LARGE-SCALE CHROMATIN STRUCTURE AND TRANSCRIPTIONAL ACTIVITY DURING FINAL DIFFERENTIATION OF BOVINE OOCYTE

Abstract

The oocyte genome is subject to several levels of regulation, and functional differentiation of large-scale chromatin structure provides an important epigenetic mechanism for the developmental control of global gene expression. Dynamic changes in chromatin structure and function occur during oocyte growth in several mammalian species. In cow, we recently characterized the morphological transitions in the germinal vesicle (GV) during final stages of oogenesys in which chromatin acquires different configurations. In particular, we identified four discrete stages of GV (from GV0 to GV3), characterized by an increase of chromatin condensation, which was related to the sequential achievement of meiotic and embryonic developmental competencies. However, in bovine species, little is known concerning the unique molecular mechanisms involved in the regulation of large-scale chromatin structure and its impact on global transcription and the expression of specific genes involved in the later aspect of oocyte differentiation as well as in the control of the subsequent embryogenesis. The aim of the present study was to analyze the total transcriptional activity and the expression of a panel of genes which could be essential for the developmental competence in bovine oocytes, collected from early and middle antral follicles and sorted on the basis of their chromatin configuration. Global transcriptional activity was analyzed by H3-Uridine incorporation and subsequent autoradiography on semithin Epon sections, while gene expression was examined by RT-PCR with specific primers designed on the basis of sequence database available. In particular, we investigated on the expression pattern of DNA methyl transferase-1, which is involved in the maintenance of methylation pattern of imprinted genes after fertilization, nucleoplasmin-1, nucleoplasmin-2 and nucleoplasmin-3, which are involved in the organization of the nuclear compartment and zygotic arrest-1 that plays an important role after fertilization during the embryonic genome activation. The results indicate that an increase in chromatin condensation is related to global transcriptional silencing in bovine oocyte. In fact, while oocytes with GV0 configuration exhibited high level of transcription, GV1 and GV2 stages showed a remarkable decrease of transcription and the acquisition of GV3 configuration was associated with global repression of transcriptional activity. The gene expression analysis revealed that all the transcripts were present in the different GV categories, regardless of the chromatin configuration, suggesting the potential action of additional levels of gene regulation. Ongoing semi-quantitative analysis aim at clarifying the regulation of gene expression during the chromatin remodeling process and may provide a better understanding of the mechanisms involved in multiple and hierarchical large-scale chromatin modifications that regulate global transcription before the onset of meiosis. (Work supported by MIUR PRIN 2005Work supported by MIUR PRIN 2005073071-003 and “L'Oreal Italy For Women in Science”) (poster)