| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2000: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1999: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1998: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
The estrogen receptor (ER) is a ligand-dependent transcription factor. ER had been assumed to exist as a single species, until a novel ER (ER beta) was recently isolated. The purpose of this study is to understand the differencetial functions of these molecules. Firstly, we investigated the localization of ER alpha and ER beta in the reproductive organs in the rat. Polyclonal antibodies were raised to each specific amino acid sequence. The Western blot with anti-ER alpha antibody showed a 66 kDa band in rat ovary and uterus, while that with anti-ER beta antibody detected a 55 kDa band in rat ovary, uterus and prostate. The ligand-independent nuclear localization of the two receptors was verified by immunocytochemistry. By immunohistochemistry, the nuclei of glandular and luminal epithelial cells in the uterus were stained with anti-ER alpha antibody, whereas only the nuclei of glandular epithelium cells were stained with anti-ER beta antibody. In rat ovary, positive signals were shown
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with anti-ER beta antibody in the nuclei of granulose cells. No specific immunostaining was observed with anti-ER alpha antibody. Although ER beta was immunostained at the proestrous, metestrous and diestrous stages, the immunoreactivity of ER beta was hardly detected at the estrous stage in rat ovary. Thus, we show differential expression of ER alpha and ER beta in rat uterus and ovary at the protein level, which may provide a clue for understanding the roles of the two receptors in reproductive organs. Secondly, to understand possible roles of estrogen during early embryogenesis, we examined the expression of both ER alpha and ER beta in mouse oocytes and preimplantation embryos by means of reverse transcription polymerase chain reaction. ER alpha mRNA was detected in whole ovaries, cumulus-oocyte complexes, denuded oocytes, 2-cell and 4-cell embryos, whereas it was undetected in 8-cell embryos. Interestingly it reappeared in morulae and blastocysts. ER beta mRNA was detected similarly to ER alpha except for the absence of ER beta mRNA in morulae. The stage specific expression of ER alpha and ER beta along with detection of the product of the estrogen responsive gene in early preimplantation embryos may indicate the possible physiological roles of estrogen in early embryogenesis. Less
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