GENETIC DETERMINANTS OF STEROID HORMONE BIOSYNTHESIS

这个项目的长期目标是确定的影响

The long term objective of this project is to determine the influence of genetic control on steroidogenesis. The aims of this application are to study the gene(s) that encode the enzyme, delta5-3beta-hydroxysteroid dehydrogenase delta5->delta4 isomerase (3betaHSD). This enzyme converts the delta5-3beta-hydroxysteroid pregnenolone to progesterone and dehydrodroepiandrosterone to androstenedione. The activity of this enzyme is essential for adrenal and gonadal steroid hormone production. Preliminary data indicate that the mouse liver also expresses this enzyme and has a high capacity for the conversion of delta5-3beta-hydroxysteroids to delta4-3-ketosteroids. An antibody generated against a purified human placental 3betaHSD protein recognizes a single protein in mouse adrenal glands and testes that is identical in molecular weight to the human placental protein. The same antibody recognizes a single protein of different molecular weight in the mouse liver. These data suggest that the liver enzyme may be distinct from the adrenal and testicular enzyme. Additional evidence indicates that the mouse genome contains more than one 3betaHSD gene. It is not known whether more than one gene is expressed. The specific aims of this proposal are designed to: 1. determine whether there are two or more genes which encode distinct 3betaHSD enzymes; 2. isolate the mouse genes that encode the 3betaHSD enzyme(s) and characterize their structure; 3. examine the regulation of 3betaHSD mRNA in mouse testes, adrenal glands and liver. A full length mouse Leydig cell 3betaHSD cDNA has been isolated and characterized. To determine whether adrenal glands and testes express a 3betaHSD gene distinct from the one expressed in liver, RNAse protection assays will be performed using radioactive riboprobes and RNA from the different tissues. Protection of different size fragments by RNA from liver than from adrenal glands and testes will provide evidence for the expression of a different gene in liver. The Leydig cell 3betaHSD cDNA will be used as a probe to isolate a 3betaHSD cDNA from a mouse liver cDNA library. Positive clones from the mouse liver library will be expressed in COS-1 cells. These experiments will determine the differences in the cDNA's and the functional significance of these differences. To determine the total number of 3betaHSD structural genes and pseudogenes, genomic clones will be isolated from an EMBL3 library and characterized. The expression of 3betaHSD mRNA during embryogenesis and pubertal development will be determined in livers, kidneys, adrenal glands and gonads of both male and female mice using a reverse transcriptase- polymerase chain reaction micromethod. To gain insight into the in vivo regulation of hepatic 3betaHSD mRNA by glucocorticoids and by testosterone and to determine whether circulating testosterone modulates adrenal expression of 3betaHSD mRNA, the effect of castration and adrenalectomy and steroid replacement therapy will be studied on the expression of hepatic 3betaHDS mRNA and on adrenal and testicular mRNA, respectively. Regulation of 3betaHSD mRNA expression will be studied in primary cultures of mouse Leydig cells. The identification of a 3betaHSD gene expressed in liver which is distinct from the 3betaHSD gene expressed in adrenal glands and testes could change our thinking about the biosynthesis of steroid hormones in general and may provide an explanation for the clinical manifestations observed in patients with 3betaHSD enzyme deficiency.