9-cis Retinoic Acid Attenuates TNF-alpha Expression in Oocytes During In Vitro Embryo Maturation and Improves Developmental Competence and Embryo Quality.

Abstract

Retinoic acid (all-trans RA and 9-cis RA) regulates cell proliferation, differentiation and morphogenesis by binding to different receptors. All-trans RA binds to nuclear retinoic acid receptors (RAR; α, β, γ) and 9-cis RA binds to retinoid X receptors (RXR; α, β, γ) in vivo. All-trans RA converts to 9-cis RA in a reversible manner and 9-cis RA activates both RAR and RXR in vitro. Presence of RA receptors from the oocyte to the hatched blastocyst and cumulus cells suggests that RA may play an important role in early embryonic development. Indeed addition of RA to in vitro maturation (IVM) medium improves developmental competence and embryo quality. Tumor necrosis factor alpha (TNF-α), a pleiotropic cytokine that exerts a variety of effects including proliferation, growth inhibition, cytotoxicity, inflammation and immunomodulation, is detected at higher levels in the follicular fluid of poor quality oocytes than in that of good quality oocytes. TNF-α influences folliculogenesis and oocyte maturation by inhibiting aromatase activity and estradiol secretion in granulose cells. Recent studies have shown that RA attenuates TNF-α production in different cell lines. Therefore, the aim of the present study was to determine whether the TNF-α level in oocytes is associated with developmental competence and embryo quality and whether 9-cis RA might improve in vitro embryo development by inhibiting TNF-α in oocytes. Oocyte TNF-α level, developmental competence, total cell number and percentage of apoptotic cells were assessed in blastocysts exposed to a physiological concentration of 9-cis RA. Cumulus oocyte complexes (COCs) collected from bovine abattoir ovaries were matured in base IVM medium supplemented with 5 mM 9-cis RA, 100 ng/mL recombinant bovine TNF-α or 5 mM 9-cis RA + 100 ng/mL recombinant bovine TNF-α. Oocytes were subsequently collected for gene expression analysis or subjected to in vitro fertilization and culture at 38.5°C and 5% CO2 in a humidified atmosphere. TUNEL staining and gene expression analysis by quantitative real-time PCR (qPCR) were conducted on day 8-blastocysts. Expression was normalized to Actb, 18S rRna and Ywhaz. Results indicated that 9-cis RA downregulated both basal and TNF-α-induced TNF-α mRNA level (2.5-fold) in oocytes. 9-cis RA increased blastocyst development rates (36.4%) and total cell number (13.2%) and reduced the percentage of apoptotic cells in blastocysts (40.1%). In addition, 9-cis RA downregulated both basal and TNF-α-induced caspase-3 (2.5-fold) and TNF-α (2.5-fold) mRNA levels in blastocysts. In conclusion, the present study demonstrates that 9-cis RA improves oocyte development competence and embryo quality by inhibiting oocyte TNF-α expression. This work was partly supported by a scholarship from the BK21 program, the KRF (KRF-2008-211-F00011), the IPET (108068-03-1-SB010) and the KOSEF (10525010001-05N2501-00110). (poster)