232 SPECIES-RELATED DIFFERENCES IN BLASTOCYST QUALITY ARE ASSOCIATED WITH DIFFERENCES IN RELATIVE mRNA EXPRESSION

Abstract

We have previously reported a species-related qualitative difference (in terms of ultrastructural morphology and cryotolerance) between bovine and ovine blastocysts produced under identical conditions of in vitro culture in synthetic oviduct fluid (SOF1, Rizos et al. 2002 Mol. Reprod. Dev. 62, 320–327). The overall objective of this study was to see if these differences were reflected at the transcript level. From each of five IVF replicates, groups of 10 bovine and 10 ovine blastocysts were used. The objective of Experiment 1 was to compare the relative transcript abundance of eight candidate genes between ovine and bovine blastocysts cultured in SOF1. Following real-time quantitative RT-PCR, transcript levels for MnSOD, survivin, and Glut-5 were significantly higher in ovine than in bovine blastocysts (ANOVA, P < 0.05), while transcripts for Cx31, IFN-tau and SOX were significantly more abundant in bovine blastocysts (P < 0.01). For the two remaining transcripts, E-cad and Na/K, there was no difference. The objective of Experiment 2 was to examine the possibility of modifying the pattern of expression in both types of blastocysts by changing the culture medium. Culture took place in SOF1 or SOF2 (Holm et al. 1999 Theriogenology 52, 683–700). Culture of bovine embryos in SOF2 resulted in a significant increase in the level of expression of MnSOD and Glut-5 (P < 0.05) compared to culture in SOF1. For all the other transcripts except survivin, there was a significant decrease in the relative abundance. Culture of ovine embryos in either SOF1 or SOF2 did not have a major influence on transcript abundance; of the eight transcripts examined, the relative abundance of only one, SOX, was significantly altered. Based on the above, the objective of Experiment 3 (3 replicates) was to determine whether the changed pattern of expression in bovine blastocysts produced in SOF2 was associated with an improvement in cryotolerance. Bovine blastocysts produced in both culture media were vitrified and warmed, and survival was assessed by re-expansion and hatching. Blastocysts produced in SOF2 had significantly higher survival rates at 24, 48, and 72 h and significantly higher hatching rates following vitrification and warming than those produced in SOF1 (P < 0.001). In conclusion, we have demonstrated that the apparent differences between ovine and bovine embryos in their adaptability to culture conditions, manifested in differences in embryo morphology and cryotolerance, are related to differences in mRNA relative abundance. This work was supported by Science Foundation Ireland under Grant No. 02/IN1/B78 and by Ministerio de Ciencia y Tecnologia from Spain under Grant No. AGL2003-05783.