In vivo evidence for the production of sulfated steroids in the frog brain

It is well established that sulfated neurosteroids are potent regulators of neuronal activity but the biosynthesis of sulfate esters of steroids in the central nervous system (CNS) has received little attention. In particular, the localization of hydroxysteroid sulfotransferase (HST), the enzyme which is responsible for the formation of sulfated steroids, has never been determined in the brain. We took advantage of the availability of an antiserum raised against rat liver HST to investigate the distribution of this enzyme in the CNS of the frog Rana ridibunda. Two populations of HST-positive neurons were localized in the anterior preoptic area and the magnocellular nucleus of the hypothalamus. Numerous HST-immunoreactive fibers were visualized throughout the telencephalon and the diencephalon. Reversed-phase high performance liquid chromatography (HPLC) analysis of frog telencephalon and hypothalamus extracts combined with radioimmunoasssay (RIA) detection showed the presence of substantial amounts of DHEAS-immunoreactive material which coeluted with synthetic DHEAS. The concentrations of DHEAS detected in the telencephalon and hypothalamus were respectively eight and five times higher than in the serum. The present study demonstrates the occurrence of HST-immunoreactive material in neurons of the frog telencephalon and diencephalon. This report also provides evidence for the presence of HST bioactivity, in vivo, in the frog brain.     

Electrophoretic analysis of DNA-binding proteins during induction of 3α,20β- and 3β,17β-hydroxysteroid dehydrogenase in wild type and mutants of Streptomyces hydrogenans

Abstract Treatment of the wild-type strain HY 0 of Streptomyces hydrogenans with estradiol, a specific inducer of 3β,17β-hydroxysteroid dehydrogenase (17β-HSD) formation, caused several soluble proteins to bind to DNA-cellulose with altered affinity. Hydrocortisone which induces biosynthesis of 3α,20β-hydroxysteroid dehydrogenase (20β-HSD), and progesterone which induces production of both 17β- and 20β-HSD, had no effect on DNA-binding properties of the proteins. In mutants with altered activity/inducibility of 17β- and 20β-HSD only one DNA-binding protein (protein 23) still showed an altered DNA affinity in response to estradiol-treatment and this in only one strain. In other mutants the DNA affinity was not altered during induction with estradiol but the DNA affinity of protein 23 varied between low, low-and-high, and high affinity, depending on the strain. In the mutant where DNA affinity was altered by estradiol treatment the change was opposite to that found in the wild type.     

Immunocytochemical localization of 17β-hydroxysteroid dehydrogenase in porcine testis

Summary The immunocytochemical localization of 17-hydroxysteroid dehydrogenase (17-HSD) in porcine testes was examined by applying an indirect-immunofluorescence method using an antiporcine testicular 17-HSD antibody. Only the Leydig cells located in the interstitial tissue exhibited a positive immunoreaction for 17-HSD: the germ cells and Sertoli cells located in the seminiferous tubules were entirely negative. These results suggest that, in porcine testis, the biosynthesis of testicular testosterone, the final step of which is the conversion of androstenedione to testosterone, takes place in the Leydig cells.Supported by grants from the Ministry of Education, Science, and Culture, Japan     

A study on the 3β- and 17β-hydroxysteroid dehydrogenase activities in the gonads of Monopterus albus (Pisces: Teleostei) at various sexual phases during natural sex reversal

