Effects of caffeine treatment on aged porcine oocytes: parthenogenetic activation ability, chromosome condensation and development to the blastocyst stage after somatic cell nuclear transfer

Abstract

The possibility of using aged porcine oocytes treated with caffeine, which inhibits the decrease in M-phase promoting factor activity, for pig cloning was evaluated. Cumulus–oocyte complexes (COCs) were cultured initially for 36h and subsequently with or without 5mM caffeine for 24h (in total for 60h: 60CA+ or 60CA– group, respectively). As a control group, COCs were cultured for 48h without caffeine (48CA–). The pronuclear formation rates at 10h after electrical stimulation in the 60CA+ and 60CA– groups decreased significantly (p<0.05) compared with the 48CA– group. However, the fragmentation rate was significantly higher (p<0.05) in the 60CA– group than in the 60CA+ and 48CA– groups. When the stimulated oocytes were cultured for 6 days, the 60CA+ group showed significantly lower blastocyst formation and higher fragmentation or degeneration rates (p<0.05) than the 48CA– group. However, the number of total cells in blastocysts was not affected by maturation period or caffeine treatment. When somatic cell nuclei were injected into the non-enucleated oocytes and exposed to cytoplasm for a certain duration (1–11h) before the completion of maturation (48 or 60h), the rate of nuclear membrane breakdown after exposure to cytoplasm for 1–2h in the 60CA– oocytes was significantly lower (p;<0.05) than in the other experimental groups. The rate of scattered chromosome formation in the same 60CA– group tended to be lower (p=0.08) than in the other groups. After the enucleation and transfer of nuclei, blastocyst formation rates in the 60CA+ and 60CA– groups were significantly lower (p<0.05) than in the 48CA– group. Blastocyst quality did not differ among all the groups. These results suggest that chromosome decondensation of the transplanted somatic nucleus is affected by both the duration of exposure to cytoplasm and the age of the recipient porcine oocytes, and that caffeine treatment promotes nuclear remodelling but does not prevent the decrease in the developmental ability of cloned embryos caused by oocyte aging.