The development of competence for meiotic maturation during oogenesis in Xenopus laevis

Meiotic maturation of large, 1.2-1.4 mm in diameter, stage VI oocytes of Xenopus laevis can be induced to mature in vitro by exposure to progesterone or by microinjection of maturation-promoting factor (MPF). Small, 0.95 mm in diameter, stage IV oocytes do not respond to progesterone but do undergo germinal vesicle breakdown (GVBD) in response to microinjection of MPF. The possibility that small oocytes are nonresponsive to progesterone due to a specific defect in an event known to occur with large oocytes is investigated. Both large and small oocytes possess a plasma membrane steroid receptor (Mr = 110,000) as measured by photoaffinity labeling with [3H]R5020, but the density of receptors in small oocytes is only 20% of that in large oocytes. Adenylate cyclase activity stimulated by guanyl-5'-yl-imidodiphosphate is equally inhibited by steroid (50%) in plasma membranes from both large and small oocytes with an apparent IC50 of 2 X 10(-7) M progesterone. Microinjection of the heat-stable inhibitor protein of cAMP-dependent protein kinase induces GVBD in large but not in small oocytes. These results indicate that the nonresponsiveness of small, stage IV oocytes to progesterone is due to a deficiency in an event(s) subsequent to cAMP fluctuations but prior to MPF action.     

Role of cAMP in modulating intrafollicular progesterone levels and oocyte maturation in amphibians (Rana pipiens)

The role of cAMP in regulating follicular progesterone levels and oocyte maturation was investigated following in vitro culture of amphibian (Rana pipiens) ovarian follicles. Intrafollicular levels of cAMP were manipulated with the use of a stimulator of cAMP synthesis (forskolin) or by exogenous addition of cAMP alone or either of these in combination with an inhibitor of cAMP catabolism (3-isobutyl-1-methyl xanthine, IBMX). Follicular progesterone content was determined by RIA and oocyte maturation was assessed cytologically. In the presence of increasing doses of forskolin (0-3 microM), cAMP (0-3 mM), or dibutyryl cAMP (dbcAMP, 0-2.5 mM) increasing but low levels of progesterone were detected. Increasing doses of IBMX (0-0.09 mM) alone had no significant effect on follicular steroid content. Exogenous cAMP, dbcAMP, or IBMX (0.09 mM) suppressed hormone-induced oocyte maturation. Simultaneous exposure of follicles to increasing doses of both forskolin (0-3 microM) and IBMX (0-0.09 mM) markedly increased intrafollicular progesterone levels to those produced by frog pituitary homogenate (FPH). A marked increase in progesterone levels also occurred when follicles were exposed to exogenous cAMP (3 mM) and IBMX (0.09 mM). These results indicate that exogenous cAMP is incorporated by follicle cells and that forskolin effects are mediated through cAMP. Changes in follicular progesterone levels (increase and decrease) over time following FPH or cAMP manipulation (cAMP + IBMX or forskolin + IBMX) were essentially identical. In contrast to cAMP, cGMP was inactive in inhibiting hormone induced GVBD or stimulating follicular progesterone accumulation. Elevation of follicular and medium levels of progesterone resulting from FPH or cAMP stimulation required the presence of the somatic follicular cells. The decrease in follicular progesterone levels with prolonged culture was not associated with a corresponding increase in progesterone levels in the medium. The decrease in follicular progesterone levels appears to reflect steroid catabolism rather than loss of steroid to the culture medium. The results suggest that the level of intracellular cAMP in the follicle cells is modulated by the relative activity of the adenylate cyclase system and phosphodiesterase and that FPH can affect both components. Thus, intracellular levels of cAMP play a key role in regulating follicular progesterone levels and FPH action on the follicle cells. The steroidogenic capacity of follicle cells can be manipulated independently of FPH stimulation.     

