63 PARTHENOGENETIC DEVELOPMENT OF PORCINE OOCYTES RECONSTRUCTED BY CENTRIFUGATION AND ELECTROFUSION AFTER METAPHASE-II CHROMOSOME TRANSFER

Abstract

Metaphase-II chromosome transfer (M-II transfer) is considered to be a useful technique for studying nucleus–cytoplasm relationships, or for generating oocytes with good developmental ability after transfer of the nucleus to the cytoplasm. The reconstructed oocytes carry the original genomic information within the metaphase chromosomes from the donor oocytes. The objective of the present study was to evaluate the parthenogenetic developmental ability of porcine M-II transferred oocytes. In vitro maturation was carried out as reported previously (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041). After culture for 44 h, cumulus cells were removed by hyaluronidase treatment and gentle pipetting. Oocytes that had extruded the first polar body were selected and centrifuged at 13 000g for 9 min to stratify the cytoplasm. The zonae pellucidae were removed after exposure to pronase, and zona-free oocytes were layered on a 300 µL discontinuous gradient (100 µL each of 45%, 30%, and 7.5%) of Percoll in TCM-HEPES supplemented with 5 µg mL^-1 cytochalasin B. After centrifugation of the oocytes on the gradient in microcentrifuge tubes at 6000g for 20 s, fragmented cytoplasm with an equal volume was obtained, stained with Hoechst 33342, and classified as cytoplasm with or without chromosomes by observation with a fluorescence microscope. One fragmented cytoplasm with chromosomes and 2 fragmented cytoplasms without chromosomes were fused by electric stimulation with a single DC pulse (1.5 kV cm^-1, 20 µs) and cultured temporarily for 1 h. The reconstructed oocytes were then stimulated again to induce parthenogenetic activation (0.8 kV cm^-1, 30 µs, 2 DC pulses) (treatment group). Zona-free mature oocytes that had not been subjected to reconstruction were activated as a control group. The oocytes in both groups were treated with 5 µg mL^-1 cytochalasin B for 2 h, and then cultured for 6 days in media (Kikuchi et al. 2002) using the WOW system (Gabor et al. 2000 Mol. Reprod. Dev.). The blastocyst formation rate in the control group (22.9 ± 5.5%) was significantly higher (P < 0.05; ANOVA and PLSD-test) than that in the treatment group (7.6 ± 1.8%). The total cell number per blastocyst in the control group (28.7 ± 4.6) was significantly higher (P < 0.05) than that in the treatment group (16.7 ± 1.0). These results suggest that reconstructed porcine oocytes following M-II transfer by centrifugation and electrofusion can develop to the blastocyst stage in vitro. This technique enables transfer of the nucleus to cytoplasm with good developmental ability without the use of a micro-manipulation system.