75 CHANGES IN GENE EXPRESSION PATTERN DURING PRE- AND PERI-IMPLANTATION STAGES IN BOVINE CLONED EMBRYOS

Abstract

In ruminants after blastocyst formation and before implantation the embryo elongates drastically. This peri-implantation stage is characterized by active proliferation of trophoblastic cells and by changes in gene expression. In cloned embryos inappropriate expression of genes during early stages could be the cause of embryonic losses. The aim of this study was to compare the expression pattern of selected developmentally important genes in Day 7 embryos produced by somatic cell nuclear transfer with gene expression at Day 17 during elongation. IVF embryos at each stage were used for comparison. For analysis we used an RT-PCR approach (conventional + quantitative real-time PCR; qPCR). For nuclear transfer we used handmade cloning with an adult fibroblast cell line that previously yielded live cloned calves. Cells were confluent, non-serum starved in passage 4. Cloned embryos were cultured in SOF medium for 7 days in the well of the well (WOW) system. Semen from a proven bull was used for IVF and the presumptive zygotes were cultured under the same conditions as cloned embryos. At Day 7, embryos (cloned or IVF) were either transferred to recipients or pooled in tens for RNA extraction using NucleoSpin RNA XS Kit (Macherey-Nagel, Düren, Germany), converted to complementary (c)DNA and amplified with specific primers in PCR reactions. Transferred embryos were recovered at elongation stage (Day 17); their RNA was extracted and treated as above. The genes studied were NANOG, FGF4, OCT4, EOMES, IFNtau, and ACTB (internal standard for quantification). Data were analyzed by Kruskal Wallis test using the InfoStat program (Buenos Aires, Argentina). Differences were considered significant at P < 0.05. Development to blastocysts was 65% for clones and 32% for IVF embryos; 19 and 6 elongated embryos were flushed back, respectively. For gene expression analysis we used 5 pools of 10 blastocyts each and 12 (6 IVF; 6 cloned) elongated embryos with embryonic disc. At pre-implantation, we found OCT4, FGF4, IFNtau, and NANOG expressed and their relative levels were quantified (except for NANOG). Expression of OCT4 was higher in the cloned embryos; expression of EOMES was not detected at this stage. At Day 17, all 5 genes were expressed and quantified via qPCR, and interesting differences were found: relative levels of expression of FGF4 decreased in both types of embryos from Days 7 to 17, whereas those for IFNtau, NANOG, and EOMES increased significantly. Expression of OCT4 remained constant in IVF embryos but not in the cloned embryos (higher at Day 7). Our results provide an initial approach to the dynamic changes occurring in the expression of developmentally important genes in the transition from expansion to elongation. The decrease in FGF4 expression as well as the increase in EOMES and NANOG expression is a new finding that could shed light on the mechanisms of trophoblastic differentiation in bovine elongation.