Effect of protein kinase C inhibitors on porcine oocyte activation.

The effect of protein kinase C (PKC) inhibitors on porcine oocyte activation by calcium ionophore A23187 was studied. Calcium ionophore applied in a 50 microM concentration for 10 min induced activation in 74% of oocytes matured in vitro. When the ionophore-treated oocytes were exposed to the effect of bisindolylmaleimide I, which inhibits calcium-dependent PKC isotypes (PKC-alpha, -beta(I), -beta(II), -gamma,) and calcium-independent PKC isotypes (PKC-delta, -epsilon), the portion of activated oocytes decreased (at a concentration of 100 nM, 2% of the oocytes were activated). Go6976, the inhibitor of calcium-dependent PKC isotypes PKC-alpha, -beta(I) did not prevent the action of the oocytes treated with calcium ionophore in concentrations from 1 to 100 microM. The inhibitor of PKC-beta(I) and beta(II) isotypes, hispidin, in a concentration of 2 microM-2 mM, was not effective either. The inhibitor of PKC-delta isotype, rottlerin, suppressed activation of the oocytes by calcium ionophore (no oocyte was activated at 10 microM concentration). The PKC-delta isotype in matured porcine oocytes, studied by Western blot analysis, appeared as non-truncated PKC-delta of 77.5 kDa molecular weight, on the one hand, and as truncated PKC-delta, which was present in the form of a doublet of approximately 62.5 and 68 kDa molecular weight, on the other hand. On the basis of these results, it can be supposed that PKC participates in the regulation of processes associated with oocyte activation. Calcium-dependent PKC-alpha, -beta isotypes do not seem to play any significant role in calcium activation. The activation seems to depend on the activity of the calcium-independent PKC-delta isoform.

Expression of heat shock protein70 in pig oocytes: heat shock response during oocyte growth.

The heat shock response of growing and fully-grown pig oocytes was analyzed in vitro by determining heat shock protein70 (HSP70) synthesis under both normal conditions (39 degrees C; 0 and 6h) and after heat shock (43 degrees C; 1, 4 and 6h). The expression of HSP70 in oocytes was detected by immunoblotting analysis. Growing oocytes measuring 80-99 microm synthesized a high number of HSP70 without heat shock effect, and these were capable of increasing the synthesis of HSP70 after heat shock to a maximum after 1h. Growing oocytes measuring 100-115 microm also synthesized HSP70 without heat shock and after it, but the HSP70 synthesis was not statistically changed by increasing duration of heat shock. In fully-grown oocytes, great amounts of HSP70 were found without heat shock treatment, and the contents of HSP70 significantly decreased after heat shock. These results indicate that growing oocytes are able to synthesize HSP70 after heat shock. This ability declines at the end of the growth period, and fully-grown oocytes are unable to induce HSP70 synthesis after heat shock. HSP70 is synthesized and stored during oocyte growth. The high HSP70 synthesis in non-heat-treated growing oocytes and a great amount of HSP70 in fully-grown oocytes support the hypothesis that HSP70 is important for oocyte growth and maturation.

Activation of pig oocytes using nitric oxide donors.

Nitric oxide (NO) plays an important role in intracellular signaling, but its role during the activation of mammalian oocytes is little understood. In our study, in vitro matured pig oocytes were cultured with NO-donors-S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitropruside (SNP). These treatments were able to induce parthenogenetic activation of pig oocytes matured in vitro. The specificity of this effect was confirmed by the activation of oocytes by exogenous endothelial nitric oxide synthase (eNOS) microinjected in the oocyte with its activator calmodulin. Relatively long exposure (10 hr) is needed for activation of pig oocytes with 2.0 mM SNAP. An active NOS is necessary for the NO-dependent activation of pig oocytes because NOS inhibitors L-NMMA or L-NAME are able to inhibit activation of oocytes with NO-donor SNAP. On the basis of our data, we conclude that the NO-dependent activating stimulus seems inadequate because it did not induce the exocytosis of cortical granules. Also, the cleavage of parthenogenetic embryos was very low, and embryos did not develop beyond the stage of eight blastomeres.

Induction and activation of meiosis and subsequent parthenogenetic development of growing pig oocytes using calcium ionophore A23187.

The pig ovary contains a large number of growing oocytes, which do not mature in vitro and cannot be readily used in various biotechnologies. This study was conducted to determine the possibility of inducing meiotic maturation in growing pig oocytes with an internal diameter of 110 microm, which had developed partial meiotic competence. Most of these oocytes spontaneously stopped maturation at the metaphase I stage (68%); a limited number proceeded to the metaphase II stage (26%). Treatment with calcium ionophore A23187 (50 microM for 5 or 10 min) after 24h in vitro culture overcame the block at the metaphase I stage, and treated growing pig oocytes matured to the metaphase II stage (66%). Oocytes in which maturation had been induced by calcium ionophore were again treated with calcium ionophore. Up to 58% of the treated oocytes were activated. Parthenogenetic development in oocytes treated with ionophore for meiosis induction and activation was very limited. The portion which reached morula stage did not exceed 8% and at most 3% developed to the blastocyst stage.