cyclic-gmp and 1-methyladenine

Oocyte maturation (meiosis reinitiation) in starfish is induced by the natural hormone 1-methyladenine (1-MeAde). Cyclic AMP seems to play a negative role in maturation since 1-MeAde triggers a decrease of the oocyte cAMP concentration and since intracellular microinjections of cAMP delay or inhibit maturation. Cyclic GMP is also inhibitory but other nucleotides such as cCMP, cIMP, and cUMP are inactive. The involvement of cAMP and cGMP in the control of oocyte maturation has been further investigated by the use of the stereoisomers of the phosphodiesterase-stable adenosine and guanosine 3',5'-phosphorothioates (cAMPS and cGMPS). The Sp isomers of cAMPS and cGMPS respectively activate cAMP-dependent protein kinase and cGMP-dependent kinase, while the Rp isomers inhibit the kinases. Extracellular addition of these cAMPS and cGMPS isomers has no effect on the oocytes. Intracellular microinjection of the kinase-activating (Sp)-cAMPS and (Sp)-cGMPS delays or inhibits 1-MeAde-induced maturation in a concentration-dependent manner (I50, 30 and 300 microM, respectively). Microinjections of (Rp)-cAMPS and (Rp)-cGMPS have no inhibitory effects and neither trigger nor facilitate maturation. Using various analogs, we found that the delaying or inhibiting effect is restricted to the compounds activating cAMP-dependent kinase, while the compounds inactive on or inhibiting the kinase have no effects on maturation. The inhibitory effect of (Sp)-cAMPS can be reversed by comicroinjection of the heat-stable inhibitor of cAMP-dependent protein kinase, by comicroinjection of the antagonist (Rp)-cAMPS, or by an increase in the 1-MeAde concentration. The negative effects of (Sp)-cAMPS or (Sp)-cGMPS are observed only when these isomers are microinjected during the hormone-dependent period. These results suggest that a cAMP-dependent inhibitory pathway participates in the maintenance of the prophase arrest of oocytes and that 1-MeAde acts both by inhibiting this negative pathway (dis-inhibitory pathway) and by stimulating a parallel activatory pathway leading to oocyte maturation. The generality of this mechanism is discussed.

Topics: Adenine; Animals; Cyclic AMP; Cyclic GMP; Female; Oocytes; Starfish; Stereoisomerism; Thionucleotides; Time Factors

Resumption of meiosis in starfish oocytes is induced by 1-methyladenine (1-MeAde) produced by ovarian follicle cells under the influence of a gonad-stimulating substance (GSS). It has also been reported that concanavalin A (Con A) and two serine proteolytic enzymes (trypsin and Pronase) can stimulate 1-MeAde production. This study was undertaken to determine if 1-MeAde production induced by these compounds is mediated through elevation of cAMP in starfish (Asterina pectinifera) follicle cells. GSS at a concentration of 0.1 mg/ml significantly stimulated 1-MeAde accumulation in extracellular medium after 1-2 hr of follicle cell incubations. GSS also caused a four- to fivefold increase in intracellular levels of cAMP. The continuous presence of GSS was required for the maintenance of elevated levels of cAMP and 1-MeAde. Basal levels of intracellular cGMP were only about 20% of those of cAMP and were not influenced by treatment with GSS. At 1 mM, 3-isobutyl-1-methylxanthine (IBMX), a potent phosphodiesterase inhibitor, stimulated both 1-MeAde and cAMP production in a concentration-dependent manner. Con A and two serine proteases also raised both cAMP and 1-MeAde production. Con A-induced (1.0 mg/ml) increases in cAMP and 1-MeAde were greater than the response to GSS (0.1 mg/ml) and were completely suppressed by treatment with alpha-methyl-D-mannoside (10 mM), a competitive inhibitor of Con A. These results strongly suggest that cAMP is a second messenger in the production of 1-MeAde by starfish ovarian follicle cells.

Topics: Adenine; Animals; Concanavalin A; Cyclic AMP; Cyclic GMP; Female; Ovary; Peptide Hydrolases; Peptides; Phosphodiesterase Inhibitors; Starfish