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.
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