Identifying Biomarkers of Autophagy and Apoptosis in Transfected Nuclear Donor Cells and Transgenic Cloned Pig Embryos

Abstract

Abstract In this study, we first investigated the effects of 3-methyladenine (3-MA), an autophagy inhibitor, and the inducer – rapamycin (RAPA) on the incidence of programmed cell death (PCD) symptoms during in vitro development of porcine somatic cell nuclear transfer (SCNT)-derived embryos. The expression of autophagy inhibitor mTOR protein was decreased in porcine SCNT blastocysts treated with 3MA. The abundance of the autophagy marker LC3 increased in blastocysts following RAPA treatment. Exposure of porcine SCNT-derived embryos to 3-MA suppressed their developmental abilities to reach the blastocyst stage. No significant difference in the expression pattern of PCD-related proteins was found between non-transfected dermal cell and transfected dermal cell groups. Additionally, the pattern of PCD in SCNT-derived blastocysts generated using SC and TSC was not significantly different, and in terms of porcine SCNT-derived embryo development rates and total blastocyst cell numbers, there was no significant difference between non-transfected cells and transfected cells. In conclusion, regulation of autophagy affected the development of porcine SCNT embryos. Regardless of the type of nuclear donor cells (transfected or non-transfected dermal cells) used for SCNT, there was no difference in the developmental potential and quantitative profiles of autophagy/apoptosis biomarkers between porcine transgenic and non-transgenic cloned embryos. These results led us to conclude that PCD is important for controlling porcine SCNT-derived embryo development, and that transfected dermal cells can be utilized as a source of nuclear donors for the production of transgenic cloned progeny in pigs.