Artificial Activation and Parthenogenetic Development of Unaged Bovine Oocytes
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概要
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The aim of this study was to activate unaged bovine oocytes matured 24 hr in vitro.<BR>In the first experiment, we demonstrated that artificial activation of in vitro matured bovine oocytes, assessed by pronuclear formation, is age-dependent, and young oocytes matured 24 hr are difficult to activate compared with aged oocytes matured 32 or 42 hr.<BR>In the next experiment, young oocytes were randomly assigned to either electrical stimulation or chemical treatment. Oocytes were electrically stimulated with two DC pulses of 1.0 kV/cm for 25, a s ec, in non-electrolyte medium containing 0.3 M mannitol (T1), electrolyte modified PBS (T2) or modified PBS with 0.01 mg/ml BSA (T3) . Oocytes were incubated for 5 min in modified PBS containing 7% ethanol (T4) or 50, E M calciumionophore (T5) . Alternatively, oocytes were incubated with 1 0μg/ml cycloheximide in 12.5 mM-Hepes TCM 199 for 10 hr (T6), 7% ethanol for 5 min followed by incubation in 10μg/ml cycloheximide for 10 hr (T7) or 50μM calcium ionophore for 5 min followed by incubation in 10μg/ml cycloheximide for 10 hr (T8) .<BR>At 18 hr (T1-5) or 8 hr (T6-8) po-stactivation, the oocytes with one or more pronuclei were classified as activated oocytes. The activation rates of T2 (82.2%, 83/101) and T3 (71.7%, 43/60) were higher (P<0.05) than that of T1 (49.5%, 48/97) . The activation rates of T4, T5, T6, T7 and T8 were 46.7% (42/90), 26.7% (43/161), 66.0% (209/291), 92.6% (113/122) and 62.9% (56/89), respectively. The activation rate of T7, was higher than those of other chemical treatments (P<0.05) .<BR>Young oocytes were parthenogenetically activated by T7 and 5 μg/ml cytochalasin D treatment, and incubated for development to blastocysts. By this treatment, 86.2% of the oocytes was activated, and 79.1% of the activated oocytes formed two pronuclei. Rates of treated oocytes that cleaved, developed to the morula≤andblastocyst≤stages were 49.2%, 7.5% and 3.0%, respectively.<BR>The results suggest that unaged bovine oocytes matured 24 hr in vitro can be efficiently activated by electrical stimulation in electrolyte medium or by treatment with ethanol-cycloheximide, and activated oocytes can develop to the blastocyst stage.
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