Activities of 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β-HSD) in Monopterus gonads were studied at different sexual phases during natural sex reversal. Before sexual transformation, positive reactions for 3β-HSD in the follicular epithelium were found in the granulosa cells of some large, maturing follicles in some females during the breeding season. Weak reaction for this enzyme was also detected in some scattered interstitial cells found occasionally in some ovaries. At the intersexual and the male phases, intense 3β-HSD activities were demonstrated exclusively in the interstitial Leydig cells. No 17β-HSD activities were observable in the gonads at any stage of development. The reaction intensity of 3β-HSD in the interstitial cells exhibited a marked increase during the process of sex change from female to the intersexual and the male phases and there is a definite correlation with the density and nuclear size of these cells. It is concluded that in Monopterus , the granulosa cells in the ovary and the interstitial cells of the intersexual and male gonads are the major sites for the biosynthesis of oestrogens and androgens, respectively, and that the intensive development of interstitial tissue with increasing steroidogenic enzyme activities at the intersexual and male phases was directly related to the increase in androgen production in vitro reported previously. The occasional presence of some 3β-HSD positive interstitial cells in the ovary suggests that interstitial cell development might precede testicular lobule formation during natural sex reversal.     

Pregnenolone and progesterone metabolism by the testes of Bufo arenarum

Sliced testis tissue from Bufo arenarum was incubated in the presence of [³H]pregnenolone. Testis fragments were also used for double isotope experiments using [³H]pregnenolone and [¹⁴C]progesterone. Specific activities were equated with the addition of radioinert pregnenolone. When yields of radiometabolites were analysed, pregnenolone was found to be a good precursor for C₁₉ steroids such as dehydroepiandrosterone, 5-androsten-3β,17β diol, testosterone, 5α-dihydrotestosterone and a C₂₁ steroid, 5α-pregnan-3,20 dione. Progesterone mainly converts to 5α-pregnan-3,20 dione, a steroid with unknown function in amphibians. The 5-ene pathway, including 5-androsten-3β,17β diol as intermediate, could be predominant for androgen biosynthesis. Testes bypass not only progesterone but also androstenedione for testosterone biosynthesis. Accepted: 17 April 1998     

Rapid estrogen regulation of DHEA metabolism in the male and female songbird brain

In the songbird brain, dehydroepiandrosterone (DHEA) is metabolized to the active and aromatizable androgen androstenedione (AE) by 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD). Thus, brain 3β-HSD plays a key role in regulating the steroidal milieu of the nervous system. Previous studies have shown that stress rapidly regulates brain 3β-HSD activity in a sex-specific manner. To elucidate endocrine regulation of brain 3β-HSD, we asked whether 17β-estradiol (E₂) regulates DHEA metabolism in adult zebra finch ( Taeniopygia guttata ) and whether there are sex-specific effects. Brain tissue was homogenized and centrifuged to obtain supernatant lacking whole cells and cell nuclei. Supernatant was incubated with [³H]DHEA and radioinert E₂ in vitro . Within only 10 min, E₂ significantly reduced 3β-HSD activity in both male and female brain. Interestingly, the rapid effects of E₂ were more pronounced in females than males. These are the first data to show a rapid effect of estrogens on the songbird brain and suggest that rapid estrogen effects differ between male and female brains.     

Gonadal morphology, enzyme histochemistry and plasma steroid levels during the annual reproductive cycle of male and female brown bullhead catfish, Ictalurus nebulosus Lesueur

The annual reproductive cycle of the brown bullhead catfish, Ictalurus nebulosus Lesueur, was investigated over a two-year period. In females, GSI increased in the spring as follicles enlarged and the granulosa became hypertrophied, dropped during spawning in August, then rose in the autumn as follicles enlarged slightly. 3β-Hydroxysteroid dehydrogenase (3β-HSD) activity was limited to thecal nests of large, vitellogenic follicles. Plasma testosterone and estradiol-17β levels increased in parallel with GSI. Levels of both steroids dropped prior to the spawning period, although a peak in estradiol-17β was evident during the spawning period. No 11-ketotestosterone was detected in female plasma. In males, GSI increased in the spring as spermatogenesis proceeded, and dropped during spawning. 3β-HSD activity was confined to Leydig cells and was most intense prior to spawning. Plasma testosterone and 11-ketotestosterone peaked during the pre-spawning period, dropped prior to spawning, then rose slowly during the autumn. A peak in estradiol-17β occurred during the spawning period. Significant differences in GSI and plasma steroid levels during the pre-spawning and spawning periods were observed between the two yearly cycles; they may be related to differences in rainfall during these periods.     

