GENE EXPRESSION OF STEROID HORMONE RECEPTORS IN BRAIN AND PERIPHERAL TISSUES RELATIVE TO REPRODUCTION

In order to examine sex steroid hormone receptor expression in the central and peripheral tissues relative to reproduction, we have analysed estrogen (ER) and progestin receptors (PR) and their mRNAs in rat brain, and human uterine and other tissues, using immunocytochemical assay (ICA), reverse transcrip-tion-polymerase chain reaction (RT-PCR) and in situ hybridization technique (ISH) Summary and conclusion are as follows; 1) ICA studies confirmed nuclear localization of ER or PR in human uterus and ovarian endometriosis. Good correlation exists between the ER-ICA and ligand binding assay (LH20 assay) or ER-enzyme immunoassay (EIA), indicating the usefulness of the ER-ICA for the semiquantitative measurement in humanuterine tissues. The ER score of ovarian endometriotic tissue was much less than that of the normal endometrium, suggesting much lower hormonal responsiveness in the pathologic tissue. 2) ERmRNA analysis: (1) Northern blot analysis using rat ERcRNA probe demonstrated 6.6 kb ERmRNA in rat brain (the hypothalamus-preoptic area, HPOA), anterior hypophysis (AP), uterus, tube, ovary, and testis. Additional subspecies of 4.2, 2.6 and 2.0 kb were identified in the testis. (2) A very sensitive and specific RT-PCR assay for ERmRNA was developed to analyse the receptor ex-pression in more detail. The RT-PCR product 287 bp was generated from tissue RNA using the primer set derived from the rat ERcDNA sequence and its authenticity was confirmed by direct sequencing. Quan-tification of the RT-PCR assay was made possible by measurement of the hybridization signals in a Bio-Im-age Analysing System, BAS-2000. A standard curve was obtained from graded dilutions of the uterine RT-PCR products. The levels of ERmRNA were as follows; AP>HPOA, amygdala (AMY) >cerebral cortex (CC), cerebellum (Ce). The existence of a small amount of ERmRNA in the “non-target” CC and Ce implied adirect action of estrogen on these brain regions. 3) PRmRNA analysis: In analogous experiments as ERmRNA, a very sensitive and specific RT-PCR assay for PRmRNA was also developed to detect and quantify PRcDNA in the tissues. Since rat PRcDNA has been not cloned and sequenced, rat PRmRNA was synthesized with the primer set derived from the human PRcDNA sequence. The RT-PCR product of 320 bp was generated from tissue RNA, and the authenticity of the rat uterine product was confirmed by direct sequencing. The levels of PRmRNA in adult rat brain were as follows; AP, HPOA, AMY, CC>Ce, with smaller