Preimplantation development is characterized by cell proliferation, differentiation, and differential gene expression. Although these processes can be regulated in somatic cells by signal transduction mechanisms that involve the production of second messengers and activation of protein kinases, remarkably little is known regarding the role of signal transduction during preimplantation development. Protein phosphorylation catalyzed the cAMP-dependent protein kinase (PKA) is apparently involved in zygotic gene activation.
The first aim of this proposal, which will over-express exogenous cyclic nucleotide phosphodiesterase by microinjection of the enzyme, will determine if lowering endogenous cAMP levels in mouse embryos inhibits zygotic gene activation. Although inhibitors of PKA inhibit transcription mediated by RNA polymerase II, the second part of this aim will determine if these inhibitors also inhibit transcription mediated by RNA polymerases I and III.
The second aim of this proposal will use immunoprecipitation, immunofluorescence, and reverse transcription-PCR to determine the temporal pattern of expression of three transcription factors (CREB, AP-1, and SRF) that are regulated by phosphorylation and that can potentially regulate genes involved in preimplantation development. A functional analysis of these transcription factors will be conducted by culturing embryos in medium containing antisense oligonucleotides to these transcription factors, as well as microinjection of inhibitory antibodies to these transcription factors, and assessing the effect of these treatments on development in vitro. The first part of the third aim of this proposal will examine the if in addition to TGF-alpha, which stimulates the rate of blastocoel expansion, other growth factors whose receptor possesses a tyrosine kinase activity can also stimulate this response. The second part of this aim will be to use an antisense RNA approach, as well as the expression of truncated forms of the EGF receptor, which will generate a transdominant mutant phenotype, to examine the function of this receptor in preimplantation development. The last part of this aim will use reverse transcription-PCR to examine growth factor-regulated expression of blastocyst genes that are likely to be involved in blastocoel expansion and implantation. Results of these experiments should provide additional insights regarding the role of protein phosphorylation in zygotic gene activation and growth factors in preimplantation development.
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