TRANSLATIONAL CONTROL OF THE MOLECULAR MECHANISMS OF THE ONSET OF EMBRYOGENESIS

Early embryogenesis is a remarkable example of how a stem cell arises from terminally differentiated cell. The gametes are non-dividing haploid cells that upon fertilization initiate the functional conversion of their genomes into a single embryonic one. Particularly, the zygote has to resume the mitotic cell cycle, to remodel the parental chromatin, to activate transcription, and to initiate the embryonic developmental program, which requires acquisition of totipotency of the early blastomeres (1, 2). Evidence gathered in several organisms demonstrates that translational activation of dormant maternal mRNAs is crucial to set in motion the molecular mechanisms of development. To investigate such mechanisms, our laboratory has focused its attention on two aspects: a) the identification of maternal mRNAs recruited for translation at the onset of embryogenesis; b) the role played by the RNA-binding protein Sam68 in this process. a) The oocyte is transcriptionally inactive throughout the meiotic divisions and the first embryonic cell cycle (2). In order to identify the maternal mRNAs involved in early embryogenesis we isolated the polysomes from two different developmental stages: the ovulated oocytes arrested in metaphase II (MII oocytes) and zygotes at the pronuclear stage (PN embryos). Purified mRNas from the polysomal fractions were analysed by microarrays. Comparative analyses were performed using four approaches: Ingenuity Pathway Analysis (IPA) software; EASE database screen, DAVID database screen and manual analysis using the NCBI database. These analyses allowed us to group RNAs in specific functional categories and pointed out some interesting differences among the two stages examined. In MII oocytes the polysomes are enriched with mRNAs that encode for proteins involved in metabolism and ribosome biogenesis, suggesting that the ovulated oocyte is mainly involved in maintaining its energy status awaiting for fertilization. On the other hand, after fertilization the polysomes become enriched with RNAs coding for proteins involved in cell cycle, chromatin remodelling and transcription. We are currently confirming these results by real time PCR on a selected number of genes. Our attention will be focused on some categories which play important roles in early embryogenesis, such as chromatin remodelling. Among these genes we found: enhancer of polycomb homolog 1 and 2 (Epc) (3), Nalp 9b (4) and Metll8. We are currently using RNAi methodology to deplete these genes from early embryos and to determine their impact on epigenetic modifications at the morula and blastocyst stages. b) Sam68 belongs to the STAR family of RNA-binding protein, which play evolutionarily conserved functions in development (5). We have investigated the expression and function of this protein during early embryogenesis. Sam68 localizes in the germinal vesicle of GV oocytes, and after the nuclear breakdown, its levels in the oocyte decrease. Residual Sam68 remains in the cytoplasm during the first meiotic division, until the cytostatic arrest. At fertilization, Sam68 localizes in the cytoplasm until the complete formation of pronuclei. During each mitotic division, Sam68 shuttles from the nucleus to the cytoplasm, and then slowly re-enters the nucleus. To understand the function of Sam68, we knocked-down its expression in vivo by microinjecting a double stranded RNA in zygotes at pronuclear stage. Embryos were cultured up to 120 hours after fertilization, when most of the control embryos had reached the blastocyst stage. Depletion of Sam68 in fertilized embryos strongly affected pre-implantation development, lowering the rate of blastocyst formation (30% versus 65% in GFP dsRNA controls) and generating profound alterations of the trophoblast layer (manuscript in preparation). Modifications in gene expression in these embryos are currently being investigated.