Calmodulin transmitted through gap junctions stimulates endocytic incorporation of yolk precursors in insect oocytes

In ovarian follicles of Oncopeltus fasciatus, and of Xylocopa virginica, calmodulin (CaM) of epithelial cell origin is required by oocytes for endocytic uptake of yolk precursor molecules. Furthermore, this 17-19 kDa protein is normally transported to the oocytes via gap junctions. Downregulation of gap junctions by treatment with 1 mM octanol or separation of the epithelial cells from their oocytes terminated precursor uptake, and this activity could be rescued by microinjection of 60 microM CaM, but not by injections of incubation medium, nor solutions of other molecular species tested. That endogenous CaM is required was confirmed by incubating otherwise untreated follicles in physiological salt solution (PSS) containing either calmidazolium or W-7, both known antagonists of CaM. By radioimmunoprecipitation, we show that the epithelial cells surrounding an oocyte synthesized 15 times as much calmodulin as did the oocytes they encircled. Neither octanol-treated follicles nor denuded oocytes incubated in medium containing calmodulin were able to resume endocytosis, arguing against an extracellular route. However, fluorescently labeled calmodulin microinjected into oocytes is shown to have crossed through gap junctions, making epithelial cells distinctly fluorescent.     

A possible role for Mg2+ ions in the induction of meiotic maturation of Xenopus oocyte

Progesterone induces in vitro the meiotic cell division of Xenopus full-grown oocytes. Microinjection into oocyte of a solution containing Mg2+ (20 mM) facilitates by one order of magnitude the dose of progesterone which induces 50% of germinal vesicle breakdown. Microinjected in the absence of hormone, Mg2+ and also Mn2+ can induce maturation with efficiencies of, respectively, 24% (SEM = 8; n = 13) and 70% (SEM = 6; n = 23). The dose-response curves of cation-induction of maturation show an optimum of 20 mM for Mg2+ and 15 mM for Mn2+ (pipet concentration); higher doses were less active. Cation-induction of maturation is inhibited when oocytes are preincubated with cholera toxin (500 ng/ml); nevertheless, it cannot be interpreted at the level of cAMP, since both Mg2+ and Mn2+ microinjections provoke an increase in the oocyte cAMP content. Mg2+ induction of maturation is more efficient when oocytes are incubated in trimethylamine at pH 8.2, which is known to increase intracellular pH suggesting an action at the level of alkali pH-sensitive enzymes. Altogether, our results indicate a positive role for Mg2+ ions in the induction of oocyte maturation and raise an attractive hypothesis about the respective roles of cAMP and Mg2+ changes involved in the mechanism of progesterone action. Our results also show that co-injection of 2-glycerophosphate and Mg2+ ions, which are both commonly used in the preparation of the MPF mitotic factors from dividing cells, induces oocyte maturation more efficiently than Mg2+ alone and drastically shortens the kinetics of germinal vesicle breakdown to 1 h 30 min to 2 h 30 min.     

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.     

Selective carboxyl methylation of structurally altered calmodulins in Xenopus oocytes

The eucaryotic protein carboxyl methyltransferase specifically modifies atypical D-aspartyl and L-isoaspartyl residues which are generated spontaneously as proteins age. The selectivity of the enzyme for altered proteins in intact cells was explored by co-injecting Xenopus laevis oocytes with S-adenosyl-L-[methyl-3H]methionine and structurally altered calmodulins generated during a 14-day preincubation in vitro. Control experiments indicated that the oocyte protein carboxyl methyltransferase was not saturated with endogenous substrates, since protein carboxyl methylation rates could be stimulated up to 8-fold by increasing concentrations of injected calmodulin. The oocyte protein carboxyl methyltransferase showed strong selectivities for bovine brain and bacterially synthesized calmodulins which had been preincubated in the presence of 1 mM EDTA relative to calmodulins which had been preincubated with 1 mM CaCl2. Radioactive methyl groups were incorporated into base-stable linkages with recombinant calmodulin as well as into carboxyl methyl esters following its microinjection into oocytes. This base-stable radioactivity most likely represents the trimethylation of lysine 115, a highly conserved post-translational modification which is present in bovine and Xenopus but not in bacterially synthesized calmodulin. Endogenous oocyte calmodulin incorporates radioactivity into both carboxyl methyl esters and into base-stable linkages following microinjection of oocytes with S-adenosyl-[methyl-3H]methionine alone. The rate of oocyte calmodulin carboxyl methylation in injected oocytes is calculated to be similar to that of lysine 115 trimethylation, suggesting that the rate of calmodulin carboxyl methylation is similar to that of calmodulin synthesis. At steady state, oocyte calmodulin contains approximately 0.0002 esters/mol of protein, which turn over rapidly. The results suggest the quantitative significance of carboxyl methylation in the metabolism of oocyte calmodulin.     

