28 GLOBAL GENE EXPRESSION ANALYSIS OF ELONGATED EMBRYOS PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER AND IN VITRO FERTILIZATION

Abstract

Despite their ability to reach to the blastocyst stage, nuclear transfer embryos showed various abnormalities with respect to pregnancy outcome. We hypothesised that once cloned embryos were transferred to a suitable recipient the conceptus and endometrial interaction during peri-implantation stage was a remarkable step that determines the pregnancy outcome. In order to elucidate the molecular mechanisms underlining this phenomenon, the present experiment was conducted to compare the transcriptome profile of Day-16 elongated embryos derived from somatic cell nuclear transfer (SCNT) and fertilized controls, namely, artificially inseminated (AI) and in vitro fertilized (IVF) using bovine genome Affymetrix array. Following total RNA isolation from 3 replicates of each group, biotin-labeled cRNA was hybridized on 9 bovine chips. Data were normalized by using guanine cytosine robust multi-array analysis (GCRMA) and analysis was performed using the LIMMA package in R software. The present findings demonstrate that the gene expression profile of SCNT elongated embryos closely resembled those of the in vitro fertilized group. Only 10 genes were found to be differentially regulated between SCNT and IVF embryos (P ≤ 0.05, fold change ≥2 and false discovery rate 20%). On the other hand 303 and 336 genes were differentially expressed in IVF vs AI and NT vs AI embryos comparisons, respectively. Majority of the transcripts were found to be down-regulated on IVF embryos, whereas only 21 transcripts were up-regulated. Moreover, 158 and 178 genes were up- and down-regulated, respectively, in NT embryos as compared with AI. The NT embryos were found to be enriched with transcripts responsible for lipid metabolism (ACOX1, ACOT4 and ACOT8) and molecular transport (B4GALT1, ERBB3, MIF and PRKAG2) compared with AI. Moreover, genes involved in cell movement (CD97, CTSL1, F2R, ITGA and ITGAV) were highly abundant in IVF embryos compared with AI. Ingenuity pathway analysis of differentially regulated genes showed that metabolic and tight junction pathways and the genes involved in these pathways were activated in NT and IVP elongated embryos, respectively, as compared with AI. In conclusion, elongated embryos from NT and IVP pregnancies show differences in expression of genes involved in various biological processes compared with their AI counterparts.