79 PSEUDOPHYSIOLOGICAL TRANSCOMPLEMENTARY ACTIVATION OF GOAT OOCYTES RECEIVING ADULT EAR CUTANEOUS FIBROBLAST CELL NUCLEI

Abstract

The aim of the study was to determine the in vitro developmental potential of caprine cloned embryos following pseudophysiological (transcytoplasmic) transcomplementary activation of oocytes reconstructed with ear skin-derived fibroblast cell nuclei. The source of nuclear recipient cells were IVM doe oocytes. The reconstruction of the previously enucleated oocytes (i.e. ooplasts) was performed by microinjection of either the somatic cell-derived karyoplasts or intact whole tiny nuclear donor cells directly into the cytoplasm. The reconstructed oocytes were incubated in Upgraded B2 INRA medium for 30 min to 1 h before their pseudophysiological activation. The activation was achieved by electrofusion of clonal cybrids with the allogeneic cytoplasts isolated from caprine IVF-created zygotes, which led to the formation of triple allocytoplasmic hybrids (allocybrids). These originate from 3 sources: (1) homologous whole nuclear donor fibroblast cells or their karyoplasts; (2) enucleated oocytes (ooplasts), and (3) zygote-derived cytoplasts. Single zygote-descended cytoplasts (the so-called zygoplasts) were inserted into the perivitelline space of previously reconstituted oocytes. The resulting zygoplast-clonal cybrid couplets were subsequently subjected to electrofusion, which was induced by application of a single DC pulse of 2.4 kV cm^-1 for 15 μs. The electrofusion of zygoplast and reconstructed oocyte plasma membranes occurred in an isotonic dielectric solution deprived of Ca²⁺ ions. The transcytoplasmically activated clonal cybrids were cultured in vitro in Upgraded B2 INRA medium for 48 h at 38.5°C in a 100% water-saturated atmosphere of 5% CO₂ and 95% air. Afterward, cleaved embryos were co-cultured with Vero cells in medium supplemented with 10% fetal bovine serum for an additional 96 to 144 h up to morula and blastocyst stages under the same thermal and atmospheric conditions. A total of 53/78 (67.9%) oocytes reconstructed with fibroblast cell nuclei were successfully fused with zygoplasts. From among 53 cultured cloned embryos, 34 (64.2%) cleaved. The rates of embryos that reached the morula and blastocyst stages were 21/53 (39.6%) and 11/53 (20.8%), respectively. In conclusion, the relatively high percentages of morulae and blastocysts were noticed among in vitro-cultured caprine cloned embryos produced by the strategy of pseudophysiological transcytoplasmic activation of oocytes reconstructed with adult dermal fibroblast cell nuclei. Therefore, the use of cytoplasmic components originating from zygotes as the stimuli for activation of nuclear-transferred oocytes appeared to be an effective procedure in the generation of goat blastocysts by somatic cell cloning.