Analysis of differential functions of estrogen receptor subtypes in uterine endometrium and embryo

雌激素受体（ER）是一种依赖配体的转录因子。一直以来，人们都认为内质网是一种单一物种，直到最近才分离出一种新的内质网（ER β）。本研究的目的是了解这些分子的不同功能。首先，我们研究了内质网α和内质网β在大鼠生殖器官中的定位。对每个特定氨基酸序列产生多克隆抗体。抗内质网α抗体Western blot在大鼠卵巢和子宫中检测到66 kDa条带，而抗内质网β抗体Western blot在大鼠卵巢、子宫和前列腺中检测到55 kDa条带。免疫细胞化学证实了这两种受体的核定位与配体无关。免疫组化方法对子宫腺上皮细胞和腔上皮细胞的细胞核进行抗er α抗体染色，而对子宫腺上皮细胞的细胞核进行抗er β抗体染色。在大鼠卵巢中，颗粒细胞核内可见抗er β抗体阳性信号。抗er α抗体未见特异性免疫染色。大鼠卵巢在发情期、孕期和死期均可进行ER β免疫染色，但在发情期几乎检测不到ER β的免疫反应性。因此，我们在蛋白水平上发现了ER α和ER β在大鼠子宫和卵巢中的差异表达，这可能为理解这两种受体在生殖器官中的作用提供线索。其次，为了了解雌激素在早期胚胎发生中的可能作用，我们通过逆转录聚合酶链反应检测了小鼠卵母细胞和着床前胚胎中ER α和ER β的表达。ER α mRNA在全卵巢、卵丘-卵母细胞复合物、去皮卵母细胞、2细胞和4细胞胚胎中均检测到，而在8细胞胚胎中未检测到。有趣的是，它再次出现在桑葚胚和囊胚中。除了桑葚胚中没有ER β mRNA外，ER β mRNA的检测与ER α相似。在着床前早期胚胎中，ER α和ER β的阶段特异性表达以及雌激素应答基因产物的检测可能提示雌激素在早期胚胎发生中的可能生理作用。少

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 … More 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