Production of monoclonal antibodies against 3α,20β-hydroxysteroid dehydrogenase from Streptomyces hydrogenans

Abstract A procedure is described for the production of monoclonal antibodies (mAbs) against 3α,20β-hydroxysteroid dehydrogenase (3α,20β-HSD) from the actinomycete Streptomyces hydrogenans ATCC 19631. Clones which were obtained after fusion of immune cells were screened by solid-phase ELISA and immunoblotting. About 5.2% of the clones secreted immunoglobulins with specificity for 3α,20β-HSD. The purified mAbs were found to belong to subclass IgG₁ and to recognize both the native enzyme as well as its identical subunits which were obtained by SDS denaturation. However, the activity of the tetrameric holoenzyme was only weakly diminished in the presence of these mAbs.     

Shift in Steroidogenesis in the Ovarian Follicles of the Goldfish (Carassius auratus) during Gonadotropin-Induced Oocyte Maturation

Both partially purified chum salmon gonadotropin and 17α-hydroxyprogesterone stimulated in vitro production of testosterone by postvitellogenic follicles of goldfish ( Carassius auratus ). Chum salmon gonadotropin further enhanced the conversion of exogenously supplied 17α-hydroxyprogesterone to 17α, 20β-dihydroxy-4-pregnen-3-one. The increased medium concentrations of 17α, 20β-dihydroxy-4-pregnen-3-one were associated with the induction of final oocyte maturation.
The capacity of postvitellogenic follicles to produce steroids in response to exogenous 17α-hydroxyprogesterone was examined in females at various stages of final oocyte maturation following the administration of human chorionic gonadotropin in vivo combined with elevation of holding temperature. The maximum production of testosterone in response to 17α-hydroxyprogesterone was obtained in follicles from initial controls. In contrast, 17α 20β-diOHprog production was very low in initial controls and markedly increased during oocyte maturation (3–6 hr following injection), followed by a significant decrease in follicles collected at 15 hr. Estradiol-17β production by the follicles was very low at any stages of gonadotropin-induced oocyte maturation. These results suggest that gonadotropin-induced shift in the biosynthetic pathway in the follicle from the secretion of predominantly testosterone to 17α, 20β-dihydroxy-4-pregnen-3-one secretion is a prerequisite step for the induction of oocyte maturation in goldfish.     

Expression of Cytochrome P450 Side-Chain Cleavage Enzyme and 3β-Hydroxysteroid Dehydrogenase in the Rat Central Nervous System: A Study by Polymerase Chain Reaction and In Situ Hybridization

Abstract: In examining steroid synthesis in the CNS, expression of the mRNAs encoding for cytochrome P450 side-chain cleavage enzyme (P450_SCC) and 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴ isomerase (3β-HSD) has been studied in the rat brain. P450_SCC transforms cholesterol into pregnenolone and 3β-HSD transforms pregnenolone into progesterone. PCR was used to amplify cDNA sequences from total RNA extracts. Classical steroidogenic tissues, like adrenal and testis, as well as the non-steroidogenic tissue lung have been used as controls. The expression of P450_SCC and 3β-HSD have been demonstrated by PCR in cortex, cerebellum, and spinal cord. In addition, primary cultures of rat cerebellar glial cells and rat cerebellar granule cells were found to express P450_SCC and 3β-HSD at comparable levels. Furthermore, three of the four known isoenzymes of 3β-HSD were identified, as determined using selective PCR primers coupled with discriminative restriction enzymes and sequencing analysis of the amplified brain products. Using RNA probes, in situ hybridization indicated that P450_SCC and 3β-HSD are expressed throughout the brain at a low level and mainly in white matter. Enrichment of glial cell cultures in oligodendrocytes, however, does not increase the relative abundance of P450_SCC and 3β-HSD mRNA detected by PCR. This discrepancy suggests that the developmental state of cultured cells and their intercellular environment may be critical for regulating the expression of these enzymes. These findings support the proposal that the brain apparently has the capacity to synthesize progesterone from cholesterol, through pregnenolone, but that the expression of these enzymes appears to be quite low. Furthermore, the identification of these messages in cerebellar granule cell cultures implies that certain neurons, in addition to glial cells, may express these steroidogenic enzymes.     

