55 The Role of Poly (ADP-Ribose) Polymerases in Porcine Cumulus–Oocyte Complex During In Vitro Maturation and Embryonic Development

Abstract

Poly(ADP-ribosyl)ation (PARylation) is related to DNA repair, chromatin modification, and apoptosis and is catalyzed by PARylation polymerases (PARP). Previous studies have shown that PARylation regulates pre-implantation development and participates autophagy mechanism in mouse and pig. However, the involvement of PARylation and pro-survival autophagy in pre-implantation development from cumulus–oocyte complexes (COC) to the blastocyst stage has not yet been documented. Thus, we investigated the role of PARylation during in vitro maturation (IVM) of porcine COC and their embryonic development. To study the effect of PARylation, COC were cultured with 3-aminobenzamide (3-ABA, PARP inhibitor) during IVM. Nuclear maturation rates of oocytes were showed no significant differences between 2 groups in all stages (from GV to MII). However, the expansion rates of cumulus cells were significantly decreased in 3-ABA–treated COC compared with control (11.05 ± 1.09 v. 48.40 ± 0.67%). When we analysed mRNA levels of maturation- and expansion-related genes in cumulus cells, levels of PTX3, CX43, and COX-2 were increased but levels of HAS2 and TNFAIP6 were decreased in treatment group. In addition, expression levels of GDF9 and BMP15 in oocytes were up-regulated in treated group. Then, we examined the development of IVF embryos from IVM oocytes in the presence and absence of 3-ABA and their quality at the blastocyst stage. We found that the developmental rates of embryos were significantly decreased in 3-ABA-treated group. In particular, the proportion of expanded blastocysts was lower in the treated embryos (2.65 ± 1.53) compared with control embryos (12.27 ± 3.05). Furthermore, the transcript levels of autophagy-related genes (ATG5, BECLIN1, and LC3) in 3-ABA-treated embryos were lowered in all stages. In addition, we found a higher rate of apoptosis in treated blastocysts compared with the control (total apoptosis index; 15.65 ± 2.73 v. 4.89 ± 0.67). Finally, SQSTM1/p62 aggregate increased in 3-ABA-treated blastocysts, indicating that the inhibition of PARylation regulates selective autophagy pathways to utilise SQSTM1/p62. Therefore, these results indicate that PARylation by PARPs during IVM of COC is deeply involved in the pro-survival autophagy and influences the development and quality of porcine embryos.

This research was supported by a Grant from the Bio & Medical Technology Development Program (2015M3A9C7030091) of the National Research Foundation (NRF) funded by the Korean government.