269 INACTIVATION OF MATURATION PROMOTING FACTOR AND MITOGEN-ACTIVATED PROTEIN KINASE IN PORCINE OOCYTES BY A SINGLE ELECTRICAL PULSE

Abstract

Activities of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) of mature oocytes should be decreased to begin subsequent development. In this study, activities of MPF and MAPK were investigated in porcine oocytes after artificial activation. To determine optimal electrical activation, porcine oocytes were exposed to 3 V AC pulse for 5 s followed by a single DC pulse of various electric field strengths (120, 150, 180, and 210 V/mm) and pulse durations (15, 30, 45, and 60 μs). For chemical activation, oocytes were exposed to 5 μM ionomycin for 5 min followed by 2 mM 6-dimethylaminopurine (6-DMAP) or 5 μg/mL cycloheximide for 4 h or 6 h. After activation, 40 to 50 oocytes were cultured in 50-μL drops of NCSU23 medium supplemented with 4 mg/mL BSA at 39°C, and 5% CO₂ in air. After 6 days of culture, blastocyst formation was observed and then numbers of blastocyst nuclei were counted after staining with Hoechst 33342. in vitro development rates and numbers of blastocyst nuclei by the field strengths were not significantly different among experimental groups (P > 0.05). However, development rates to the blastocyst stage of porcine oocytes exposed to 15 and 30 μ­s were 27.4 and 24.4%, respectively, which were significantly higher than that (12.5%) of 60 μs (P < 0.05). Mean numbers of blastocyst nuclei in 15- and 30-μs groups (38.6 ± 13.4 and 37.9 ± 11.4, respectively) were significantly higher than that (25.8 ± 10.5) of the 60-μs group (P < 0.05). Blastocyst development after optimal electrical pulse exposure was compared with that after different chemical treatments. Electrical stimulation induced 22.9% of blastocyst formation, which was significantly higher (P < 0.01) than those induced by the chemical stimulators (3.4 and 2.7%). Based on these results, changes of constituent proteins (cdc2 and ERK) of MPF and MAPK after artificial activation were analyzed by immunoblotting using anti-PSTAIRE monoclonal antibody and anti-MAP kinase polyclonal antibody. Activities of both cdc2 and ERK in pig oocytes were reduced 4 h after electrical stimulus, but were maintained at optimal levels after treatment with ionomycin + 6-DMAP. Our results indicate that an optimal single electrical pulse is effective on the inactivation of MPF and MAPK in porcine oocytes, eventually resulting in activation of porcine oocytes produced in vitro.