177 EMBRYOS PRODUCED IN VITRO FROM PREPUBERTAL LAMB AND ADULT SHEEP OOCYTES DISPLAY DIFFERENT GENE EXPRESSION PATTERNS

Abstract

Breeding from prepubertal females reduces the generation interval and increases the rate of genetic gain in animal breeding programs. Despite considerable interest in this technology, its efficiency remains too low. Reduced in vitro and in vivo developmental competence of oocytes derived from prepubertal animals have been reported in association with morphologic, metabolic, and biochemical differences. The objective of this study was to compare the relative transcript abundance of a panel of developmentally important genes in embryos produced in vitro from prepubertal lamb and adult sheep oocytes. Cumulus–oocyte complexes derived from ovaries of regularly slaughtered 1-month-old prepubertal and adult sheep were matured in vitro in TCM-199 with 10% heat-treated oestrus sheep serum (OSS), 10 μL mL^–1 of FSH/LH and 100 μm cysteamine, in 5% CO₂ in air at 38.5°C for 24 h. Matured oocytes were fertilized with frozen–thawed ram semen in SOF medium + 2% OSS for 22 h at 38.5°C and 5% CO₂, 5% O₂, and 90% N₂ atmosphere. Zygotes were cultured in SOF + AA + 0.4% BSA in 5% CO₂ and 5% O₂ up to blastocyst stage. Three groups of 10 blastocysts for each class (4 replicates) were used to quantify the relative expression of 15 genes by reverse transcription followed by real-time PCR. The relative quantification of the transcripts was performed with the 2-ddCt method (Livak and Schmittgen 2001 Methods 25, 402–408), after normalization against the β-actin expression levels. The analysis of gene expression evidenced higher relative abundance for Aquaporin 3, P34^Cdc2, cyclin B, Oct4, H2A.Z, and Nanog transcripts in sheep embryos than in prepubertal-derived ones (ANOVA; P < 0.05), while interferon τ and insulin-like growth factor (IGF) 2 mRNAs were significantly more abundant in lamb-derived embryos (ANOVA; P < 0.01). No differences were observed for the remaining analyzed transcripts (BAX, IGF2R, heat shock protein 90, NaKATPase, E-cadherin, PAP, and glyceraldehyde 3-phosphate dehydrogenase). Overall, results show that embryos produced in vitro from prepubertal and adult oocytes display different patterns of expression at the blastocyst stage. Such difference may be related to the generally observed reduced in vitro and in vivo developmental competence. Increased understanding of the gene expression status during pre-implantation development may provide valuable insights into the molecular basis underlying the very early stages of life and an opportunity for optimizing in vitro embryo production systems.