Regulation of trophoblast development in the bovine embryo

Altered placental development in embryos produced by somatic cell nuclear transfer (SCNT) is associated with a high number of pregnancy losses. Several studies have described morphological development of the bovine placenta, whereas the molecular regulation remains unknown. The objectives of this thesis were to; (1) characterize the trophoblast gene Ascl2 in the bovine; (2) determine the expression of Ascl2, as well as other trophoblast genes, in early gestation of embryos produced by artificial insemination, in vitro fertilization and SCNT; and (3) determine if the epigenetic alterations associated with SCNT embryos is due to altered expression of epigenetic modifying genes (Dnmt1, Dnmt3b, HAT1 and HDAC1), as well as development of IVF and SCNT trophoblast cell lines as a potential model for bovine trophoblast development.; Bovine Ascl2 mRNA shares high homology with human and mouse Ascl2, especially in the DNA binding domain and bHLH regions. Expression Ascl2 mRNA was exclusively in the cotyledon tissue with the greatest abundance of Ascl2 mRNA in day 17 filamentous embryos, during the time of rapid trophoblast proliferation. Reduction in Ascl2 mRNA abundance was detected in day 8 parthenogenetic blastocysts which suggests a paternal regulation of the maternally expressed gene. Prior to implantation (days 8 and 17), Ascl2 mRNA appears to have biallelic expression, but is paternally silenced after implantation (days 40 and 60). In conclusion, the Ascl2 is highly conserved across species and is specifically expressed in the bovine placenta. Bovine Ascl2 appears to be maternally expressed after implantation, but the paternal genome plays a role in regulating bovine Ascl2 expression.; Ascl2 mRNA was greater in NT embryos compared to AI, while Hand1 was greater in AI embryos compared to NT. IFN-tau mRNA abundance did not differ among groups. PAG-9 mRNA was detectable in AI embryos but not in NT embryos. At day 40, NT fetal cotyledons had higher Ascl2 and Hand1 than did AI tissues. Day 40 NT cotyledons had the fewest functional binucleate cells, followed by IVF and AI. Thus, genes critical for normal placental development are altered in NT bovine embryos leading to abnormal trophoblast differentiation and contributing to pregnancy loss.; In day 8 SCNT blastocysts, abundance of Dnmt1mRNA was less than IVF counterparts. In day 40 placental tissues no difference in mRNA was detected in any of the epigenetic modifying genes studied. In SCNT primary trophoblast stem (TS) cells abundance of Dnmt3b mRNA was less and HAT1 RNA was more. In addition SCNT TS cells grew at a faster rate and expressed more Ascl2 mRNA, than IVF TS cells. These results suggest that the expression of epigenetic modifying genes may not be the cause of altered reprogramming associated with SCNT. In addition, the ability of TS, developed from SCNT embryos, to maintain elevated levels of Ascl2 mRNA was similar to that found in embryos and cotyledonary tissues, suggesting that this cell line are a model for investigating bovine trophoblast development in culture.; By understanding the underlying molecular events involved in bovine trophoblast development, we can gain insight into the regulatory mechanism involved in successful placentation and how these events may be manipulated to improve assisted reproductive techniques such as somatic cell nuclear transfer.