Retinoic acid (RA) exhibits diverse functions in cell differentiation, proliferation, and pattern formation. The actions of RA are mediated through retinoic acid receptors (RARs) which are composed of two gene families (RAR and RXR). The presence of multiple receptor types for one ligand indicates that each kind of receptor has its own biological function. The broad, long-term objective of this project is to understand the roles of RA and RARs in early pregnancy.
The specific aims of this proposal are: 1) to examine the cullular distribution of RARalpha, beta, and gamma in preimplantation mouse embryos, 2) to study the effect of RA on preimplantation embryos, 3) to study the effect of RA on RAR gene expression in preimplantation embryos, 4) to analyze the function of RARalpha, beta, and gamma in F9 mouse teratocarcinoma cells and embryos. To achieve these, the stage specificity and cellular specificity of RAR gene expression will be examined in preimplantation embryos using immunohistochemistry. Different amounts of RA will be added to growing two-cell embryos, in culture media, and their beneficial and detrimental effects will be assessed. The RA-regulated RAR gene expression, a presumed mechanism. by which RA controls its own effects, will be examined in embryos using reverse transcriptase-competitive polymerase chain reaction. Therefore, the effects of RA on its own receptor gene expression during embryonic development can be determined. Furthermore, the function of each RAR type in both F9 mouse teratocarcinoma cells and preimplantation embryos will be examined. Antisense DNA specific for RARalpha, beta, or gamma will be introduced into F9 cells to block the expression of RAR. The successful blockage of each receptor type will be confirmed by degradation of RNA using Northern blot hybridization, reverse transcriptase-polymerase chain reaction, or by absence of the receptor protein using immunohistochemistry. After a specific RAR expression is blocked, its impact on F9 cell differentiation will be examined. Similarly, after an antisense oligonucleotide specific to each RAR type is introduced, into early embryos, its effect on embryogenesis will be determined. These approaches will allow us to reveal which RAR type is essential for specific embryonic development and cell differentiation. Since RA exerts diverse effects in embryos, the outcomes from our proposed studies should be able to reveal the biochemical/molecular mechanisms by which RA and RARs regulate embryogenesis, cell differentiation and gene expression.
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