192 THE EFFECT OF ZWITTERONIC BUFFERS AND PBS ON GENE TRANSCRIPTION OF BOVINE IN VIVO- AND IN VITRO-DERIVED EMBRYOS

Abstract

Choice of buffer used for the IVF procedures affects embryo developmental rates (De la Fuente et al. 2006 Reprod. Fertil. Dev. 18, 187). Also, it has been shown that the 3 zwitterionic buffers tested in this study, TES (T), MOPS (M), and HEPES (H) (pKa values at 20°C: 7.2–7.5) interact with DNA (Stellwagen et al. 2000 Anal. Biochem. 287, 167). Our objective was to evaluate the effect of T, M, H, and PBS buffers on the expression of the following genes, Fgf-4 (fibroblast growth factor 4 precursor), Lama1 (laminin alpha 1), Ube2a (ubiquitin-conjugating enzyme), Gsta4 (glutathione S-transferase A4), Il6 (interleukin 6), Sod1 (superoxide dismutase), Prss11 (IGF binding), and Hspb1 (Heat shock protein binding 1), on bovine in vivo and in vitro embryos. Genes were selected based on their sensitivity to adverse in vitro embryo culture conditions by microarray analysis (data not shown). All buffers were prepared at a concentration of 10 mM in TALP medium and the final pH was adjusted to 7.2. Bovine follicular fluid was aspirated from abattoir-derived ovaries and evenly divided into 4 tubes. Collected oocytes (5 replicates) from each tube were processed separately through entire IVM, IVF, and IVC procedures using washing medium buffered with: PBS (n = 490), Group 1; H (n = 438), Group 2; M (n = 440), Group 3; and T (n = 394), Group 4. All buffers contained 4% BSA. Oocytes were matured in TCM-199 supplemented with 10% FCS and 10 ng mL^-1 epidermal growth factor, and inseminated in Fert-TALP containing 25 mM bicarbonate, 22 mM sodium lactate, 1 mM sodium pyruvate, 6 mg mL^-1 BSA-FAF, and 10 µg mL^-1 heparin with 1 × 10⁶ mL spermatozoa. After 24 h of oocytes–sperm co-incubation, presumptive zygotes were cultured in SOFaa medium with 8% BSA at 39°C under paraffin oil and 5% CO₂ in humidified air. Cumulus–oocyte complexes and zygotes were held in designated buffers <16 min before oocyte maturation, <7 min after IVM and before IVF, and <18 min after IVF and before culture. The total time of oocyte/embryo exposure to each buffer was <41 min. Day 7 blastocysts from 5 replicates and 4 treatments were frozen for mRNA analysis. Day 7 in vivo blastocysts exposed for <45 min to PBS were used as controls. Poly(A) mRNA was prepared from 20 groups of pools of 5–7 embryos. The quantification of all gene transcripts was performed by real-time quantitative RT-PCR in 3 replicates. Data on mRNA expression were analyzed by one-way repeated-measures ANOVA. Results showed that, except for Hspb1 and Ube2a genes, the levels of expression of the 6 remaining transcripts were higher (P < 0.05) in controls than in in vitro embryos irrespective of buffer used. In addition, higher expression of Hspb1 and lower expression of Ube2a and Lama 1 were identified in PBS and T than in M and H (P < 0.05). Expression of Fgf-4 and Gsta4 among in vitro embryos were lower (P < 0.05) in PBS than in the remaining 3 buffers. There was no difference in expression of Il6, Sod1, and Prss11 genes in any buffer among in vitro-derived embryos. It can be concluded that mRNA transcription of in vitro-derived embryos is affected by the choice of the buffer used for the IVF procedure.