ESTROGEN REGULATION OF THE RAT LH BETA GENE

促黄体生成素（LH）在性腺发育中起关键作用， 配子发生和性类固醇如雌二醇的产生 (E2)因此，其生理调节对于正常的 生育 LH由所有垂体共同的α亚单位组成 糖蛋白激素，和一个独特的LHBeta亚基，它是由 E2在体内以阳性和阴性方式存在。 我们已经证明 大鼠LH-β基因可以通过雌激素被E2直接刺激 反应元件（ERE）的程度，这是调节发情周期 下丘脑促性腺激素释放激素（GnRH）。 我们有 最近发现并克隆了一种较小的大鼠雌激素受体 (ER)mRNA，仅在垂体中且仅在雌性中检测到。 垂体特异性ER mRNA具有N端截短的编码区， 序列，和一个独特的5素数序列，并显着刺激 在E2。 具有适当翻译产物大小的ER蛋白是 存在于女性垂体中。 我们还分离出了其他的cDNA 编码ER mRNA剪接变体和同种型， 显着的特异性，并含有DNA结合域。 我们的目标 是为了确定生物活性和生理调节， 垂体特异性ER 克隆的cDNA的特征在于： 通过引物延伸分析的测序和mRNA，以及组织 表情 垂体特异性和变体ER mRNA和蛋白水平 将在整个发情周期中定量，并在给药后 雌激素和孕酮的雌性大鼠。 野生型和 变体ER蛋白表达将通过Western印迹法测定， 垂体提取物，然后用-结构域特异性检测蛋白质 ER抗体，或通过免疫沉淀。变异ER mRNA转录本 将通过原位杂交定位于特定的垂体细胞类型 杂交方法 将测试变体ER蛋白结合 E2和DNA，包括卵黄蛋白原、LHBeta和催乳素的ERE 基因，并与野生型ER形成同源二聚体或异源二聚体。 的 变异ER单独刺激基因活性或影响 野生型ER这样做能力将在瞬时 Cos或293细胞中的表达测定， 载体的变体和野生型ER以几种比例。 分泌和 将在共转染研究中测量基因活化应答， GH 3细胞。 这些实验将确定变体ER表达是否 可能影响组织对E2的反应，如果这发生在启动子中， 具体方式。ER蛋白量的变化-不同的 生物活动或特异性可能具有深刻的生理学意义， 影响，从而促进了许多生物学变化 在生殖周期中，如启动和限制 发情前期的高潮

Lutropin (LH) plays a critical role in gonadal development and gametogenesis, and the production of the sex steroids such as estradiol (E2); its physiological regulation is thus important for normal fertility. LH consists of the alpha-subunit common to all pituitary glycoprotein hormones, and a unique LHBeta subunit which is regulated by E2 in both a positive and negative manner in vivo. We have shown that the rat LH-beta gene can be directly stimulated by E2 via an estrogen response element (ERE) to a degree which is modulated by estrous cycle stage of pitUitary tissue, and by the hypothalamiC peptide GnRH. We have recently identified and cloned a smaller form of rat estrogen receptor (ER) mRNA which is detected only in the pituitary and only in females. The pituitary-specific ER mRNA has a N-terminal truncated coding sequence, and a unique 5 prime sequence, and is dramatically stimulated by E2. An ER protein of the appropriate translation product size is present in female pituitaries. We have also isolated additional cDNAs encoding ER mRNA splice variants and isoforms which do not exhibit such dramatic specificity and which contain the DNA binding domain. Our goal is to determine the biological activity and physiological regulation of the pituitary-specific ERs. Cloned cDNAs will be characterized by sequencing and mRNA by primer extension analysis, and for tissue expression. Levels of pituitary-specific and variant ER mRNA and protein will be quantitated throughout the estrous cycle, and after treatment of female rats with progesterone and estrogen. The level of wild type and variant ER protein expression will be determined by Western blotting of pituitary extracts followed by detection of protein with -domain-specific ER antibodies, or by immunoprecipitation. Variant ER mRNA transcripts will be localized to specific pituitary cell types by in situ hybridization. Variant ER protein will be tested for its ability to bind E2 and DNA, including EREs for the vitellogenin, LHBeta and prolactin genes, and to form homodimers or heterodimers with the wild type ER. The ability of variant ER to stimulate gene activity alone, or to influence the ability of wild type ER to do so will be evaluated in transient expression assays in Cos or 293 cells cotransfected with expression vectors for variant and wild type ER at several ratios. Secretion and gene activation responses will be measured in cotransfection studies in GH3 cells. These experiments will determine if variant ER expression could influence tissue responses to E2 and if this occurs in a promoter- specific manner. Variations in amounts of ER proteins-with different biological activities or specificities could have profound physiological implications, and thus contribute to the numerous biological changes during the reproductive cycle, such as the initiation and limitation of the proestrus surge.