Metabolism of steroids in pure cultures of neurons and glial cells: Role of intracellular signalling

In the brain, the 5-reductase converting testosterone (T) is present both in neurons and in glial cells, even if it prevails in neurons; the 3-hydroxysteroid-dehydrogenase (3-HSD), the enzyme converting dihydrotestosterone (DHT) into 3-diol, is particularly concentrated in type 1 astrocytes. In glial cells, since the 5-reductase is activated by a cAMP analogue, PKA seems to be invlved in the control of this enzyme, postulating that nervous inputs utilizing cAMP as the second messenger might modify the activity of this enzyme in glial cells. Moreover, the results indicate that, in type 1 astrocytes, both the 5-reductase and the 3-HSD are stimulated by the co-culture with neurons and by the addition of neuron-conditioned medium, suggesting that secretory products released by neurons might intervene in the control of glial cell function.     

Plasma concentrations of ovarian steroids in relation to oocyte final maturation and ovulation in female plaice sampled at sea

Blood plasma concentrations of free 17 β -oestradiol, free testosterone and glucuronidated testosterone were strongly positively related to the percentage of vitellogenic oocytes remaining in the ovaries of plaice Pleuronectes platessa caught at sea–being at their highest in pre-spawning (stage IV) females (i.e. those in which the oocytes were close to fully grown, but had not yet entered the stage of final maturation). In contrast, the concentrations of free and sulphated 17,20 β -P, 3α aL ,17, 20 β -P-5 β , and 3 α ,17,21-P-5 β were at their lowest in stage IV females. Free 17,20 β -P (the putative maturation-inducing steroid) became only slightly elevated (less than twofold) during spawning (i.e. in stage V and VI females with hydrated and/or ovulated eggs). Sulphated 17,20 β -P and 3 α ,17,21-P-5 β became slightly more elevated (three- to fourfold). However, sulphated 3 α , 17,20 β -P-5 β concentrations increased 30-fold and were at their highest in fish in which only 40% of vitellogenic oocytes remained in the ovaries. Sulphated 17,20 β -P, 3 α , 17, 20 β -P-5 β and 3 α ,17,21-P-5 β concentrations were significantly positively related to hyaline oocyte batch size; and sulphated 17,20 β -P and sulphated 3 α , 17,20 β -P-5 β were significantly negatively related to the degree of hydration of the hyaline oocytes. None of the steroid concentrations, however, was related to the time of capture. More ovulated females were found in the afternoon than at any other time of the day.     

Antiserum to 5alpha-dihydrotestosterone: production, characterization and use in radioimmunoassay

5α-Dihydrotestosterone (5α-DHT) was rendered antigenic by covalent attachment to bovine serum albumin (BSA) through position 1 of the steroid. Nucleophilic attack by β-mercaptopropionic acid on the 1,2-dehydro derivative of 5α-DHT yielded the corresponding 1α-thioether alkanoic acid which was coupled to bovine serum albumin by use of the carbodiimide reagent. The method should be generally applicable to 3-oxosteroids. Immunization of rabbits with 5α-DHT-1α-carboxyethyl-thioether-BSA gave rise to antisera of high affinity for 5α-DHT (K_a= 1.4 × 10⁹ 1/mol) that showed little cross reaction with 17β-hydroxy-5β-androstan-3-one (3%), and with a variety of 17-oxoandrostane compounds (≤0.5%). However the serum cross-reacted significantly with testosterone (10%) and with 5α-androstene-3α, 17β-diol (16%). A radioimmunoassay procedure for the determination of 5α-DHT in plasma is described. Chromatographic purification of the plasma extracts proved necessary for obtaining valid results. The plasma level of 5α-DHT(pg/ml; ean ± S.D.) was 364±79 (n = 7) in normal human adult males and 188 ± 62 (n = 5) in normal non-pregnant women.     

