) and the anti-E2 tamoxifen (tam) on forskolin (F)-dependent meiotic arrest in hamster oocytes were investigated. The hypotheses tested were that 1) the arresting action of F is enhanced by E2 and suppressed by tam and 2) the extent of heterologous metabolic coupling and the concomitant transfer of cumulus cell cAMP into the oocyte is increased and decreased by E2 and tam, respectively. E2 was tested with the ID25 F (where ID25 is the dose of F previously shown to arrest the meiosis of 25% cultured oocytes; intact, 3 microM; denuded, 10 microM) and tam was tested with the ID75 F (intact, 10 microM; denuded, 100 microM). E2 induced reversible dose-dependent increases in the percent germinal vesicle (%GV; determined cytogenetically) of both intact and denuded oocytes in the presence of ID25 F (intact: ID50 E2 = 18.0 microM; denuded: ID50 E2 = 17.2 microM) but, in contrast to intact oocytes, E2 also exerted a dose-dependent action on denuded oocytes in the absence of F (ID50 = 26.1 microM). E2 induced dose-dependent increases in the cAMP content (determined by RIA) of intact oocytes (cAMP-oo) and of cumulus masses (cAMP-cm) and in the ratio of cAMPooo:cAMP-cm but failed to elevate F-stimulated cAMP in denuded oocytes. Heterologous metabolic coupling, as assessed by determination of the fraction of radiolabeled uridine marker that was transferred from the cumulus cells to the oocyte, was not significantly enhanced by E2. In contrast to denuded oocytes, tam induced dose-dependent decreases in the %GV and cAMP content of intact oocytes in the presence of ID75 F and significantly depressed heterologous metabolic coupling. While tam failed to antagonize the E2 action on denuded oocytes in the presence of ID25 F, in intact oocytes cultured with E2 and the ID25 F, the anti-E2 significantly decreased the %GV, the cAMP-oo and cAMP-cm, and the extent of heterologous metabolic coupling. These data show that while E2 can directly arrest the maturation of denuded hamster oocytes with no associated elevation of cAMP-oo, E2-enhancement of arrest in intact oocytes is correlated with both elevation of cAMP within the oocyte-cumulus complex and maintenance of heterologous metabolic coupling and is accompanied by an increase in cAMP-oo.     

Effects of follicle stimulating hormone and purines on rat oocyte maturation

The presented data demonstrate a dose-dependent inhibition of spontaneous meiosis of cumulus-enclosed rat oocytes by guanosine, hypoxanthine, and adenosine. The inhibition by adenosine was transient whereas guanosine and hypoxanthine exerted a persistent effect over 24 h of incubation. The order of potency of the substances was guanosine greater than hypoxanthine greater than adenosine and the inhibition was reversible. The inhibitory effect was reduced when the cumulus cells around the oocyte were removed. The inhibition during the first 12 h of incubation was potentiated by FSH. However, at 24 h of incubation FSH partially overcame the inhibitory effect by hypoxanthine but did not influence the inhibitory effect by guanosine. Also 8BrcAMP potentiated the inhibitory effect observed by guanosine, hypoxanthine, and adenosine, suggesting that the potentiating effect of FSH was mediated via cAMP. Our data demonstrate that adenosine, hypoxanthine, and guanosine synergized with FSH in inhibiting spontaneous rat meiosis, as previously shown in mouse. FSH could partially overcome the inhibitory effect exerted by hypoxanthine but did not counteract the inhibitory effect of guanosine.