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.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Research Project (R01)
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Human Embryology and Development Subcommittee 1 (HED)
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University of California Los Angeles
Schools of Medicine
Los Angeles
United States
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Wan, Y J; Wang, L; Wu, T C (1996) Murine endodermal F9E cells, derived from the teratocarcinoma line F9, contain high basal levels of retinoic acid receptors (RARs and RXRs) but are not sensitive to the actions of retinoic acid. Differentiation 60:211-8
Wan, Y J; Pan, T; Wang, L et al. (1995) 9-cis-retinoic acid is more effective than all-trans-retinoic acid in upregulating expression of the alpha-fetoprotein gene. J Mol Endocrinol 14:101-8
Wan, Y J; Wang, L; Wu, T C (1995) Different response to retinoic acid of two teratocarcinoma cell lines. Exp Cell Res 219:392-8
Jih, M H; Wu, T C (1995) Altered regulation of pituitary luteinizing hormone secretion by GnRH and inhibin in the aged persistent-estrous female rat. Mech Ageing Dev 84:15-27
Wan, Y J; Wang, L; Wu, T C (1995) Retinoic acid induces activin receptor IIB mRNA in F9 embryonal carcinoma cells. J Mol Endocrinol 14:247-54
Wan, Y J; Wang, L; Wu, T C (1994) The expression of retinoid X receptor genes is regulated by all-trans- and 9-cis-retinoic acid in F9 teratocarcinoma cells. Exp Cell Res 210:56-61
Wu, T C; Jih, M H; Wang, L et al. (1994) Expression of activin receptor II and IIB mRNA isoforms in mouse reproductive organs and oocytes. Mol Reprod Dev 38:9-15
Wan, Y J; Wang, L; Wu, T C (1994) Dexamethasone increases the expression of retinoid X receptor genes in rat hepatoma cell lines. Lab Invest 70:547-52
Wu, T C; Wang, L; Wan, Y J (1993) Detection of estrogen receptor messenger ribonucleic acid in human oocytes and cumulus-oocyte complexes using reverse transcriptase-polymerase chain reaction. Fertil Steril 59:54-9
Wu, T C; Lee, S M; Jih, M H et al. (1993) Differential distribution of glycoconjugates in human reproductive tract. Fertil Steril 59:60-4

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