Calcium-dependent regulation of progesterone production by isolated rat granulosa cells: effects of the calcium ionophore A23187, prostaglandin E2, dl-isoproterenol and cholera toxin

The role of calcium in the regulation of ovarian steroidogenesis was investigated in granulosa cells from estradiol-treated immature rats. Incubation of granulosa cells with various calcium channel blockers (verapamil, cobalt or manganese) and a calcium chelator (EGTA) resulted in marked decreases in progesterone production in response to follicle-stimulating hormone (FSH), cholera toxin, prostaglandin E2, dl-isoproterenol and dibutyryl cyclic AMP (Bt2cAMP). Cyclic AMP production, however, was unaffected by treatment with EGTA and verapamil at concentrations which attenuated steroidogenesis (0.1-1.0 mM and 125 microM, respectively). Two inhibitors of the calcium-dependent regulatory protein, calmodulin [trifluoperazine, 40 microM and 1[bis-(p-chlorophenyl)methyl] 3-[2,4-dichloro-beta-(2,4- dichlorobenzyloxy )-phenethyl]imidazolium chloride, ( R24571 ) 20 microM] significantly inhibited both cyclic AMP and progesterone production elicited by these stimulatory agents. Over the concentration range of 62.5 ng/ml-1.0 micrograms/ml, the calcium ionophore A23187 increased basal progesterone production in a dose-dependent manner, with half-maximal stimulation at approximately 0.14 microgram/ml. Maximal steroidogenic response to the calcium ionophore (1 microgram/ml) however, was only 50% of that evoked by FSH (0.33 microgram/ml). A23187 (0.5 microgram/ml) significantly enhanced progesterone production stimulated by a low concentration of FSH (0.025 microgram/ml) but failed to potentiate the maximally stimulatory action of the gonadotropin (0.33 microgram/ml). These findings support our earlier suggestion that the calcium-calmodulin system plays a central role in the gonadotropic regulation of ovarian steroidogenesis and suggest that a transmembrane flux of extracellular calcium may be an important and common step in the mechanism of stimulation of granulosa cell progesterone production.     

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.     

Calmodulin triggers the resumption of meiosis in amphibian oocytes

The calcium-binding protein, calmodulin, has been purified from Xenopus laevis oocytes. This 18,500-dalton protein, pl 4.3, has two high-affinity calcium-binding sites per mole protein having a dissociation constant of 2.8 x 10(-6) M. Full-grown Xenopus oocytes, arrested in late G2 of the meiotic cell cycle, resumed meiosis when microinjected with 60-80 ng (3-4 pmol) of calmodulin in the form of a calcium-calmodulin complex. The timing of the meiotic events in these recipient oocytes was the same as that normally induced by progesterone. Xenopus ovarian calmodulin stimulated bovine brain phosphodiesterase (PDE) 3- to 10-fold in a calcium-dependent manner, but it had no apparent effect on ovarian PDE activity. A calcium-calmodulin-dependent protein kinase has been isolated from Xenopus oocytes using a calmodulin-Sepharose 4B affinity column. The possible role for this kinase in regulating the G2-M transition in oocytes has been discussed.     

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.     

ATP-gamma-S (adenosine 5'-0(3-thiotriphosphate)) blocks progesterone-induced maturation of the Xenopus oocyte

ATP-gamma-S microinjection into Xenopus oocyte prevents progesterone induced maturation. Inhibition is time and dose dependent; 50% inhibition occurs when 50 nl of 0.5 mM ATP-gamma-S solution are microinjected/oocyte 1 hr prior to the hormonal trigger. ATP-gamma-S inhibited oocytes can be induced to mature (100%) following microinjection of extracts containing maturation promoting factor (MPF). Our results suggest that the maturation protein(s) has been stabilized in ovo by ATP-gamma-S microinjection, in its phosphorylated inhibitory form.     

