In vitro or in vivo post-fertilization embryo culture affects the gene expression patterns of ovine prepubertal embryos

Production of embryos in vitro from prepubertal females has enormous potential for research and commercial applications. However, embryos derived from those donors showed reduced in vitro development rate and viability after transfer in recipients. Although the oocyte origin can play a crucial role in determining the developmental competence, the post-fertilization embryo culture environment may affect the blastocyst yield and quality. Aim of this study was to examine the relative transcript abundance of a panel of developmentally important genes in in vitro and in vivo cultured prepubertal ovine embryos. Cumulus oocyte complexes derived from ovaries of regularly slaughtered prepubertal sheep were matured in vitro in TCM199 with 10% heat-treated OSS, 10 μl/ml 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. Thirty hours post-fertilization, early cleaved embryos (two cell stage) were divided in two groups for culture either in vitro, in SOF + aa + 0.4% BSA in 5% CO2 and 5% O2, or in vivo, in ewe oviduct. Seven days post-fertilization, three groups of 8 blastocysts for each class (3 replicates) were recovered and used for gene expression analysis by reverse transcription followed by Real Time PCR. Higher relative abundance for NaKATPase, IGF2R and Nanog transcripts was observed in in vitro cultured embryos than in in vivo cultured ones (ANOVA; P<0.05), while no differences were detected for OCT4, Aquaporin 3, E-cadherin and IGF2 mRNAs in the two groups. Since embryos belonging to both classes were produced by in vitro fertilization of in vitro matured oocytes, the post-fertilization embryo culture environment itself seems to affect the gene expression pattern of several important genes at the blastocyst stage.