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T. Sobajima, F. Aoki and K. Kohmoto

Mitogen-activated protein kinase (MAP kinase) plays a role in the cascade of protein kinase activation in cultured cells. To investigate the involvement of MAP kinase in meiotic maturation, we measured MAP kinase activity, using myelin basic protein as a substrate, with histone H1 kinase activity, in mouse oocytes. MAP kinase activity was low 1 h after isolation from follicles (when oocytes lost their germinal vesicle), increased abruptly at 2 h, and remained high until the second metaphase (13 h after isolation from follicles). Histone H1 kinase activity increased gradually from 2 to 7 h after isolation. When immature oocytes were treated with puromycin, MAP kinase activity did not increase after isolation from follicles. In the presence of 3-isobutyl-1-methylxanthine, the treatment of immature oocytes with okadaic acid, a specific inhibitor of protein phosphatase 1 and 2A, induced germinal vesicle breakdown and activation of MAP kinase. These results suggest that MAP kinase is involved in the regulation of meiotic maturation, and that the activation of MAP kinase requires protein synthesis and is inhibited by the protein phosphatase during meiotic maturation in mouse oocytes.

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F. Aoki, K. Ishida, M. Okuno and K. Kohmoto

Flagellar bending was analysed using photographs of hyperactivated hamster and mouse spermatozoa. The flagellar waveform consists of several bends; the centre of each bend was therefore located on the flagellum, and the angle of the bend measured. The direction of the bend was determined by using the asymmetry of hook-shaped head to assess the asymmetry of flagellar waveform. The bend that occurred in the same direction as the curve of head was defined as the reverse bend and the bend in the opposite direction as the principal bend. In hamster spermatozoa, flagellar bending was asymmetric to the direction of the reverse bend after incubation for 5 min. After incubation for 4 h the asymmetry had increased, as the angles of the reverse bends had increased in all regions of the flagellum but the principal bend had not. In mouse spermatozoa incubated for 5 min, flagellar bending was relatively symmetric. In the hyperactivated mouse spermatozoa incubated for 3 h, the angle of the principal bend increased in the distal region and those of the reverse bends increased in almost all regions of the flagellum. Since the increase in the reverse bend was relatively high, flagellar bending became asymmetric to the direction of the reverse bend as in hamster spermatozoa. These increases in asymmetry were also evident in the measurement of the total changes in angular direction between the proximal and distal end of flagella in both species. The increase in asymmetry could provide an explanation for the changes in the motility patterns seen in spermatozoa after the onset of hyperactivation. The mechanism of hyperactivation is discussed in relation to the changes in flagellar bending pattern.

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T. S. Choi, M. Mori, K. Kohmoto and Y. Shoda

Summary. Mouse oocytes matured in vitro in chemically defined medium were not penetrated by spermatozoa. The time required for dissolution of the zona pellucida of such oocytes by α-chymotrypsin was much longer than that for ovulated oocytes. Addition of fetal calf serum to the medium for maturation of oocytes improved the incidence of sperm penetration and shortened the time of enzymic dissolution of the zona pellucida. These results suggest that the low rate of fertilization of oocytes matured in vitro is mainly due to qualitative changes of the zona pellucida, which could be overcome by a factor or factors in fetal calf serum.