Changes in reproductive parameters during the spawning migration of pink salmon, Oncorhynchus gorbuscha (Walbaum)

The hormones 17β-estradiol, 17α-hydroxy-20β-dihydroprogesterone(17α, 20β-P), 11-ketotestosterone, testosterone, gonadotropin and also vitellogenin, were determined during the spawning migration of wild pink salmon in the Fraser and Thompson Rivers in British Columbia. This stock of pink salmon takes approximately 2 weeks to migrate the 333 km upstream to the spawning grounds. Both sexes were at an advanced stage of sexual development when they entered fresh water. In females both the 17β-estradiol and vitellogenin levels fell precipitously during the migration, to be very low at spawning, whereas the 17α,20β-P level rose rapidly, to be highest at arrival on the spawning grounds. The gonadotropin level also rose rapidly during the migration, and was highest in spent fish. Testosterone was at a high level throughout, although this level decreased steadily during migration. In many respects similar endocrine changes were observed in the male. For example, in the case of androgen levels, both testosterone and 11-ketotestosterone fell steadily during migration but were still relatively high at spawning, whereas both gonadotropin and 17α, 20β-P levels rose markedly as migration progress. However, although the qualitative changes were often similar between the sexes, the levels of 17α, 20β-P, testosterone, and gonadotropin were considerably higher throughout in females than in males. It is concluded that this stock of pink salmon is at an advanced stage of sexual development when it enters fresh water. The endocrine changes observed during this study represent those controlling the final stages of reproduction, specifically final oocyte maturation and ovulation in females, and the final stages of spermatogenesis and spermiation in males.     

Localization of type 5 17β-hydroxysteroid dehydrogenase mRNA in mouse tissues as studied by in situ hybridization

The mouse enzyme type 5 17-hydroxysteroid dehydrogenase (17-HSD) catalyzes the conversion of androstenedione to testosterone and, to a lesser degree, the conversion of estrone to estradiol. In order to determine the exact sites of action of type 5 17-HSD, we studied the cellular localization of the mRNA of the enzyme in mouse tissues by using in situ hybridization. Specific hybridization signal was found in the liver, ovary, adrenal cortex, and kidney. In the liver of mice of both sexes, a strong signal was observed in all hepatocytes. In the ovary, specific labeling was detected in the granulosa and theca interna cells in growing follicles and in luteal cells. In the female adrenal cortex, intense labeling was restricted to the zona reticularis, whereas no type 5 17-HSD mRNA expression could be found in the male adrenal cortex. In the kidney of mice of both sexes, type 5 17-HSD mRNA was expressed in epithelial cells in both the proximal and distal convoluted tubules. The data indicate that androgens and estrogens are formed via the action of type 5 17-HSD in specific cell types in the liver, ovary, adrenal cortex, and kidney.This work was supported by Genome Canada and Genome Québec.     

Production of 5α- and 5β-dihydrotestosterone by isolated human axillary bacteria

Abstract The metabolism of testosterone by coryneform bacteria in vitro has been studied. Metabolites identified after derivatization by capillary gas chromatography and further by combined gas chromatography-mass spectrometry were 17β-hydroxy-5α-androstan-3-one and 17β-hydroxy-5β-androstan-3-one. The mass spectral characteristics of the methyl oxime trimethylsilyl ethers of all the 17-hydroxy-androstan-3-one and 3-hydroxy-androstan-17-one isomers are recorded.     

Histochemical localization of 3 beta-hydroxysteroid dehydrogenase in the testes of chicken-pheasant hybrids.

