178 TRANSCRIPTIONAL DIFFERENCES IN GENES RELATED TO GLUCOSE METABOLISM BETWEEN COMPETENT AND INCOMPETENT BOVINE IMMATURE CUMULUS-OOCYTE COMPLEXES SELECTED BY BCB

Abstract

In vitro maturation is a key step of in vitro embryo production, being the main factor responsible for the low blastocyst yield. In vitro maturation requires the selection of competent immature cumulus-oocytes complexes (COC), which is usually accomplished based on morphological criteria and follicle size. Competent immature COC have finished their growth phase and show decreased G6PDH activity. Brilliant cresol blue (BCB) is a dye that is degraded by G6PDH and, therefore, can be used to distinguish COC that have finished their growth phase (BCB+) from those that are still growing and are less competent (BCB-). The objective of this study was to determine the metabolic differences between BCB- and BCB+ COC by performing a transcriptional analysis of genes related to glucose metabolism. The COC obtained from slaughterhouse ovaries were selected based on BCB staining. The BCB+ and BCB- COC were fertilized and cultured in vitro to determine the differences in developmental ability. For gene expression analysis, BCB+ and BCB- COC were denuded by vortexing and 5 groups of 10 oocytes; their corresponding cumulus cells per group were snap-frozen until analysis. Messenger RNA was extracted by Dynabeads (Dynal Biotech, Lake Success, NY, USA) and relative mRNA abundance was analysed by quantitative PCR using PPIA1 as housekeeping. Statistical differences were determined based on ANOVA (P < 0.05). The genes analysed were G6PDH, its positive regulator SIRT2, 2 glucose transporters SLC2A1 and SLC2A5, 2 genes involved in anaerobic glycolysis GAPDH and LDHA, 2 genes related with Krebs cycle CS and ATP5A1, and 1 gene related to glutathione metabolism GPX1. As expected, the BCB+ group showed a higher cleavage rate (85.6 ± 1.8 v. 74.2 ± 1.3%, BCB+ v. BCB-; P < 0.05) and blastocyst yield (Day 9: 33.3 ± 3.8 v. 16.1 ± 1.4%, BCB+ v. BCB-; P < 0.05) compared with BCB-. Genes SIRT2, GAPDH, and LDHA were significantly up-regulated in BCB- cumulus cells (SIRT2: 1 ± 0.04 v. 1.45 ± 0.21; GAPDH: 1 ± 0.17 v. 1.46 ± 0.15; LDHA: 1 ± 0.22 v. 1.65 ± 0.12; BCB+ v. BCB-; P < 0.05), whereas no significant differences were found in the other genes and in oocytes. In conclusion, the differences in G6PDH activity between BCB+ and BCB- COC are not controlled by G6PDH transcript abundance, but seem to be mediated by SIRT2 regulation of G6PDH activity. The BCB- cumulus cells showed an up-regulation of GAPDH and LDHA, suggesting a higher activity of anaerobic glycolysis in BCB- COC compared with BCB+.