209 9-cis RETINOIC ACID INHIBITS EXPRESSION OF AKR1B1 TRANSCRIPT IN THE BOVINE OOCYTES AND PRE-IMPLANTATION EMBRYOS

Abstract

Quantitative real-time PCR has enabled quality evaluation of oocyte and pre-implantation embryo through monitoring expression of several molecular markers that are involved in metabolic activity, stress response, reprogramming, and other biological events. The aldo-keto reductase family 1 member B1 (AKR1B1) transcript is potentially involved in pregnancy failure through metabolism of progesterone and synthesis of prostaglandin F_2α in the bovine uterine endometrium. High expression of the transcript in blastocysts correlates inhibition of embryo implantation and/or embryo resorption. Maturation of immature oocyte in presence of 9-cis retinoic acid (9-cis RA) increases in vitro bovine embryo development rates and embryo quality. These beneficial effects of 9-cis RA are mediated through multiple mechanisms, including FSH/LH receptor expression, polyadenylation, growth factor signalling, oxidative-stress protection, or decreasing oocyte TNFα gene expression and inhibiting cumulus cell apoptosis during maturation. The present study aimed to evaluate the effect of 9-cis RA on expression pattern of AKR1B1 transcript in the oocyte matured in vitro and embryos (8-cell and Day 8 blastocyst) produced from in vitro matured oocytes in presence or absence of 9-cis RA. Bovine cumulus–oocyte complexes, isolated from ovaries collected at the abattoir, were matured in vitro in the presence of zero (control) or 5 nM 9-cis RA in the maturation medium (TCM199 + 10% fetal bovine serum + 1 µg mL^–1 β-oestradiol + 10 µg mL^–1 follicle stimulating hormone + 0.6 mM cystein and 0.2 mM Na-pyruvate). After maturation, the oocytes were subjected to standardized in vitro embryo production protocol or oocyte samples were collected for gene expression analysis. The expression of AKR1B1 transcript was quantified in zona-free oocytes, 8-cell embryos, and Day 8 blastocysts by real-time PCR using SYBER green. Not less than 4 biological replicates (oocytes: 50 to 60 per replicate and 8-cell embryos/day-8 blastocyst: 3 to 5 per replicate) were done for each group. The expression was normalized against a minimum of 2 out of 4 reference transcripts (18S rRNA, β-actin, glyceraldehyde-3-phosphate dehydrogenase and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) analysed each time with AKR1B1. The best combination of reference genes was automatically calculated by the CFX manager V1.1 program (Bio-Rad) based on M-value. The differences in gene expression levels were tested by Student’s t-test. Results indicated that 9-cis RA decreased expression of AKR1B1 transcript in the oocyte (1.0- v. 2.0-fold; P < 0.05), 8-cell-embryos (1.0- v. 10.1-fold; P < 0.03), and blastocyst (1.0- v. 2.1-fold; P < 0.03) compared with control. In conclusion, the present study indicates that 9-cis RA inhibits AKR1B1 transcript expression in oocytes and pre-implantation embryos.