Gene expression of steroid hormone receptors in brain and peripheral tissues relative to reproduction.

Abstract

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 differential distribution than that of ERmRNA, and roughly paralled the levels of PR with exception of the CC where the PR level is low. The expression mechanism of PR in the CC may be different from that in the other brain regions.The early postnatal development of PRmRNA was found. The increment of the cerebral PRmRNA might be associated with the early drastic increase of PR in the neonatal rat brain cortex.4) ER-and PRmRNAs analysis in human uterus:(1) Primary structure of the uterine ERmRNA, amplified by the RT-PCR and determined by direct sequencing of the product, showed that ERmRNA expressed in the normal human endometrium has Gly-400 (GGG) and Thr-594 (ACA).(2) Northern blot analysis of ER-and PRmRNAs using riboprobes demonstrated greater message levels in the endometrium than the myometrium, respectively. The results were in agreement with estrogendependent induction of PR.5) In situ hybridization studies.We have detected ER- and PRmRNAs and mapped out the distribution of the meassages in the intact female rat brain using rat ER- and PRcRNA synthetized. The distribution of both mRNAs was similar, with higher density in the arcuate nucleus and the ventrolateral part of the ventromedial nucleus of hypothalamus, indicating a possible coexpression in the same neurons.