269 EXPRESSION PATTERN OF STRESS MARKER GENES IN BOVINE OOCYTES MATURED IN VITRO IN MEDIA WITH DIFFERENT HORMONE COMPOSITION

Abstract

Evaluation and prediction of oocyte quality are 2 critical issues in assisted reproductive technologies. The lack of reliable and objective predictors of oocyte developmental competence, and subsequent embryonic development, negatively affects the efficiency of assisted reproductive technologies. The aim of this work was to study the effect of different bovine oocyte maturation protocols on the relative transcript abundance from genes involved in cellular stress (70-kDa heat shock proteins), endoplasmic reticulum (ER) stress (Bip: immunoglobulin heavy chain binding protein and proteasome subunit β-5 protease), and apoptosis (Bax and Caspase-3). Oocytes were matured in 5 different media that varied in the composition of hormones and cysteamine: 1) porcine FSH (Folltropin^®, Bioniche Animal Health, Belleville, Ontario, Canada) and cysteamine, 2) epidermal growth factor (EGF), 3) EGF and cysteamine, 4) recombinant human FSH (rhFSH), and 5) rhFSH and cysteamine. Three groups of 10 matured oocytes in each of the 5 different maturation media were analysed by real-time RT-PCR. The results were normalized to the expression of the endogenous control (glyceraldehyde-3-phosphate dehydrogenase). Messenger RNA abundance data were analysed using the InfoStat software (Universidad Nacional de Córdoba, Argentina). One-way ANOVA, followed by Tukey multiple comparison test, was used for the analysis of differences in mRNA abundance. The abundance of the β-5 subunit of the proteasome mRNA increased 70% (P < 0.05) in oocytes matured in medium supplemented with porcine FSH and cysteamine compared with that in the oocytes matured in the other maturation media supplemented with rhFSH and EGF with or without cysteamine. The expression of the ER chaperone, Bip, increased 120% (P < 0.05) in oocytes that were matured in the same medium, compared with that in those matured in medium supplemented with rhFSH and cysteamine. The results suggest that maturation of oocytes in medium supplemented with porcine FSH and cysteamine induced ER stress, which is reflected by the increased abundance of the chaperone Bip and β-5 protease. Given that the abundance of the cytoplasmic chaperone Hsp70 was not altered in any of the treatments, there was no evidence of widespread cellular stress. However, it seemed that these conditions were not stressful enough to induce apoptosis because Bax and Caspase-3 markers were not modified. This study could complement morphological analysis to help develop an effective and accurate technique to diagnose oocyte quality during assisted reproductive technologies.