255 PREIMPLANTATION DEVELOPMENT AND EXPRESSION OF Bax AND Hsp-70 GENES IN BOVINE EMBRYOS PRODUCED FROM OOCYTES MATURED IN CHEMICALLY DEFINED IVM MEDIUM WITHOUT GONADOTROPHINS

Abstract

The culture environment affects both the maternal and the embryonic expression of genes, and is likely to alter both oocyte and embryo developmental competence. Search for better and less variable culture conditions simulating the in vivo environment has led to the development of defined culture media, with lower negative impact on the molecular reprogramming of oocytes and embryos. This study aimed to evaluate the embryo development and relative expression of Hsp-70 and Bax transcripts in bovine blastocysts produced from oocytes matured in chemically defined IVM medium supplemented with insulin-like growth factor (IGF)-I, insulin, and polyvinyl alcohol (PVA) or polyvinylpyrrolidone 40 (PVP) without FSH and LH. Immature cumulus–oocyte complexes (COCs) from slaughtered cows were matured for 24 h in α-MEM plus IGF-1 and insulin and supplemented with 0.1% PVA (treatment 1; n = 410) or 0.1% PVP (treatment 2; n = 350). Neither FSH nor LH was used in both treatments. The control group consisted of COCs matured in TCM plus FSH and 10% estrous cow serum (n = 400). In vitro fertilization took place in Fert-Talp droplets for 22 h. Presumptive zygotes were co-cultured with cumulus cells in CR2aa supplemented with 10% fetal calf serum under mineral oil in a humidified atmosphere of 5% CO₂ at 38.5°C. Preimplantation embryo development was determined by cleavage, blastocyst, and hatching rates. For RNA extraction, blastocysts at Day 8 post-fertilization were frozen in liquid nitrogen and subsequently thawed. 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); 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 specific primers for Hsp-70 and Bax genes, using as endogenous reference the expression of H2a gene. Data of embryo development were analyzed by chi-square test and calculations of relative quantification were performed by REST^© (Relative Expression Software Tool; Pfaffl et al. 2002 Nucl. Acids Res. 30, e36), using the value found in the control group as calibrator. Cleavage rate was higher (P < 0.05) for the control group (67%) than for treatments 1 (54%) and 2 (59%). Nevertheless, there was no difference (P > 0.05) in blastocyst (40.2%, 45.0%, and 43.5%) and hatching (78%, 77%, and 75%) rates among treatments 1 and 2 and the control group, respectively. Similarly, no difference (P > 0.05) in relative expression of Hsp-70 and Bax transcripts (0.69 ± 0.69 and 0.53 ± 0.32 for treatment 1, and 1.08 ± 0.75 and 0.70 ± 0.47 for treatment 2) in comparison to the calibrator group was detected. These results show that bovine oocytes can be matured in serum-free and gonadotrophin-free medium supplemented with PVA or PVP without altering post-cleavage development and relative expression of some genes associated with stress and apoptosis.