Regulation of mouse oocyte maturation: Involvement of cyclic AMP phosphodiesterase and calmodulin
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Trifluoperazine
Germinal vesicle
Oocyte activation
Cyclic nucleotide phosphodiesterase
EGTA
Artificial oocyte activation method is essential for the current reproductive technology such as somatic cell nuclear transfer (SCNT) and round spermatid injection (ROSI). A variety of oocyte activators have been found such as ethanol, electric stimuli and Ca ionophore. In mouse, Sr2+-induced oocyte activation is widely used for SCNT. However, efficient Sr2+-induced activation requires Ca2+-free media(Ca(-)) (CuthBertson KS et al., 1981). Recently we demonstrated that addition of 2 mM ethyleneglycol-bis- (2-aminoethylether)-N,N,N'f,N'f-tetraacetic acid (EGTA), a calcium-selective chelator, allow oocytes to be efficiently activated by strontium even in the Ca2+-containing media which support full-term development after SCNT (Kishigami et al., 2007). In this study, using this chelating activation method, we compared activation rates of ICR-derived oocytes in a variety of commonly used media such as CZB, KSOM, M16, Whitten media, and TYH. We confirmed successful oocyte activation in all these media but found different oocyte activation rates. The highest rate (90%) was achieved by EGTA added Ca2+-containing M16media. On the other hand, the lowest rate (74%) was observed in Ca2+-free KSOM activation mainly because of oocyte degeneration. Thus, oocyte activation in different media with EGTA results in different oocyte activation rates. Further we also investigate whether another calcium-selective chelator, BAPTA can be used for oocyte activation instead of EGTA. BAPTA has a similar Kd for calcium to EGTA but the faster calcium-binding kinetics of BAPTA. Addition of 2-4 mM BAPTA in M16 produced a lower rate of activated oocytes (56%). We also found activated B6D2F1 oocytes with BAPTA normally developed into blastocysts with a lower developmental rate than ones with EGTA (65% and 80% respectively per cleavage). These results suggest that calcium-selective chelators with different kinetic properties affect efficiency of oocyte activation and the following embryonic development. (poster)
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The effect of Ca2+ on the adenylate cyclase activity associated with membranes prepared from mouse parotid gland has been examined. Ca2+ stimulated then inhibited adenylate cyclase activity, with values for half-maximal stimulation and inhibition of 0.6 and 10 microM, respectively. Maximal activation (1.4-fold) was observed at 2 microM free Ca2+. These membranes contained 1.2 microgram calmodulin/mg protein. Exogenous calmodulin (0.2-1.2 microgram) activated, in a concentration-dependent manner, adenylate cyclase activity, with maximal activation being 2.5-fold at 12 micrograms calmodulin. Preparation of membranes in 2 mM ethyleneglycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacet ic acid (EGTA) resulted not only in a significant decrease in calmodulin levels (0.5 microgram calmodulin/mg protein) but also in a loss of the ability of Ca2+ to stimulate the enzyme. Exogenous calmodulin restored the ability of Ca2+ to stimulate the adenylate cyclase activity associated with EGTA-treated membranes. Trifluoperazine (50 microM) blocked the ability of Ca2+ to activate adenylate cyclase activity in control membranes. The effect of trifluoperazine could be reversed by exogenous calmodulin (0.5 or 5.0 micrograms). These data indicate that calmodulin mediates the activation of parotid gland adenylate cyclase by Ca2+ and that Ca2+, at concentrations which stimulate and inhibit amylase secretion, can activate and inhibit adenylate cyclase activity.
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Cyclic nucleotide phosphodiesterase activity towards cyclic AMP and cyclic GMP was studied in extracts of rat islets of Langerhans. Biphasic Eadie plots [Eadie (1942) J. Biol. Chem. 146, 85-93] were obtained with either substrate suggesting the presence of both ‘high’- and ‘low’-Km components. The apparent Km values were 6.2 +/- 0.5 (n = 8) microM and 103.4 +/- 13.5 (6) microM for cyclic AMP and 3.6 +/- 0.3 (12) microM and 61.4 +/- 7.5 (13) microM for cyclic GMP. With cyclic AMP as substrate, phosphodeisterase activity was increased by calmodulin and Ca2+ and decreased by trifluoperazine, a specific inhibitor of calmodulin. With cyclic GMP as substrate, phosphodiesterase activity was decreased by omission of Ca2+ or addition of trifluoperazine. Addition of exogenous calmodulin had no effect on activity. The data suggest that Ca2+ may influence the islet content of cyclic AMP and cyclic GMP via effects on calmodulin-dependent cyclic nucleotide phosphodiesterase(s).
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Xenopus oocytes were exposed to trifluoperazine, a drug that binds to the calcium-regulating protein, calmodulin. The drug induced meiotic maturation even in the absence of progesterone. When trifluoperazine was microinjected directly into oocytes the maturation of the cells was partially inhibited in that the white spot, indicative of germinal vesicle breakdown, did not appear even though the germinal itself was absent in dissected oocytes.
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Without using sperm, artificial oocyte activation is essential for current assisted reproductive technologies, particularly somatic cell nuclear transfer and round spermatid injection. Strontium has been widely used as an activator of oocytes especially in the mouse, by which efficient oocyte activation requires Ca2+-free medium. In this study, we examined whether Sr2+ can efficiently activate oocytes in Ca2+-containing culture media when calcium is chelated. Ethylene glycol-bis (β-aminoethyl ether) -N, N, N', N'-tetraacetic acid (EGTA) was added to three standard culture media (CZB, M16 and KSOM) for mouse embryos because it preferentially binds Ca2+ rather than Sr2+. We found that treatment with 5 mM Sr2+ and 2 mM EGTA left fewer than 1% of oocytes at the MII stage, which is comparable to that of Ca2+-free medium. As a result, addition of 2 mM EGTA along with 5 mM Sr2+ in either CZB, M16 or KSOM made more than 80% of available activated oocytes, which was comparable to or better than 72% in a Ca2+-free Sr2+ medium, since EGTA-Sr2+ activation led to significantly less oocyte degeneration than Ca2+-free Sr2+ activation. Furthermore, we demonstrated that this activation method can support the birth of cloned embryos. Thus, addition of EGTA to typical Ca2+-containing culture media can easily produce activation media that does not interfere with embryonic development.
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A calmodulin-binding assay was established in rat striatal particulates which were depleted of endogenous calcium and calmodulin by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) treatment. The binding of 125I-labeled calmodulin to this preparation was saturable and time-dependent. The dependence of calmodulin binding upon temperature and concentration was also demonstrated. Calcium was a prerequisite for calmodulin binding and it facilitated the binding in a dose-dependent manner. Lanthanum, a known calcium antagonists in other tissue systems, mimicked the effect of calcium on calmodulin binding. When both ions were present at low concentrations, their effects on calmodulin binding was additive. Lanthanum, but not calcium, inhibited calmodulin release from a non-EGTA-treated preparation. This difference in action between calcium and lanthanum suggests that they mediate calmodulin binding in an independent manner. Scatchard analysis of calcium-calmodulin binding to rat striatal particulates revealed that there are two populations of binding sites: a higher affinity (apparent KD = 1.3 X 10(-7) M) and a lower affinity (apparent KD = 2.9 X 10(-7) M) binding site. Trifluoperazine, a phenothiazine antipsychotic drug, at 10(-4) M antagonized calmodulin binding only at the higher affinity binding sites. These sites may play an important role in mediating the action of trifluoperazine in the caudate nucleus.
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