25 MATERNAL-TO-EMBRYONIC TRANSITION FOLLOWING NUCLEAR TRANSFER OR PARTHENOGENETIC ACTIVATION

Abstract

The successful development of embryos generated by somatic cell nuclear transfer (SCNT) requires the ooplasm to reprogram the nucleus. This establishes the gene expression pattern necessary for full development by mechanisms that are currently being clarified. The ooplasm action on somatic nuclei shows many common aspects to the process that leads to the creation of a functional embryonic genome from the differentiated sperm and egg genomes. In order to investigate this aspect we studied a critical phase of early embryonic development: the maternal to embryonic transition (MET). We compared the pattern and level of gene expression between bovine embryos derived from in vitro fertilization (IVF), from nuclear transfer of adult fibroblasts (NT), or from parthenogenetic activation (PG). The study was performed in cattle because MET, in this species, occurs over four cell cycles, making it easier to detect even small deviations. Oocytes, matured for 22 h and fertilized in vitro or after cumulus removal, were enucleated and fused to fibroblast cells. Nuclear transfer and Met II oocytes were activated at 24-26 h of maturation with ionomycin (5 µM) for 5 min and 6DMAP (2 mM) for 4 h and then cultured in mSOFaa. Embryos were harvested at the required time for analysis at the 2-, 4-, 8-, and 16-cell; morula; and blastocyst stages and stored snap-frozen in a minimal volume of medium in groups of 5-10 embryos. Semiquantitative RT-PCR was used to study the expression of Nanog, Oct-4, Zar-1, and Par-3, because these genes are directly involved in early embryo development and have a specific expression pattern during MET. Data were analyzed with one-way ANOVA followed by Student-Newman-Keuls All Pairwise Multiple Comparison. No difference in pre-implantation development was observed among the three groups. The Nanog expression pattern was unchanged in all three groups, becoming detectable from the 8-16-cell stage onward. Oct-4 mRNA was detected at all stages in every group, but only in NT embryos did a significant increase occur at the 16-cell stage, suggesting the onset of an anticipated embryonic transcription. the Zar-1 expression pattern, with the characteristic de-novo transcription peak at the 4-cell stage, was observed in both IVF and NT embryos but not in PG embryos. In this group, Zar-1 mRNA levels were significantly higher at the 2- and 4-cell stage than in all of the following stages. The Par-3 gene showed the biggest differences among groups: IVF embryos expressed this gene from the 8-cell stage onward, whereas NT embryos showed high levels of Par-3 mRNA already at the 2-cell stage. Surprisingly, PG embryos showed no detectable Par-3 levels at any stages. The results indicate that, although in vitro development was not affected, gene-specific expression differences during MET occurred among groups. Relating the specific functions exerted by each of these genes in early development to the changes observed following the different manipulations provides useful data toward a better understanding of the role of these genes and of the mechanisms of nuclear reprogramming.

This work was supported by FIRB RBNE01HPMX, FIRST 2004, and ESF-EuroStells.