85 CARBOXYETHYLGERMANIUM SESQUIOXIDE CONFERS ANTIOXIDATIVE PROTECTION FOR PORCINE IN VITRO-FERTILIZED EMBRYOS

Abstract

Reactive oxygen species (ROS) are generated both from the environment as well as from within the embryos during normal embryo development. However, excessive production of ROS creates the defective effect on embryo development. The purpose of the present study was to determine the effect of antioxidant, carboxyethylgermanium sesquioxide (Ge-132), during in vitro culture (IVC) on embryonic development in porcine in vitro-fertilized (IVF) embryos. The different concentrations of Ge-132 (0, 100, 200, and 400 μg mL^–1) were treated to the zygotes (n = 233, 234, 233, and 234, respectively). All of the experiments were repeated at least three times. To characterise putative porcine embryonic stem cell (ESC) derived from Ge-132 treated IVF blastocysts, several assays including an assessment of intensive alkaline phosphatase activity, embryoid body formation and expression analysis of pluripotency by immunofluorescence staining were performed. In the result of embryo developmental competence, the 100- and 200-μg mL^–1 Ge-132 significantly (P < 0.05) increased cleavage rates (67.4% and 67.1%, respectively; P < 0.05) compared with the control group (61.6%). The 100 μg mL^–1 Ge-132 had significantly (P < 0.05) higher blastocyst formation rates (32.6%) than the control group (26.5%). The Ge-132 supplement during IVC significantly (P < 0.05) increased GSH levels in embryos compared with those in the control, which led to the reduced ROS level. Two putative ESC lines were successfully derived from porcine IVF embryos cultured in 100 and 200 μg mL^–1 Ge-132 treatment. The established putative porcine ESCs showed alkaline phosphatase activity, pluripotency-associated markers such as OCT4 and NANOG and the capacity for embryoid body formation, which confirm the differentiation potentials. Together, these results suggest that Ge-132 supplementations during IVC enhanced the developmental competence of porcine IVF embryos, most likely by increasing intracellular GSH synthesis, reducing ROS level, and it might help the derivation of putative porcine ESCs.

This work was supported, in part, by a grant from the Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ011077, PJ011288), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2013R1A2A2A04008751). These are the results of a study by the Leaders INdustry-University Cooperation Project 2015-B-0013–010118, supported by the Ministry of Education, Republic of Korea.