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)     

Decreases in heterologous metabolic and dye coupling, but not in electrical coupling, accompany meiotic resumption in hamster oocyte-cumulus complexes

The temporal relationship between resumption of meiosis and reduction in either heterologous intercellular coupling, or magnitude of oocyte or cumulus cell resting potential in hamster oocyte-cumulus complexes was investigated. Coupling was assessed qualitatively by lucifer yellow dye transfer and quantitatively by transfer of radiolabeled uridine metabolites or electrical current after culture of complexes in various systems previously characterized either to maintain meiotic arrest or to permit meiotic resumption. In each of the three systems which permitted meiotic resumption, cumulus to oocyte metabolic and dye coupling and oocyte to cumulus dye coupling decreased progressively with time after release from meiotic arrest. In contrast, no similar temporal changes in metabolic or dye coupling were observed in any complex after culture in either of the two systems which maintained meiotic arrest. Analysis of the extent of heterologous ionic coupling revealed that in neither direction was a decrease in ionic uncoupling consistently associated with reinitiation of meiosis. Furthermore, while the resting potential of both the oocyte and cumulus cell underwent changes characteristic of each system employed, the level of neither cell membrane potential was specific to meiotic status. These results support the hypothesis that meiotic maturation in hamster oocytes is accompanied by disruption of the integrity of intercellular, non-ionic coupling between the oocyte and its adherent cumulus cells. The data show, however, that no specific alteration either in the extent of ionic coupling or in the oocyte or cumulus cell resting potential is prerequisite for meiotic resumption in this species.     

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)     

Metabolic coupling and ligand-stimulated meiotic maturation in the mouse oocyte-cumulus cell complex.

Cumulus cells are metabolically coupled to the mammalian oocyte via heterologous gap junctions. One function attributed to the gap junctional communications is the transfer of regulatory signals that direct the meiotic state of the oocyte. However, the precise role of these junctions in meiotic maturation is still unclear. The aim of this study was to test the hypothesis that meiotic resumption is induced by the transfer of a stimulatory signal(s) from the cumulus cells to the oocyte through the gap junctional coupling pathway. We have previously shown that the mitogenic lectin concanavalin A (Con A) induces oocyte maturation in isolated cumulus cell-enclosed oocytes (CEO) when meiotic arrest is maintained with a number of different inhibitory agents [Biol Reprod 1990; 42:413-423]. In the present study, Con A stimulated maturation in dibutyryl cAMP (dbcAMP)-arrested CEO but not in denuded oocytes cocultured with cumulus cells. Heptanol, a known gap junction uncoupler, effectively prevented Con A- and FSH-induced maturation of intact CEO and dramatically reduced metabolic coupling between cumulus cells and the oocyte. However, this alcohol had no effect on denuded oocytes (DO) or on dbcAMP-arrested CEO in the absence of stimulating ligand. Con A and FSH produced only a minimal loss of coupling. When the effects of heptanol were compared with those of the n-alkanols hexanol and decanol, the efficacies of these agents as suppressors of Con A-stimulated oocyte maturation was directly related to their relative abilities to suppress metabolic coupling.(ABSTRACT TRUNCATED AT 250 WORDS)     

Generation of mouse oocytes defective in cAMP synthesis and degradation: endogenous cyclic AMP is essential for meiotic arrest

Although it is established that cAMP accumulation plays a pivotal role in preventing meiotic resumption in mammalian oocytes, the mechanisms controlling cAMP levels in the female gamete have remained elusive. Both production of cAMP via GPCRs/Gs/adenylyl cyclases endogenous to the oocyte as well as diffusion from the somatic compartment through gap junctions have been implicated in maintaining cAMP at levels that preclude maturation. Here we have used a genetic approach to investigate the different biochemical pathways contributing to cAMP accumulation and maturation in mouse oocytes. Because cAMP hydrolysis is greatly decreased and cAMP accumulates above a threshold, oocytes deficient in PDE3A do not resume meiosis in vitro or in vivo, resulting in complete female infertility. In vitro, inactivation of Gs or downregulation of the GPCR GPR3 causes meiotic resumption in the Pde3a null oocytes. Crossing of Pde3a^−/− mice with Gpr3^−/− mice causes partial recovery of female fertility. Unlike the complete meiotic block of the Pde3a null mice, oocyte maturation is restored in the double knockout, although it occurs prematurely as described for the Gpr3^−/− mouse. The increase in cAMP that follows PDE3A ablation is not detected in double mutant oocytes, confirming that GPR3 functions upstream of PDE3A in the regulation of oocyte cAMP. Metabolic coupling between oocytes and granulosa cells was not affected in follicles from the single or double mutant mice, suggesting that diffusion of cAMP is not prevented. Finally, simultaneous ablation of GPR12, an additional receptor expressed in the oocyte, does not modify the Gpr3^−/− phenotype. Taken together, these findings demonstrate that Gpr3 is epistatic to Pde3a and that fertility as well as meiotic arrest in the PDE3A-deficient oocyte is dependent on the activity of GPR3. These findings also suggest that cAMP diffusion through gap junctions or the activity of additional receptors is not sufficient by itself to maintain the meiotic arrest in the mouse oocyte.     

