Isolation of a new mouse 3beta-hydroxysteroid dehydrogenase isoform, 3beta-HSD VI, expressed during early pregnancy.

The enzyme 3beta-hydroxysteroid dehydrogenase (3beta-HSD) is a key enzyme in the biosynthesis of steroid hormones. To date, this laboratory has isolated and characterized five distinct 3beta-HSD complementary DNAs (cDNAs) in the mouse (3beta-HSD I through V). These different forms are expressed in a tissue- and developmentally-specific manner and fall into two functionally distinct enzymes. 3beta-HSD I and III, and most likely II, function as dehydrogenase/isomerases, whereas 3beta-HSD IV and V function as 3-ketosteroid reductases. This study describes the isolation, characterization, and tissue-specific expression of a sixth member of this gene family, 3beta-HSD VI. This new isoform functions as an NAD+-dependent dehydrogenase/isomerase exhibiting very low Michaelis-Menten constant (Km) values for pregnenolone (approximately 0.035 microM) and dehydroepiandrosterone (approximately 0.12 microM). 3beta-HSD VI is the earliest isoform to be expressed during embryogenesis in cells of embryonic origin at 7 and 9.5 days postcoitum (pc), and is the major isoform expressed in uterine tissue at the time of implantation (4.5 days pc) and continues to be expressed in uterine tissue at 6.5, 7.5, and 9.5 days pc. 3beta-HSD VI is expressed in giant trophoblasts at 9.5 days pc and is expressed in the placenta through day 15.5 pc. In the adult mouse, 3beta-HSD VI appears to be the only isoform expressed in the skin and also is expressed in the testis, but to a lesser extent than 3beta-HSD I. Mouse 3beta-HSD VI cDNA is orthologous to human 3beta-HSD I cDNA. Human type I 3beta-HSD has been shown to be the only isoform expressed in the placenta and skin. The demonstration that mouse 3beta-HSD VI functions as a dehydrogenase/isomerase and is the predominant isoform expressed during the first half of pregnancy in uterine tissue and in embryonic cells suggests that this isoform may be involved in local production of progesterone, which is needed for successful implantation of the blastocyst and/or maintenance of early pregnancy.

A novel mouse kidney 3 beta-hydroxysteroid dehydrogenase complementary DNA encodes a 3-ketosteroid reductase instead of a 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase.

The enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta HSD) is essential for the biosynthesis of all steroid hormones. The enzyme catalyzes the conversion of delta 5-3 beta-hydroxysteroids to delta 4-3-ketosteroids. We report the isolation of a novel mouse 3 beta HSD cDNA, 3 beta HSD IV, and describe the tissue-specific expression of its mRNA, the enzyme characteristics of the 3 beta HSD IV protein, and the structural and functional relationships it has to other 3 beta HSD isoforms previously characterized in the mouse and rat. The predicted amino acid sequence of mouse 3 beta HSD IV is 77% and 73% identical to that of mouse 3 beta HSD I and III, respectively. Comparison of the nucleotide and amino acid sequences of the four isoforms characterized to date show that 3 beta HSD IV is more distantly related to I, II, and III than these three forms are to each other. 3 beta HSD IV mRNA is only expressed in mouse kidney. In situ hybridization of mouse kidney indicates that expression is found only in the cortex and appears to be associated with the proximal tubules. Transiently expressed 3 beta HSD IV protein could not convert the delta 5-3 beta-hydroxysteroids, pregnenolone or dehydroepiandrosterone, to their respective delta 4-3-ketosteroids, progesterone or androstenedione, nor did it have the capacity to convert 5 alpha-androstane-3 beta, 17 beta-diol to dihydrotestosterone, characteristic enzymatic activities of expressed mouse 3 beta HSD I and III. 3 beta HSD IV could only catalyze the conversion of dihydrotestosterone to 5 alpha-androstanediol in the presence of the cofactor NADPH. Thus, 3 beta HSD IV is a 3-ketosteroid reductase rather than a 3 beta-hydroxysteroid dehydrogenase/isomerase despite its homology to the other members of the 3 beta HSD family. Mouse 3 beta HSD IV is functionally and structurally most closely related to rat 3 beta HSD III, an isoform expressed predominantly in male rat liver.

