In Vitro Development of Porcine Nuclear-Transferred Embryos Derived from Fibroblast Cells Analysed Cytometrically for Apoptosis Incidence and Accuracy of Cell Cycle Synchronization at the G0/G1 Stages / Rozwój In Vitro Klonalnych Zarodków Świni Zrekonstruowanych Z Jąder Komórkowych Fibroblastów Cytometrycznie Analizowanych Pod Kątem Częstotliwości Występowania Śmierci Apoptotycznej I Efektywności Synchronizacji Cyklu Mitotycznego W Fazach G0/G1

Abstract

Abstract The study was undertaken to examine whether various strategies, including contact inhibition and serum starvation, that were used for artificial synchronization of mitotic cycle of porcine fibroblast cell lines affect differently the distribution of cell cycle stage frequencies and the occurrence of apoptotic cell death in the analysed cell samples. In vitro cultured (contact-inhibited or serumstarved) somatic cells were subjected to flow cytometric diagnostics of mitotic cycle together with the detection of late-apoptotic cell fractions with hypodiploid number of nuclear DNA molecules. Moreover, impact of the methods applied to synchronize the cell division cycle of different types of nuclear donor fibroblast cells (adult cutaneous and foetal fibroblasts) on the preimplantation developmental outcomes of cloned pig embryos was investigated. The developmental capabilities of nuclear-transferred (NT) embryos that were reconstituted with contact-inhibited or serum-depleted adult cutaneous fibroblast cells to reach the morula and blastocyst stages remained at the levels of 169/278 (60.8%) and 76/278 (27.3%) or 121/265 (45.7%) and 46/265 (17.4%), respectively. The proportions of NT embryos originating from contact-inhibited or serum-deprived foetal fibroblast cells that completed their development to the morula and blastocyst stages were 223/296 (75.3%) and 108/296 (36.5%) or 165/261 (63.2%) and 67/261 (25.7%), respectively. In conclusion, the flow cytometric analysis of cultured porcine adult cutaneous and foetal fibroblast cells revealed the high efficiency of the artificial synchronization of mitotic cycle at the G0/G1 stages as a consequence of applying the methods of either contact inhibition or serum deprivation. For both types of fibroblast cells used to reconstruct the enucleated oocytes, the strategies that were utilized to synchronize the cell division cycle of nuclear donor cells considerably influenced the in vitro developmental abilities of NT pig embryos. Developmental competencies to reach the morula/blastocyst stages for cloned embryos that had been reconstructed with contact-inhibited or serum-starved foetal fibroblast cell nuclei were significantly higher than those for embryos that had been reconstructed with contact-inhibited or serum-starved adult cutaneous fibroblast cell nuclei.