Synopsis Histochemical studies on the activity of 3-hydroxysteroid dehydrogenase (3-HSD) in the testes of adult chicken-pheasant hybrids and domestic fowl of similar age were carried out using dehydroepiandrosterone as the substrate. The reaction for 3-HSD was positive in the interstitial tissue and negative within seminiferous tubules of domestic fowl. In chicken-pheasant hybrids, a strong positive reaction for 3-HSD was observed in the interstitial tissue and within the seminiferous tubules suggesting that, in hybrid testes, both Leydig cells and cells of seminiferous tubules may be capable of steroid biosynthesis. Since the plasma testosterone levels in these birds were found to be extremely low, it is hypothesized that either the chicken-pheasant hybrids do not release testosterone in sufficient amounts in the circulation or the type of steroid produced by the 3HSD-positive cells may be different from testosterone that is required for the maintenance of normal fertility and the development of secondary sexual characteristics.     

Localization of testosterone and 3β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase in cynomolgus monkey (Macaca fascicularis) testes

The enzyme 3β-hydroxysteroid dehydrogenase /Δ⁵-Δ⁴-isomerase (3β-HSD) is essential for the biosynthesis of all classes of steroid hormones, including androgens. We localized testosterone and 3β-HSD by light microscopic immunocytochemistry in the testes of adult cynomolgus monkeys. Immunoreactive testosterone was located as intense deposits in the labeled cytoplasm of Leydig cells, and located weakly in the interstitial tissues, basement membranes, and the regions near tubular walls within tubules. Immunoreactive 3β-HSD was located in the cytoplasm of all Sertoli cells and was especially intense in the parts near tubular walls and located weakly to intensely in the cytoplasm of some Leydig cells. This is the first immunocytochemical evidence that Sertoli cells of cynomolgus monkeys, as well as Leydig cells, are involved in biosynthesis of androgens.     

Comparative C19-radiosteroid metabolism by MA 160 and HeLa cell lines

Summary Since the designation of the human MA 160 line as prostatic epithelial cells has been questioned and the possibility of HeLa cross contamination raised, this comparative study of C₁₉-radiosteroid transformation in MA 160 and HeLa monolayer cultures was done to determine whether these cells possess the distinguishing features of reductive and oxidative androgen metabolism expected in male and female genital organs, respectively. We compared the radiometabolite patterns produced by incubating [¹⁴C]testosterone (300nM) and [³H]testosterone (3nm) and 5α-dihydrotestosterone (17β-hydroxy-5α-androstan-3-one) with cultures of prostatic MA 160 and HeLa Parent, TCRC-1, TCRC-2 and ATC 229 cells. C₁₉-Radiosteroid metabolite patterns from MA 160 cell incubations also were compared with patterns generated by MA 196 fibroblasts from abdomnal skin of the same donor. MA 160 cells metabolized radiotestosterone predominantly to 5α-dihydrotestosterone, 5α-androstane-3α,17β-diol and 5α-androstane-3β,17β-diol. The diol epimers were the principal metabolites of 5α-dihydrotestosterone radiosubstrate. In contrast, radiotestosterone metabolism by MA 196 and HeLa Parent, TCRC-1 and TCRC-2 cells was overwhelmingly to the 17-oxosteroids 4-androstene-3,17-dione and androsterone. Another pathway was operative in HeLa 229 and, to a minor extent, in TCRC-1, which converted radiotestosterone to 4-androstene-3α,17β-diol and 5α-androstane-3α,17β-dol, with little formation of 5α-dihydrotestosterone. MA 160 cells thus metabolize radiotestosterone preponderantly to 5α-reduced 17β-hydroxysteroids as expected for prostatic epithelial cells, whereas HeLa cells show heterogeneity in metabolizing the labeled hormone by the alternative 17-oxosteroid and Δ⁴ pathways. This work was supported by Public Health Service Research Grants CA 13417 and CA 12924 from the National Cancer Institute, AM 11011 from the National Institute of Arthritis, Metabolism and Digestive Diseases, and by appropriations of the Commonwealth of Massachusetts, Item No. 4532-9003-01.