Enzyme characteristics of two distinct forms of mouse 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase complementary deoxyribonucleic acids expressed in COS-1 cells.

The enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-Isomerase (3 beta HSD) catalyzes the conversion of delta 5-3 beta-hydroxysteroids to delta 4-3-ketosteroids, an essential step in the biosynthesis of all biologically active steroid hormones. We previously reported the isolation of three distinct mouse cDNAs for 3 beta HSD (3 beta HSD I, II, and III) and tissue-specific expression of their mRNAs. 3 beta HSD I is expressed only in gonads and adrenal glands, and 3 beta HSD II and III are expressed in both liver and kidneys. In the current study, we present data which demonstrate that transiently expressed 3 beta HSD I and 3 beta HSD III proteins can catalyze the conversion of the delta 5-steroids, pregnenolone and dehydroepiandrosterone, to their respective delta 4-3-ketosteroids, progesterone and androstenedione. They also can dehydrogenate the 3 beta-hydroxy group of the 5 alpha-reduced steroid 5 alpha-androstanediol to yield dihydrotestosterone in the presence of the cofactor NAD+. The Km values of the expressed 3 beta HSD I (for each of these substrates) were all below 0.2 microM. Km values of 3 beta HSD III were greater for all substrates, with the greatest increase observed for pregnenolone, which was over 10-fold greater. Both forms of expressed protein can catalyze the reduction of dihydrotestosterone to 5 alpha-androstanediol in the presence of the cofactor NADH, but with considerably higher Km values (5.5 microM for form I and 6.8 microM for form III). The observed maximum velocity of form I was much higher for all substrates examined. RNase protection and immunoblot analysis of expressed 3 beta HSD I and III indicate that the difference in maximum velocity reflect differences in the steady state levels of mRNA and amounts of protein. In addition, the expressed 3 beta HSD III protein analyzed by Western blot has a lower mobility than the 3 beta HSD I protein, both similar in mol wt to the 3 beta HSD proteins detected in mouse liver and adrenal glands, respectively. These data demonstrate that an isoform of 3 beta HSD expressed in liver and kidney has the capacity to convert delta 5-3 beta-hydroxysteroids to delta 4-3-ketosteroids. The data suggest that a homologous human 3 beta HSD isoform could play an important role in cases of genetic deficiency of the gonadal and adrenal isoform.

Hypothalamic, pituitary and testicular function during sexual maturation of the male rat.

Hypothalamic content of gonadotrophin-releasing hormone (GnRH), serum LH and FSH, capacity of the testis to synthesize testosterone in vitro, and testicular 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase and 17 beta-hydroxysteroid dehydrogenase were measured in groups of rats at approximately 5 day intervals from birth to day 64 and at days 74 and 89. The capacity of the testes to synthesize testosterone in vitro was measured in the presence of a saturating dose of rat LH. Gonadotrophin-releasing hormone increased steadily from 0-17 ng per hypothalamus at birth to a maximum of 7 ng at day 52 and then remained constant. LH concentrations were highly variable and often exceeded adult values between days 10 and 32. After day 32 a steady rise was observed which reached adult values between days 37 and 42. FSH concentrations markedly increased from 255 ng/ml observed at birth and day 10 to a peak value of 1000 ng/ml at day 32. Subsequently there was a steady decline in FSH values until day 74 when the concentration returned to values found at birth. 5-ene-3 beta-Hydroxysteroid dehydrogenase-isomerase activity exhibited a rapid increase between days 12 and 19 followed by an even greater rate of increase between days 19 and 32 when adult levels were attained. 17 beta-Hydroxysteroid dehydrogenase activity was very low between birth and day 22. Enzyme activity began to increase at day 22 with a rapid increase in activity observed between days 37 and 58. The increase in capacity to synthesize testosterone closely followed the increase in 17 beta-hydroxysteroid dehydrogenase activity. The study demonstrates that during sexual maturation in the male rat, changes in serum LH and FSH do not reflect changes in hypothalamic GnRH. The appearance of Leydig cells as monitored by 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase activity precedes by approximately 20 days the increase in testicular capacity to synthesize testosterone in vitro. The latter coincides with the increase in 17 beta-hydroxysteroid dehydrogenase activity. These results suggest that 17 beta-hydroxysteroid dehydrogenase is a limiting factor in the ability of the testis to respond to LH stimulation.