Factors influencing resumption of meiotic maturation and cumulus expansion of porcine oocyte-cumulus cell complexes in vitro

The present study was undertaken to examine effects of various combinations of epidermal growth factor (EGF), transforming growth factor-b?₁ (TGF-b?₁), follicle-stimulating hormone (FSH), luteinizing hormone (LH), androstenedione (A₄), and estradiol-17b? (E₂) on meiotic maturation and cumulus expansion in the pig using an in vitro model system. Oocyte-cumulus cell complexes (OCC) were cultured in the media containing the abovementioned agents for 24 hr and were observed for germinal vesicle breakdown (GVBD), indicative of initiation of meiotic maturation, and for expansion of their cumulus cells. Treatment with EGF significantly increased (P < 0.05) incidence of GVBD, with maximal stimulation occurring at 1 ng/ml (55% vs. 12% in the control). Concentrations of EGF as low as 100 pg/ml significantly stimulated GVBD over control (37% vs. 12%). Addition of EGF (1 ng/ml) and FSH (1.5 μg/ml) together and LH (2 μg/ml) and FSH (1.5 μg/ml) together resulted in significantly higher (P < 0.01) GVBD levels than were observed in response to EGF, FSH, or LH alone. Addition of E₂ (1 μg/ml) had no effect by itself but significantly decreased the incidence of GVBD in the presence of FSH and of LH + FSH. Addition of A₄ (1 μg/ml) significantly reduced the percentage of oocytes undergoing GVBD when added alone or with FSH. Although both EGF and LH stimulated cumulus expansion, FSH was more effective in stimulating cumulus expansion than EGF or LH. TGF-b?₁ had no effect on GVBD or cumulus expansion. These studies indicate that these hormones may have differing roles in oocyte maturation and that their interactions may be part of an intricate system regulating the maturation of oocytes during follicular development in vivo. © 1993 Wiley-Liss, Inc.     

Cows are not mice: the role of cyclic AMP, phosphodiesterases, and adenosine monophosphate-activated protein kinase in the maintenance of meiotic arrest in bovine oocytes

Meiotic maturation in mammalian oocytes is initiated during fetal development, and is then arrested at the dictyate stage - possibly for several years. Oocyte meiosis resumes in preovulatory follicles in response to the lutenizing hormone (LH) surge or spontaneously when competent oocytes are removed from follicles and cultured. The mechanisms involved in meiotic arrest and resumption in bovine oocytes are not fully understood, and several studies point to important differences between oocytes from rodent and livestock species. This paper reviews earlier and contemporary studies on the effects of cAMP-elevating agents and phosphodiesterase (PDE) enzyme inhibitors on the maintenance of meiotic arrest in bovine oocytes in vitro. Contrary to results obtained with mouse oocytes, bovine oocyte meiosis is inhibited by activators of the energy sensor adenosine monophosphate-activated protein kinase (AMPK, mammalian gene PRKA), which is activated by AMP, the degradation product of cAMP. It is not clear whether or not the effects were due to AMPK activation, and they may depend on culture conditions. Evidence suggests that other signaling pathways (for example, the cGMP/nitric oxide pathway) are involved in bovine oocyte meiotic arrest, but further studies are needed to understand the interactions between the signaling pathways that lead to maturation promoting factor (MPF) being inactive or active. An improved understanding of the mechanisms involved in the control of bovine oocyte meiosis will facilitate better control of the process in vitro, resulting in increased developmental competence and increased efficiency of in vitro embryo production procedures.     

Effect of N-2, 2'-O-dibutyryl cyclic AMP upon the interconvertible forms of cyclic AMP phosphodiesterase from human platelets.

