Effect of co-culture canine cumulus and oviduct cells with porcine oocytes during maturation and subsequent embryo development of parthenotes in vitro

Abstract

In the estrus stage, canine oocytes are surrounded by cumulus cells and undergo maturation in the oviduct for 2-3 days after ovulation. We hypothesized that canine oviduct cells (cOC) and canine cumulus cells (cCC) during this stage might affect the maturation of oocytes and thereby improve subsequent embryo development. Therefore, the objective of this study was to compare the effects of a cOC and cCC co-culture on oocyte in vitro maturation (IVM) and subsequent embryo development, and to analyze the gene expressions in a molecular fashion what co-culture actually gives the specific pathways in which the co-culture cells act to improve maturation and embryo development. The effect of co-culture using cOC and cCC on porcine oocyte IVM was investigated. Thereafter, oocytes were activated using electrical stimulation and embryo developmental competence was estimated. The expression of the genes related to oocyte maturation, embryo development and apoptosis were analyzed. Also, reactive oxygen species (ROS) levels after IVM was analyzed. The IVM rate and embryo development including cleavage, blastocyst formation rates, and total blastocyst cell numbers from cOC group were significantly higher than other groups (P < 0.05). The expression of SMAD2/3 and growth differentiation factor 9 (GDF9) was significantly increased in cOC and oocytes from the cOC group compared with other groups. Moreover, the levels of GDF9, prostaglandin-endoperoxide synthase 2 (PTGS2), WNT3A and matrix metalloproteinase 2 (MMP2) were significantly up-regulated in blastocysts from the cOC group. The concentration of ROS was significantly lower in the supernatant of cOC groups compared with other groups. Also, the expression of BCL2 was significantly increased in porcine cumulus cells and oocytes from cOC group. The present study demonstrated that co-culture with cOC improved in vitro porcine oocyte maturation and subsequent embryo development competence. Also, co-culture with cOC during IVM induces a suitable environment for oocyte maturation by enhancing the mRNA level of SMAD2/3 and GDF9, and for embryo development by elevating the expression level of PTGS2, WNT3A and MMP2. In addition, the decreased ROS level in cOC co-culture could have a beneficial influence on oocyte maturation.

Keywords: Co-culture; Cumulus cell; Embryo development; Gene expression; Oocyte maturation; Oviduct cell.