129 Knockdown of connexin 43 effects on developmental competence in porcine parthenotes

Abstract

Connexin 43 (Cx43) is one of the gap junction proteins that are compounds of transmembrane proteins and transports the small-molecular-weight chemicals up to 2 kDa. Lacking of Cx43 influences the junctional protein and induces autophagy and apoptosis in somatic cells. However, the function of Cx43 in porcine early embryos is still unknown. With the aim to find out the molecular mechanism of Cx43 on the developmental competence of early porcine embryos, dsRNA technology was carried out and inhibitor was used as the positive control. Additionally, it is difficult to obtain pig embryos of homogeneous quality due to the relatively high incidence of polyspermy during IVF. Therefore, diploid parthenotes have been frequently used to study early development in the pig. Connexin 43 dsRNA (1 μg μL⁻¹) was microinjected into the parthenogenetically activated porcine zygotes. Blastocyst rate [5 repeats; treatment, 8.8 ± 1.6% (n = 1356) v. control, 38.6 ± 4.3% (n = 1082)] and total cell numbers in the blastocyst [4 repeats; treatment, 20.7 ± 3.5 (n = 85) v. control, 39.8 ± 4.1(n = 97)] were significantly reduced following Cx43 knocking down. Results from fluorescein isothiocyanate-dextran and Western blot assay show that knockdown (KD) of Cx43 significantly increased membrane permeability through down-regulation of genes that are components of both adherence and tight junction in the porcine blastocyst. Reactive oxygen species were significantly increased in the Cx43 KD group compared with the control. In addition, KD of Cx43 activated Caspase 3 and significantly increased ATG8 expression and induced autophagy and apoptosis. Results suggest that KD of Cx43 influences pre-implantation porcine embryo development via increasing membrane permeability and reactive oxygen species generation and by inducing autophagy and apoptosis.