9-cis retinoic acid improves developmental competence and embryo quality during in vitro maturation of bovine oocytes through the inhibition of oocyte tumor necrosis factor-alpha gene expression

Abstract

Retinoic acid (RA; all-trans RA and 9-cis RA) enhances embryo developmental competence and quality through multiple mechanisms affecting the oocyte and preimplantation embryo. Folliculogenesis and oocyte maturation are influenced by tumor necrosis factor-alpha (TNF-alpha) via inhibition of aromatase activity and estradiol secretion in granulosa cells. Retinoic acid inhibits TNF-alpha production in various cell lines. The aim of the present study was to determine whether oocyte TNF-alpha concentrations regulate developmental competence and embryo quality and if the beneficial effects of 9-cis RA are mediated through attenuation of oocyte TNF-alpha production. Bovine cumulus oocyte complexes collected from abattoir ovaries were matured in maturation medium in the absence (control) or presence of 5 nM 9-cis RA (RA), 100 ng/mL of recombinant bovine TNF-alpha (TNF), or 5 nM 9-cis RA + 100 ng/mL of recombinant bovine TNF-alpha (RA + TNF). Oocytes were subsequently collected for gene expression analysis or subjected to in vitro fertilization and culture. Apoptosis and gene expression were analyzed in d-8 blastocysts. Results indicated that 9-cis RA down-regulated (P < 0.01) both basal and TNF-alpha-induced TNF-alpha mRNA in oocytes (1.0-fold in control, 0.4-fold in RA, 2.1-fold in TNF, and 0.7-fold in RA + TNF). The 9-cis RA increased (P < 0.001) blastocyst development rates (37.1 +/- 6.9 vs. 23.6 +/- 8.0%) and total cell number (138.4 +/- 19.2 vs. 120.2 +/- 24.5) and reduced (P < 0.001) the percentage of apoptotic cells (3.3 +/- 2.0 vs. 5.6 +/- 2.3%) compared with controls. Expression of caspase 3 (0.4- vs. 1.0-fold) and TNF-alpha (0.4- vs. 1.0-fold) mRNA was downregulated (P < 0.05) in RA-treated blastocysts compared with controls. Moreover, 9-cis RA rescued (P < 0.001) development rates (24.5 +/- 11.1 vs. 15.6 +/- 9.0%), increased total cell number (124.6 +/- 36.5 vs. 106.9 +/- 31.1), and reduced apoptosis (5.8 +/- 2.0 vs. 8.1 +/- 3.1%) in blastocysts exposed to TNF-alpha (TNF group). Caspase 3 (0.8-fold in RA + TNF vs. 2.2-fold in TNF) and TNF-alpha (0.3-fold in RA + TNF vs. 2.8-fold in TNF) mRNA expression was attenuated (P < 0.05) in TNF-alpha-treated blastocysts. In conclusion, the present study suggests that 9-cis RA exerts its beneficial roles on oocyte developmental competence and embryo quality by attenuating oocyte TNF-alpha mRNA expression.