192 9-cis-RETINOIC ACID AFFECTS OOCYTE COMPETENCE MARKER GENE EXPRESSION IN THE BOVINE IN VITRO-MATURED OOCYTES AND CUMULUS CELLS

Abstract

Oocyte competence is the ability of an oocyte to undergo pre- and post-implantation development and to deliver a healthy offspring. A close association between the oocyte and the cumulus cells (CC) affects oocyte competence. Expression of several genes in the CC, known as oocyte competence markers, correlates with subsequent embryo development and quality. Addition of 9-cis-retinoic acid (9cisRA) to maturation medium increases oocyte competence through multiple mechanisms, including FSH/LH receptor expression, polyadenylation, growth factors signaling, oxidative-stress protection, or decreasing oocyte TNF-α gene expression. However, the effect of 9cisRA on the expression of oocyte competence markers in the oocytes and CC has not been determined. Therefore, the present study evaluated the effect of 9cisRA on the expression of oocyte competence marker genes in the oocytes and in the CC. Bovine cumulus–oocyte complexes, isolated from ovaries collected at the abattoir, were matured in vitro in the presence of 0 or 5 nM 9cisRA in the maturation medium (TCM-199 supplemented with 10% fetal bovine serum, 1 μg mL^–1 of β-oestradiol, 10 μg mL^–1 of follicle stimulating hormone, 0.6 mM cystein and 0.2 mM Na-pyruvate). After maturation, expression of target transcripts was quantified in CC and zona-free oocytes by SYBER green real-time PCR. The expression was normalized against a minimum of 2 out of 4 reference genes analyzed each time with target genes. The best combination of reference genes was automatically calculated by the CFX manager V1.1 program (BioRad) based on M-value during the analysis of gene expression data. A minimum of 5 biological replicates (50–60 oocytes/replicate) were performed for statistical analysis using a Student's t-test. Results indicated that 9cisRA increased (P < 0.05 to P < 0.001) expression of gremlin 1, prostaglandin G/H synthase 2, pentraxin 3, midkine, CD9 and thioredoxin mRNA in oocytes (3.0-, 2.8-, 3.1-, 2.0-, 4.0- and 2.4-fold) and CC (4.0-, 3.2-, 1.9-, 1.7-, 4.0- and 2.4-fold) compared to controls. In contrast, the aldose reductase 1b1 mRNA was down-regulated both in oocytes (1.0- vs 2.0-fold; P < 0.05) and in CC (1.0- vs 1.9-fold; P < 0.04) compared to the respective controls. In conclusion, the present study indicates that 9cisRA influences mRNA expression of oocytes and cumulus cells. This might be another explanation of the improved embryo development and quality in response to 9cisRA during in vitro maturation.

This work was partly supported by a scholarship from the BK21 program, the KRF (KRF-2008-211-F00011), the Next-generation BioGreen 21 Program (No. PJ007990012011), IPET (110020-3 and 109016-3) and the KOSEF (10525010001-05N2501-00110).