Early effect of progesterone on levels of cyclic adenosine 3':5'-monophosphate in Xenopus oocytes.

Progesterone treatment of Xenopus oocytes in vitro causes progression through meiotic cell division. The role of altered intracellular levels of cAMP on the initiation of meiotic cell division has been studied. Basal levels of cAMP averaged 1.5 pmol in oocytes from eight females, and exposure to progesterone caused a rapid drop in cAMP to about 40 to 60% of basal. Half-maximal decreases occurred within 15 to 60 s, and cAMP returned to near basal values by 20 min after progesterone. Theophylline inhibition of progesterone-induced cell division was characterized by a small increase in basal levels of cAMP and a reduced drop in cAMP due to the hormone. Cholera toxin, an activator of adenylate cyclase, was found to be a potent inhibitor of progesterone-induced meiosis, with half-maximal inhibition at 8 times 10(-12) M. In addition, the purified A subunit of cholera toxin was an effective inhibitor of progesterone action when microinjected into oocytes, with half-maximal inhibition occurring at an approximate internal concentration of 1 X 10(-7) M. Cholera toxin alone increased cAMP levels by 20%, but upon addition of progesterone, the level increased transiently to 200% of basal, indicating that the inhibition was due to elevated levels of cAMP. The results support a model in which the initiation of meiotic cell division is regulated by cAMP and protein phosphorylation.     

Involvement of cAMP in initiating maturation of the brittle-star Amphipholis kochii oocytes: induction of oocyte maturation by inhibitors of cyclic nucleotide phosphodiesterase and activators of adenylate cyclase

The maturation of brittle-star (Amphipholis kochii) oocytes, i.e., the reinitiation of meiosis accompanied by germinal vesicle breakdown (GVBD) and the acquisition of fertilizability, was induced by acid (pH 3.0) seawater containing 10 mM cAMP. Oocyte maturation was also induced by seawater of normal pH (pH 8.0) that contained either an inhibitor of cyclic nucleotide phosphodiesterase (25 mM theophylline, 25 mM caffeine) or an activator of adenylate cyclase (100 microM forskolin, 0.6 microM cholera toxin). Experiments in which the oocytes were treated with forskolin or theophylline for various periods of time demonstrated that there was a positive correlation between the oocyte cAMP level measured by radioimmunoassay and the extent of GVBD induced in each treatment: both increased as the treatment period became longer and about a threefold increase in cAMP level induced 50% GVBD. These results indicate that an increase in cAMP level initiates maturation of the brittle-star oocytes.     

Progesterone-inhibited phosphorylation of an unique Mr 48,000 protein in the plasma membrane of Xenopus laevis oocytes

The meiotic maturation of Xenopus laevis oocytes is induced in vitro by progesterone which interacts at the cell surface level. A cell-free membrane preparation (P-10,000) incorporated 32P from [gamma-32P]ATP, mostly into two proteins, Mr approximately 56,000 and approximately 48,000 (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Progesterone, added in vitro, specifically inhibited the phosphorylation of the Mr approximately 48,000 protein (named p48). Half-maximal inhibition of p48 phosphorylation occurred with progesterone approximately 8 microM, in good correlation with hormone concentration inducing oocyte maturation. The effect was not due to stimulation of protein phosphatase activity. The potent maturation inducers testosterone and deoxycorticosterone also inhibited p48 phosphorylation, whereas biologically inactive steroids or cholesterol did not. p48 phosphorylation was not affected by cAMP, cGMP, polyamines, calmodulin, and phospholipids + diolein. EGTA had a stimulatory effect which was reversed by added Ca2+. The inhibitory effects of progesterone and Ca2+ were additive, suggesting two distinct sites of action. Phospho-p48 was not detected in yolk platelets, microsomes, and cytosol of oocytes. Contrary to p48 itself, the p48 kinase activity was loosely associated with P-10,000. Progesterone inhibited p48 phosphorylation produced by either cytosol or exogenous pure catalytic subunit of cAMP-dependent protein kinase. Conversely, phosphorylation of casein and histones by protein kinase activity present in P-10,000 was not modified by progesterone. It is then suggested that progesterone regulates p48 phosphorylation by affecting the protein substrate in the membrane, rather than by inhibiting the protein kinase enzyme itself. The data demonstrate a direct effect (not mediated by change of protein synthesis) of steroids on p48 phosphorylation in the plasma membrane, and they suggest that this protein could be implicated in the initial action of progesterone on oocyte maturation.     

