Control of gene expression at the onset of bovine embryonic development.

摘要

Bovine early development which is supported by maternal RNAs and proteins from the oocyte begins with fertilization of the oocyte by a sperm to form a zygote. We used different molecular approaches to determine timing and control of gene expression at the beginning of bovine development. Treatment of zygotes and embryos with a specific inhibitor of RNA polymerase II dependent transcription (alpha-amanitin) at different times during the first four cell cycles all resulted in inhibition of blastocyst formation indicating that transcription during zygotic and early embryonic stages is essential for embryo development beyond the 9- to 16-cell stages. Immunoblotting experiments using antibodies specific to the unphosphorylated form of RNA ploymerase II showed that bovine immature oocytes and early embryos including 2-cells were transcriptionally competent. Transcriptional activity during oocyte maturation and early embryogenesis was determined by labeling the cells with [35S]UTP followed by total RNA isolation and autoradiography. Bovine zygotes and 2-cell embryos were transcriptionally active and some of these transcripts were translated as determined by labeling cells with [3H]uridine and [ 35S]methionine followed by total RNA isolation and two-dimensional polyacrylamide gel electrophoresis respectively. This experiment also showed that the first and second rounds of DNA replication, and changes in the acetylation of core histones play important roles in regulation of early gene expression. Finally, an attempt was made to determine expression patterns of histone deacetylases at mRNA and protein levels in oocytes and early embryos. Results of this experiment showed that histone deacetylases are expressed in bovine oocytes and embryos suggesting that control of early gene expression in bovine is mediated in part by transcriptional repressors such as histone deacetylases. Collectively, data presented in this thesis provided a model for zygotic/embryonic gene expression in bovine where bovine maternal to embryonic control of development at transcriptional and translational levels is evident as early as the zygotic stage.