The influence of dibutyryl cyclic AMP upon the equilibrium existing between three interconvertible forms - 2.5 S, 4.8 S and 7 S - of cyclic AMP phosphodiesterase from human platelets was investigated. It shifted the equilibrium towards the lighter form. It also exerted a protective effect against the thermo-inactivation of the enzyme. It is suggested that the analogue-induced equilibrium shift towards the dissociated high Km form of the phosphodiesterase might reflect a regulatory mechanism occurring also with natural cyclic nucleotides.     

Assay and importance of adhesive interaction between hamster (Mesocricetus auratus) oocyte-cumulus complexes and the oviductal epithelium

Adhesion between the oocyte-cumulus complex and infundibulum plays an important, but poorly understood, role in oocyte pick-up. The purposes of this study were to determine which components of the oocyte-cumulus complex and oviductal epithelium function in adhesion, to measure adhesion under physiological conditions, and to examine the effect of modulation of adhesion on oocyte-cumulus complex pick-up rate. Oocyte-cumulus complexes containing an expanded matrix were readily transported into the oviduct, while unexpanded complexes lacking an extracellular matrix were not picked up, indicating that the matrix is necessary for pick-up. Transmission electron microscopy revealed that during pick-up, adhesion occurred specifically between the ciliary crowns of the oviduct and the granules and filaments of the cumulus matrix. An assay was developed using vacuum from a low-flow peristaltic precision pump, modified for bi-directional flow, to measure the strength of adhesion between the oocyte-cumulus complex and the oviductal epithelium, and adhesion was measured during physiological conditions. The lectin wheat germ agglutinin and the polycation poly-L-lysine were then used to modulate adhesion, and the effects of increasing or decreasing adhesion on oocyte pick-up rate and ciliary beat frequency were examined. The data show that 1) the matrix of the oocyte-cumulus complex and the ciliary crowns of the oviduct function in adhesion during pick-up and that adhesion is necessary for pick-up, 2) adhesion can be assayed quantitatively and is very uniform among control infundibula, and 3) decreasing or increasing adhesion decreases oocyte pick-up rate and in some cases prevents pick-up without affecting ciliary beat frequency.     

Comparative studies on sporulation-promotive actions of cyclic AMP,theophylline and caffeine in Saccharomyces cerevisiae

Cyclic AMP, theophylline and caffeine promoted sporulation when added to a presporulation medium containing glucose. Caffeine promoted sporulation even when added to a presporulation medium containing acetate as the carbon source, but cyclic AMP and theophylline did not. Caffeine did not increase the intracellular cyclic AMP level, while theophylling did significantly when added to a presporulation medium containing glucose Caffeine inhibited the vegetative DNA synthesis with little effect on RNA and protein synthesis, resulting in the increase in cell volume, dry weight, and RNA and protein contents, but cyclic AMP and theophylline did not show such effects.     

Changes in the content of cyclic AMP and cyclic GMP during the development of Xenopus laevis.

The levels of the three major DNA-dependent RNA polymerases (enzymes I, II and III) present in the dimorphic fungus Mucor rouxii have been investigated during the transition from yeast-like cells to mycelial growth. Increases in the specific activity of crude extracts were observed at 2 h and at 6 h after induction of mycelium formation by aeration of yeast-like cells. These increases could be attributed to changes in the specific activities of enzymes I and II. Alterations were also found in the relative amounts of enzymes I and II: prior to aeration, 31% of the total polymerase activity of crude extracts was present as enzyme I; after 2 h of aeration, the specific activity of this enzyme doubled and the relative amount increased to 64% of the total activity. After 6 h of aeration, the relative amounts of enzymes I and II were 25 and 65%, respectively, and the specific activity of enzyme II had nearly doubled. The amounts and specific activities of enzyme III did not change significantly during the transition.     

Differential effects of testosterone and dibutyryl cyclic AMP on the meiotic maturation of mouse oocytes in vitro