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.     

Inhibition of maturation and metabolism in rat oocytes by cyclic amp.

It is known that dibutyryl cyclic AMP (dbcAMP) and theophylline inhibit the spontaneous maturation of isolated mouse oocytes. The present study demonstrates that dbcAMP (0.01-1.0 mM) as well as cyclic AMP (cAMP, 10 mM) and a phosphodiesterase inhibitor (IBMX, 0.01-1.0 mM) prevent spontaneous maturation of isolated rat oocytes. As reported earlier an increase in oxygen consumption by the oocyte was found following maturation. When the oocytes were cultured in the presence of dbcAMP or cAMP no change in respiration occurred during culture. These results argue against the theory that cAMP acts as a direct mediator of the action of luteinizing hormone (LH) on oocyte maturation. Furthermore they suggest that changes in oocyte energy metabolism are closely related to the maturation process.     

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.     

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.     

Tamoxifen-induced alterations in meiotic maturation and cytogenetic abnormalities in mouse oocytes and 1-cell zygotes

Alterations in the rate of oocyte meiotic maturation (OM) and the timing of the metaphase-anaphase transition may predispose oocytes to premature centromere separation (PCS) and aneuploidy. Tamoxifen has the potential for perturbing the rate of OM since it can function as a calcium antagonist by binding to calmodulin and inhibiting the formation of a calcium-calmodulin complex which is needed for activating calmodulin-dependent cAMP phosphodiesterase and initiating OM. The objective of this study was to test the hypothesis that tamoxifen alters the rate of OM and predisposes oocytes to PCS and aneuploidy. Different does of tamoxifen were administered by oral gavage to female mice preovulation. Metaphase II oocyte and 1-cell zygote chromosomes were C-banded and cytogenetically analysed. Tamoxifen treatment resulted in a modest, but significant (p < 0.05), increase in oocytes with PCS. Similar frequencies of hyperploidy and oocytes with unpaired, single chromatids (SC) were found. Metaphase I, diploid and premature anaphase (PA) oocytes were not detected. Hyperploidy, polyploidy, PCS, PA and SC were not detected in zygotes. These data indicate that the levels of tamoxifen-induced PCS found in mouse oocytes did not predispose zygotes to aneuploidy. Tamoxifen did, however, reduce the proportion of females exhibiting oestrus.     

Biphasic effect of calcium on luteinizing hormone-stimulated cyclic adenosine 3',5'-monophosphate production in granulosa cells of the fowl (Gallus domesticus).

Both cAMP and Ca2+ play important roles in the steroidogenic action of LH in hen granulosa cells. However, the interaction of these intracellular messengers is not fully understood. In the present study we used two calcium ionophores (ionomycin and A23187), as well as trifluoperazine (TFP), an inhibitor of calmodulin, to investigate LH- and forskolin-induced cAMP production in granulosa cells isolated from the largest (F1) preovulatory follicle of White Leghorn laying hens. Between 0.1 and 1.0 microM, both ionophores significantly potentiated cAMP responses to LH in the presence of 0.1 mM extracellular Ca2+. When calcium was omitted from the medium, ionophores had no effect. When either calcium was raised above 1 mM, or the concentration of ionophores was increased above 1 microM, LH-induced cAMP production was drastically inhibited. In the presence of 0.5-2.0 mM calcium, A23187 inhibited forskolin-promoted cAMP synthesis. TFP, while having no effect on basal cAMP, suppressed LH-induced responses and the potentiating effect of ionomycin. It is concluded that for full activation of the adenylate cyclase/cAMP system by LH, Ca-calmodulin is required at a site upstream from the catalytic component of the enzyme. However, high intracellular Ca2+ and/or other effects of ionophores (such as uncoupling of oxidative phosphorylation) inhibit LH-induced cAMP production.