285 TRANSCRIPT LEVEL OF Hsp70 AND IGF2R GENES IN BOVINE INDIVIDUAL BLASTOCYSTS DERIVED FROM OOCYTES MATURED WITH FBS OR FATTY ACID-FREE BSA

Abstract

The efficiency of in vitro embryo production in cattle varies between 30 and 40% of blastocysts derived from oocytes matured in vitro. Despite a rigorous selection, some embryos at blastocyst stage displaying normal morphology are not competent to develop after hatching (Maddox-Hyttel et al. 2003 Reproduction (Suppl. 61), 103–116). Therefore, a lot of attention has been focused on embryo quality. Supplements to culture media are one of the factors significantly contributing to this phenomenon. Serum (FBS) and albumin (fatty acid-free BSA, fafBSA) are widely used protein supplements; however, their effect on embryo quality is still variable (Rizos et al. 2003 Biol. Reprod. 68, 236–243). The aim of the present study was to investigate whether good-quality blastocysts (hatched or expanded) derived from oocytes matured in media supplemented with FBS or fafBSA differ in transcript level of 2 genes: heat shock protein (Hsp70) and receptor for insulin-like 2 factor (IGF2R). Bovine Day 8 blastocysts were produced in vitro from oocytes aspirated from slaughterhouse ovaries after maturation in TCM-199 medium supplemented with 10% FBS or 6% fafBSA, as previously described (Makarevich and Markkula 2002 Biol. Reprod. 66, 386–392). On Day 8 post-insemination (pi), good-morphology blastocysts were allocated into 3 groups: (1) hatched, (2) expanded of excellent quality, and (3) expanded of good quality, and individually frozen in liquid nitrogen. Each embryo was processed individually through RNA extraction and cDNA synthesis. Transcript quantitation protocol included: real-time PCR with SYBR Green I, β-actin gene as an internal standard, and relative standard curve method. Data analysis was performed by 2-way ANOVA. In each reaction, an equivalent of 0.125 embryo (2.5 µL of cDNA) was used, and 43 blastocysts were analyzed. All analyzed embryos were positive for the Hsp70 transcript, whereas IGF2R mRNA was detected in only 58% of blastocysts regardless of the maturation medium. A large variation in relative abundance (RA) was observed among individual embryos: coefficients of variation were 114.5 and 323.6% for IGF2R and Hsp70, respectively. Due to the distribution of Hsp70 RA, log transformation was performed. Real-time PCR data revealed a maximum 100-fold variation for the reference gene. Hatched blastocysts were characterized by a significantly lower RA for both analyzed genes. The 2 classes of expanded blastocysts did not differ in transcript level. With regard to protein supplements, only the RA for Hsp70 gene was significantly affected. This transcript was more abundant in embryos derived from fafBSA-supplemented IVM medium. The present results confirmed previously the described phenomenon concerning a large variability in mRNA content in single pre-implantation embryos. Moreover, because embryos able to hatch significantly differed in RA from their expanded counterparts, it is possible to relate embryo quality to transcript level.