273 OPTIMIZATION OF PROTOCOLS FOR ACTIVATION OF GOAT OOCYTES WITH IONOMYCIN IN COMBINATION WITH 6-DIMETHYLAMINOPURINE

Abstract

The protocol of ionomycin in combination with 6-dimethylaminopurine (6-DMAP) is commonly used for activation of oocytes and reconstructed embryos of different species. Since numerous abnormalities and impaired development have been observed when oocytes are activated with 6-DMAP, this protocol needs optimization. Effects of concentration and treatment duration of both drugs on activation kinetics and parthenogenetic development of goat oocytes were examined in this study. When goat oocytes matured in vitro in TCM-199 were treated with 5 M ionomycin in PBS for different periods before exposure to 6-DMAP in CR1aa, the activation rate obtained with ionomycin treatment for 1 min (95.2%) was significantly (P < 0.05, Duncan multiple comparison test) higher than with ionomycin treatments for 3, 5, 7, or 9 min. When oocytes were treated with different concentrations of ionomycin for 1 min before exposure to 6-DMAP, activation rates obtained with 0.625, 1.25, 2.5, 5, 10, and 20 M ionomycin (87–95%) did not differ significantly but were significantly higher than that achieved with 0.3125 M ionomycin. Progressive reduction of time for 6-DMAP exposure showed that the duration of 6-DMAP treatment can be reduced to 1 h from the 2nd up to the 4th hour after ionomycin treatment, to produce activation rates greater than 85%. When oocytes were treated with different concentrations of 6-DMAP for the 3rd hour (a total of 1 h, 3 h after the exposure to ionomycin), activation rates with 4 and 2 mM 6-DMAP (>90%) were significantly higher than those with 1 and 0.5 mM. Therefore, the best protocol for goat oocyte activation would be a 1-min exposure to 2.5 M ionomycin followed by 2 mM 6-DMAP treatment for the 3rd hour. When oocytes matured in vitro for different times were stimulated with the best protocol, activation rates of the 27-, 30-, and 33-h oocytes (85, 85, and 91%) were significantly higher than those of the 24-, 26-, and 39-h oocytes. When activated oocytes were co-cultured in CR1aa with cumulus cell monolayers, the highest rates of cleavage (92%) and morulae/blastocysts (23%) were obtained with oocytes activated by the best protocol, and any increase in the intensity of ionomycin treatment and in the duration of 6-DMAP exposure impaired the development of the parthenotes. During anaphase II, chromosomes (the dyads) did not separate into two units in oocytes that were activated by long exposure to 6-DMAP, but they did in oocytes that were activated by short or no exposure to 6-DMAP; as a result, only one pronucleus developed in most of the former but two pronuclei were formed in most of the latter cases. Laser scanning confocal microscopy showed that microtubules also behaved differently in these two groups of activated oocytes. It is therefore concluded that to obtain better activation and development, goat oocytes matured in vitro for 27–33 h should be used, and these should be activated by a 1-min exposure to 2.5 M ionomycin followed by 2 mM 6-DMAP treatment for the 3rd hour.

This study was supported by the “973” Project of China Sci. Technol. Ministry (No. G200016108).