93 STRESS INDICATOR GENES EXPRESSION PATTERN IN BOVINE BLASTOCYSTS PRODUCED BY IN VITRO FERTILIZATION AND SOMATIC CELL NUCLEAR TRANSFER

Abstract

Reproductive biotechnologies, such as IVF and somatic cell nuclear transfer (SCNT) enable improved reproductive efficiency of animals. However, the birth rate from in vitro derived embryos still lags behind their in vivo counterparts. It is critical to develop an accurate evaluation and prediction system of embryo competence, for commercial purposes and for scientific research. Previous works have demonstrated that in vitro culture systems induce alterations in the relative abundance (RA) of diverse transcripts compromising embryo quality. The aim of this work was to analyse the RA of a set of genes involved in cellular stress (HSP70), endoplasmic reticulum (ER) stress (BiP and PSMB5) and apoptosis (Bax and Caspase-3), in bovine blastocysts produced by IVF, SCNT and their in vivo counterparts. Poly(A) RNA was isolated from 3 pools of 10 blastocysts per treatment and analysed by real-time PCR (RT–PCR). The results were normalized to the expression of the endogenous control (GAPDH). Data of mRNA expression was analysed using the InfoStat software (Universidad Nacional de Córdoba, Córdoba, Argentina). ANOVA, followed by Tukey multiple comparison test, was used for the analysis of differences in mRNA expression assayed by quantitative RT–PCR. Differences of P < 0.05 were considered significant. The RA of 3 stress indicators shown to be significantly increased in SCNT embryos compared with in vivo-derived blastocysts: by 23.9-fold for Bax, 12.1-fold for PSMB5 and 7.9-fold for Bip. No significant differences in RA of HSP70 and Caspase-3 gene transcripts were found. The increased expression of PSMB5 and BiP suggest that the SCNT embryos were under conditions of ER stress. When ER stress conditions persist, initiation of apoptotic processes is promoted. Furthermore, the significantly elevated transcript level of Bax could imply an initial activation of the apoptotic mechanisms, but the low levels of Caspase-3 revealed that the apoptotic caspase-cascade signalling system was not activated, hence not eliciting apoptosis, so far. With regard to IVF-derived embryos, no significant differences were detected in the RA of investigated genes compared with in vivo-produced blastocysts. Even though the embryos were morphologically comparable, mRNA expression pattern was dissimilar. Differential mRNA expression in bovine blastocysts obtained by different reproductive techniques produces blastocysts of divergent quality. We propose a new set of stress-indicator genes that give a widespread idea of bovine blastocyst quality. This study could complement morphological analysis to help develop an effective and accurate technique to diagnose embryo quality, towards improving the efficiency of artificial reproductive techniques.