PubMed · 3003517
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J N Ihle, Y Weinstein, J Keller, L Henderson, E Palaszynski. 1985. Interleukin 3.. https://doi.org/10.1016/s0076-6879(85)16042-8
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BACKGROUND: 20alpha-Hydroxysteroid dehydrogenase (HSD) is a member of the aldo-keto reductase (AKR) superfamily and catalyses the reaction of progesterone to the inactive form 20alpha-hydroxyprogesterone. Progesterone plays an important role in the maintenance of pregnancy, and, in rodents, plasma progesterone levels decrease abruptly just before parturition. The induction of 20alpha-HSD is thought to be responsible for the decrease in plasma progesterone at term. High homology between human 20alpha-HSD [AKR 1C1] cDNA with other AKRs had caused difficulty in gene isolation and expression analysis. Thus, the metabolism of progesterone in the human reproductive system remained unclear. RESULTS: By hybridization with rat 20alpha-HSD [AKR 1C8] cDNA and high-stringency polymerase chain reaction (PCR) with gene-specific primers, we were able to isolate the human 20alpha-HSD, bile acid-binding protein (BABP) [AKR 1C2], prostaglandin F synthase (PGFS) [AKR 1C3], and dihydrodiol dehydrogenase (DD) 4 [AKR 1C4] genes. These genes had similar exon-intron organizations and shared a high homology. The four recombinant enzymes encoded by these genes showed distinct substrate specificity. By reverse transcription-PCR analysis, human 20alpha-HSD, BABP and PGFS mRNAs were expressed ubiquitously, while DD4 mRNA was restricted to the liver. Promoter activities of the 20alpha-HSD, BABP and PGFS genes were high, both in ovarian granulosa cells and hepatocytes. Radiation hybridization analysis revealed that all these genes were located close together in chromosome 10. CONCLUSION: The human gene encoding for the progesterone-metabolizing enzyme 20alpha-HSD in the female reproductive system was cloned, and its expression and gene localization were elucidated. BABP, PGFS and DD4 genes, which were highly homologous to the 20alpha-HSD gene, were also cloned, and their structure and function were characterized.
Hydroxysteroid dehydrogenases (HSDs) regulate the occupancy and activation of steroid hormone receptors by converting potent steroid hormones into their cognate inactive metabolites. 3alpha-HSD catalyzes the inactivation of androgens in the prostate by converting 5alpha-dihydrotestosterone to 3alpha-androstanediol, where excess 5alpha-dihydrotestosterone is implicated in prostate disease. By contrast, 20alpha-HSD catalyzes the inactivation of progestins in the ovary and placenta by converting progesterone to 20alpha-hydroxyprogesterone, where progesterone is essential for maintaining pregnancy. Mammalian 3alpha-HSDs and 20alpha-HSDs belong to the aldo-keto reductase superfamily and share 67% amino acid sequence identity yet show positional and stereospecificity for the formation of secondary alcohols on opposite ends of steroid hormone substrates. The crystal structure of 3alpha-HSD indicates that the mature steroid binding pocket consists of 10 residues located on five loops, including loop A and the mobile loops B and C. 3alpha-HSD was converted to 20alpha-HSD by replacing these loops with those found in 20alpha-HSD. However, when pocket residues in 3alpha-HSD were mutated to those found in 20alpha-HSD altered specificity was not achieved. Replacement of loop A created a 17beta-HSD activity that was absent in either 3alpha- or 20alpha-HSD. Once loops A and C were replaced, the chimera had both 3alpha- and 20alpha-HSD activity. When loops A, B, and C were substituted, 3alpha-HSD was converted to a stereospecific 20alpha-HSD with a resultant shift in k(cat)/K(m) for the desired reaction of 2 x 10(11). This study represents an example where sex hormone specificity can be changed at the enzyme level.
The present studies were conducted to evaluate the effects of DL111-IT [3-(2-ethyl phenyl)-5-(3-methoxy phenyl)-1H-1,2,4 triazol] on ovaries of pregnant rats. Pregnant rats were i.m. treated with DL111-IT 2.5 mg kg(-1) day(-1) or camellia oleum (vehicle control) 0.2 ml/day from day 6 of pregnancy for 1, 3 or 5 days. Blood and ovaries were collected 24 h after the last injection. Ovarian fresh weight and protein contents, activities of the 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) in ovaries, and cell apoptosis of corpus luteum (including hematoxylin-eosine stain, in situ 3'-end labeling and nucleosomal banding) were estimated. Compared with that in the control group, ovarian fresh weight declined 11% and 22% after DL111-IT-3 days and -5 days; protein content dropped 29% after 5-day administration. DL111-IT for 3 days provoked a marked decrease of serum progesterone, by 31% of the control; the activity of 3beta-HSD decreased 34.4% after i.m. DL111-IT for 5 days, while that of 20alpha-HSD increased dramatically after only one injection of DL111-IT (P < 0.01). Histological analysis and in situ 3'-end DNA labeling indicated that DL111-IT induced the pyknosis of cells and the formations of apoptotic bodies and intense oligonucleosomes in luteal cells of pregnant rats. The cell apoptosis induced by DL111-IT was further confirmed by evaluation of nucleosomal DNA fragmentation by agarose gel electrophoresis in cultured luteal cells exposed to DL111-IT for 24 h. In conclusion, all results, including shrunken luteal cells, decreased concentration of protein content and serum progesterone, changed activities of 3beta-HSD and 20alpha-HSD and formation of DNA fragments in luteal cells, showed the luteolytic effect of DL111-IT in pregnant rats.