76 DEVELOPMENTAL CAPABILITY OF TRANSGENIC NUCLEAR-TRANSFERRED PIG EMBRYOS PRODUCED USING THE NOVEL METHOD OF PSEUDOPHYSIOLOGICAL ACTIVATION

Abstract

The physicochemical stimuli, which are commonly used for artificial activation of porcine nuclear-transferred (NT) oocytes, can affect detrimentally or cytotoxically the clonal cybrids and thereby inhibit the development or decrease the quality of cloned embryos. Therefore, we have recently developed a novel method of pseudophysiological transcomplementary (transcytoplasmic) activation to stimulate the developmental program of porcine oocytes reconstructed by somatic cell nuclear transfer. The mechanism underlying this original technique of activation is transcytoplasmic influx of sperm-derived proteins triggering intracellular calcium oscillations, which is mediated via heterologous (rabbit) zygote-descended cytoplasts. The purpose of our study was to estimate the in vitro developmental competences of porcine transgenic cloned embryos following pseudophysiological activation of oocytes receiving pWAPhGH-GFPBsd gene construct-nucleofected fetal fibroblast cell nuclei. In the cloning procedure, IVM pig oocytes were used as recipient cells for cell nuclei of positively selected transgenic fibroblast cells. The reconstruction of enucleated oocytes was performed by intracytoplasmic injection of either the somatic cell-derived karyoplast or whole tiny nuclear donor cell. The activation of porcine NT oocytes was achieved by electrofusion of them with the xenogeneic cytoplasts isolated from in vivo-derived rabbit zygotes (i.e. with the so-called zygoplasts), which led to the formation of triple xenocytoplasmic hybrids (xenocybrids). The rabbit zygotes had been flushed postmortem from the separated oviducts of superovulated postpubertal female donors 18 to 20 hafter administration of hCG and copulation. Single rabbit zygote-descended cytoplasts were inserted into the perivitelline space of previously reconstructed pig oocytes. The resulting zygoplast-NT oocyte couplets underwent fusion, which was induced by generation of 2 successive DC pulses of 1.2 kV cm^-1 for 60 μs. The electrofusion medium consisted of 0.3 M Ca²⁺-deprived mannitol supplemented with 0.1 mM MgSO₄ and 0.2 mg mL^-1 fatty-acid-free BSA. The transcytoplasmically activated xenocybrids were cultured in vitro for 6 to 7 days up to morula/blastocyst stages. A total of 183/207 (88.4%) oocytes reconstructed with nucleofected fibroblast cell nuclei were successfully fused with zygoplasts. Out of 183 cultured NT embryos, 138 (75.4%) were cleaved. The rates of transgenic NT embryos that reached the morula and blastocyst stages yielded 106/183 (57.9%) and 65/183 (35.5%), respectively. In conclusion, the original method of pseudophysiological activation of porcine NT oocytes turned out to be relatively efficient, which has been confirmed by the high percentages of pWAPhGH-GFPBsd transgenic embryos developing in vitro to morula and blastocyst stages.