Marcadores moleculares para competência ovocitária em células do Cumulus bovinas

Abstract

The present study aimed to quantify the expression of candidate genes in cumulus cells (CC) from oocytes with high and low potential to develop in vitro up to the blastocyst stage. Initially, the effect of the individual culture system and the biopsy on embryo development was evaluated. Cumulus-oocyte-complexes (COCs) were distributed into 3 groups: control (COCs were cultured in groups); WOW (COCs were cultured individually in the WOW system); and micro droplet (COCs were individually cultured in micro droplet of 20 μL). Then, embryo production was compared between the control and the individual system (micro droplet) in which the COCs were submitted or not to biopsy. Expression levels of GPC4, IGFBP4, FSHR, GHR, EGFR, FGF11, SLC2A1, SLC2A3, SPRY1, VCAN and KRT8 genes were quantified by real time PCR (qPCR) in CC biopsies. Each biopsied COC was individually tracked, by culturing them in a micro droplet, and categorized based on his fate: embryo at blastocyst stage at D7, cleaved and arrested and not cleaved. Average blastocyst rates were lower in individual culture (WOW=17,9 % n=95; microdrop= 26,3% n=95) than in the control group (45,0 %, n=209) (P<0.05), and no effect of the biopsy was observed for both groups (P>0.05). From the 11 genes evaluated 3 showed differential expressions. Higher expression of GHR (p<0.10) and VCAN (P<0.10) was observed in CCs that formed embryos than those not cleaved. The GPC4 gene was overexpressed (P=0,007) in CC from the formed embryos compared to cleaved and arrested ones. It can be concluded that biopsy do not affect embryo development. The expression of GHR, VCAN and GPC4 genes can be used as markers to distinguish COCs associated with good quality embryos from COCs with limited developmental potential.