Fully grown, meiotically immature mouse oocytes were isolated and cultured under varying conditions with the aim of determining a) whether the inhibitory effects of testosterone on oocyte meiotic maturation require the synthesis of new oocyte proteins and b) if the meiosis-inhibiting effects of testosterone and dibutyryl cyclic AMP (dbcAMP) are distinct and can be differentiated. We found that the inclusion of puromycin in culture medium containing testosterone has no effect on the meiosis-inhibiting potency of testosterone or upon the reversibility of testosterone effects. We conclude that testosterone inhibits oocyte meiosis by a mechanism that is independent of protein synthesis. We also found that oocytes exposed to testosterone recover more rapidly, as evidenced by the timing of germinal vesicle breakdown (GVBD) following placement in a control medium, than do oocytes exposed to dbcAMP. Through further investigation of this phenomenon we have determined the sequence of testosterone and dbcAMP effects relative to the time course of GVBD. A testosterone-sensitive event occurs 20 min prior to GVBD, while the dbcAMP-sensitive event precedes GVBD by 41 min. The nature of this difference may involve the differential interaction of testosterone and dbcAMP with a set of puromycin-sensitive proteins that are required for GVBD. When oocytes were initially cultured in medium containing both puromycin and either testosterone or dbcAMP and then moved to medium containing puromycin alone the incidence of GVBD was reduced relative to oocytes never exposed to puromycin. This observation suggests that mouse oocytes contain proteins that are required for GVBD and that experience a high turnover rate. The degree of reduction in GVBD was a function of the length of puromycin exposure and was significantly greater in dbcAMP- than in testosterone-exposed oocytes. If oocytes were initially cultured in medium containing puromycin and dbcAMP, the rate of GVBD upon removal of dbcAMP was initially slow but increased with time. This observation is consistent with the hypothesis that dbcAMP inhibits oocytes at a point prior to the functioning of the puromycin-sensitive proteins. However, if oocytes were cultured in medium containing puromycin and testosterone the rate of GVBD following testosterone removal was not significantly reduced relative to oocytes that were not exposed to puromycin. This observation suggests that testosterone acts to inhibit meiosis at a site beyond the function of the puromycin-sensitive proteins or that testosterone causes a reduction in the turnover rate of these proteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Growth inhibition and the regulation of cyclic AMP by the triphenylethylene anti-estrogen tamoxifen in the quail oviduct.

The aim of the present study was to investigate the regulation of cAMP by tamoxifen in quail oviduct. A single injection of tamoxifen to immature female quails induced a transient activation of adenylate cyclase. Enzyme activity began to increase 3 h after the injection, peaked at 6 h and then dropped to control level at 12 h. The same time-response curves were observed following the injection of estradiol benzoate or estradiol benzoate + tamoxifen. Moreover, adenylcyclase exhibited the same sensitivity to exogenous activators (guanylylimidodiphosphate and forskolin) in the different treated groups. Phosphodiesterase activity was left unchanged during the prereplicative period and cAMP concentration was significantly increased at 6 h (+ 44.3%). Then, cAMP concentration continued to increase (+ 73.8% at 24 h) while cAMP phosphodiesterase and adenylcyclase activities remained at control levels. Injected concurrently with estradiol benzoate, tamoxifen completely inhibited the growth promoting effect of estradiol. Tamoxifen also inhibited the activation of adenylcyclase and cAMP phosphodiesterase induced by the hormone alone during the proliferative phase of the tissue. Moreover, the combined treatment led to a sustained elevation of cAMP in the oviduct, whereas estradiol benzoate alone decreased the level of cAMP. These results and those of our previous studies showing a significant correlation between the growth inhibitory potency of triphenylethylene derivatives in vivo and their efficiency to inhibit calmodulin-dependent cAMP phosphodiesterase in vitro, strongly suggest that the differential regulation of cAMP levels by estradiol and tamoxifen is essential for the growth promoting or growth inhibiting activities of these molecules.     

Changes in the cyclic AMP content during growth and development of Acetabularia.

The 3',5'-adenosine monophosphate (cyclic-AMP) content of the unicellular alga Acetabularia has been examined at various developmental stages. It has been found that very young algae, less than 10mm in length, have a high cAMP content [more than 7 pmoles per 100 mg wet weight (WW)], but that with the growth of the algae, the cAMP content decreases rapidly, reaching the low level of 0.5--1.0 pmoles per 100mg WW. The cAMP content remains at this level until cap differentiation, after which an increase in cAMP content accompanies cap enlargement. It has been shown that these results are unlikely to be affected by changes in the cAMP content induced by variations in circadian rhythm. Treatment with theophylline (2.10(-3) M), a phosphodieterase inhibitor, results in an increase in the cAMP content and delays growth and cap formation. Experiments on the effects of theophylline upon the circadian rhythm of oxygen evolution have shown that the continuous presence of theophylline in the culture medium does not induce a phase shift in the rhythm. The cAMP content of anucleate Acetabularia shows development stage variations parallel to that of the whole algae.