Expression of Hsp-70 and Bax genes in bovine blastocysts derived of oocytes matured in chemically defined medium supplemented with synthetic macromolecules

Abstract

Analysis of expression patterns of developmentally important genes essential in early developmental provides a useful tool to assess the normality of produced embryos and to optimize ART. The in vitro production of bovine embryos has been shown to be correlated with significant up- or downregulation of genes critical for undisturbed fetal development. Bax and Hsp-70 genes are strongly transcripts in IVP embryos and their products are involved in processes such as apoptosis and stress adaptation, respectively. This study aimed to quantify Hsp-70 and Bax gene expression in bovine blastocysts produced from oocytes matured in chemically defined IVM system. An experimental study using a bovine model was performed. Immature cumulus oocyte complexes from slaughtered cows were matured for 24 h in three treatments: T1) α-MEM + IGF-I + Insulin + 0.1% PVA; T2) α-MEM + IGF-I + Insulin + 0.1% PVP-40; T3) control: TCM + FSH + 10% oestrous cow serum. Twenty two hours after IVF, zygotes were cultured in CR2aa with 10% fetal calf serum in a humidified atmosphere of 5% CO2 at 38.5°C. Blastocysts on day 8 post-fertilization were frozen in liquid nitrogen and thawed for RNA extraction. Total RNA extraction was performed using Rneasy Micro kit (Qiagen, Valencia, CA, USA) and first strand synthesized using Superscript III First Strand Synthesis kit (Invitrogen, Chicago, IL, USA). Relative quantification was performed in duplicate using Real Time PCR (ABI Prism 7000 Applied Biosystem, Foster City, CA, USA) and reactions consisted of a mixture of iTaq SYBR Green Supermix with ROX (Bio-Rad, Waltham, MA, USA) with cDNA equivalent to 0.8 embryos and gene specific primers. Expression of H2a gene was used as endogenous reference. Calculations of relative quantification were performed by REST © (Relative Expression Software Tool), using the value found in TCM group as calibrator. The blastocyst rates in T1, T2, and T3 were 40%, 48% and 38%, respectively. The relative expression of Hsp-70 and Bax transcripts were, respectively, 0.69 ± 0.69 and 0.53 ± 0.32 for T1, and 1.08 ± 0.75 and 0.7 ± 0.47 for T2 in comparison to the calibrator group (T3), with no significant differences between groups (P > 0.05). These results indicate that oocyte maturation in chemically defined media supplemented with PVA or PVP-40, without FCS and gonadotropins, do not alter the relative abundance of Hsp70–1 and Bax transcripts in resulting early embryos.