Release of free and conjugated forms of the putative pheromonal steroid 11-oxo-etiocholanolone by reproductively mature male round goby (Neogobius melanostomus Pallas, 1814)

Previous studies of the round goby (Neogobius melanostomus Pallas, 1814), an invasive fish species in the Laurentian Great Lakes of North America, have shown that this species has the ability to both synthesize and smell steroids that have a 5 beta-reduced and 3 alpha-hydroxyl (5 beta,3 alpha) configuration. An enzyme-linked immunoassay (EIA) for 3 alpha-hydroxy-5 beta-androstane-11,17-dione (11-O-ETIO) has been used to show a substantial rise in the rate of release of immunoreactive compounds into the water when males are injected with salmon gonadotropin releasing hormone analogue. Similar increases were noted for 11-ketotestosterone and 17,20 beta-dihydroxypregn-4-en-3-one. Partitioning of the extracts between diethyl ether and water showed the presence of both free and conjugated immunoreactive 11-O-ETIO. Only conjugated immunoreactivity was found in urine (implying that free steroid is released via the gills). The identity of the conjugates was probed by using HPLC, EIA, and mass spectrometry and removal of sulfate and glucosiduronate groups. Immunoreactivity in the conjugated fraction was found to be due mainly to 3 alpha,17beta-dihydroxy-5 beta-androstan-11-one 17-sulfate. However, the evidence was also strong for the presence in water extracts of substantial amounts of 3 alpha-hydroxy-5 beta-androstane-11,17-dione 3-glucosiduronate (which could be detected only by EIA after removal of the glucosiduronate group with beta-glucuronidase). There were also small amounts of 3 alpha-hydroxy-5 beta-androstane-11,17-dione 3-sulfate and 3 alpha,17beta-dihydroxy-5 beta-androstan-11-one 17-glucosiduronate. These studies give some idea of the types, amounts, and ratios of 11-O-ETIO derivatives that are released by reproductive N. melanostomus and will aid further research into the putative pheromonal roles of 5 beta,3 alpha-reduced androgens in this species.     

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.     

The seminal vesicle synthesizes steroids in the round goby Neogobius melanostomus

In this study, we examine the possible contribution of the seminal vesicles of the male round goby to the production of putative steroidal pheromones. A previous study showed that the testes of the round goby are rich in steroid-producing Leydig-like cells; and when incubated in vitro, convert tritiated androstenedione to at least six other steroids, including one not previously identified in fish--namely 3alpha-hydroxy-5beta-androstane-11,17-dione (11-oxo-etiocholanolone, 11-oxo-ETIO). The seminal vesicles of reproductively mature males were examined by conventional histology, transmission electron microscopy and immunocytochemistry (utilizing an antibody against 3beta-hydroxysteroid dehydrogenase--a key enzyme in vertebrate steroid synthesis). All three procedures identified Leydig cells in the proximal and medial regions of the seminal vesicles. In vitro incubation of seminal vesicles with tritiated androstenedione demonstrated biosynthesis of 11-oxo-androstenedione, 11-oxo-testosterone (more commonly known as 11-ketotestosterone) and 11 oxo-ETIO. These data indicate that the seminal vesicles, as well as the testes are involved in the synthesis of steroidal compounds that may function as pheromones.     

Synthesis and pyrogenic effect of 3α,7α-dihydroxy-5β-androstan-17-one and 3α-hydroxy-5β-androstane-7,17-dione

The first chemical synthesis of 3α,7α-dihydroxy-5β-androstan-17-one and 3α-hydroxy-5β-androstane-7,17-dione is reported. In this method, the 17β-side chain of commercial chenodesoxycholic acid was degraded in 6 steps after selective protection of the hydroxyl groups : 3α-OH by a $\text{tert}$-butyldimetfaylsilyl group and 7α-OH by an acetoxy group. The capacity of 3α,7α-dihydroxy-5β-androstan-17-one and 3α-hydroxy-5β-androstane-7, 17-dione to release a pyrogen by human leukocytes was investigated by two independent methods : supernatants from leukocytes incubated with a steroid are injected to rabbits whose fever is measured, or tested by the Limulus Test (a pyrogen detection technique). The 7-keto substituted etiocholanolone still possessed pyrogenic activity, while the 7α-hydroxyl substituted one did not.     

Synthesis of 5α-Androstane-3α,16α,17β-triol from 3β-hydroxy-5-androsten-17-one

5α-Androstane-3α, 16α 17β-triol was synthesized from 3β-hy-droxy-5-androsten-17-one. The procedure Involved catalytic hydrogenation of 3β-hydroxy-5-androsten-17-one to 3β-hydroxy-5α-androstan-17-one. This was followed by conversion of the 3β-hydroxy group to 3α-benzoyloxy group by the Mitsunobu reaction. Further treatment with isopropenyl acetate yielded 5α-androsten-16-ene-3α, 17-diol 3-benzoate 17-acetate. This was then converted to 3α, 17-dihydroxy-5α-androstan-16-one 3-benzoate 17-acetate via the unstable epoxide intermediate after treatment with $\text{m}$-cloroperoxybenzoic acid. LiAlH₄ reduction of this compound formed 5α-androstane-3α, 16α, 17β-trlol. ¹H and ¹³C NMR of various steroids are presented to confirm the structure of this compound.     

Biotransformation studies of prednisone using human intestinal bacteria Part II: Anaerobic incubation and docking studies

Anaerobic incubation of prednisone 1 with human intestinal bacteria (HIB) afforded nine metabolites: 5β-androst-1-ene-3,11,17-trione 3, 3α-hydroxy-5α-androstane-11,17-dione 4, 3β,17α,20-trihydroxy-5α-pregnan-11-one 5, 3α,17α-dihydroxy-5α-pregnane-11,20-dione 6, 3α,17α-dihydroxy-5β-pregnane-11,20-dione 7, 3β,17β-dihydroxy-5α-androstan-11-one 8β, 3β,17α-dihydroxy-5α-androstan-11-one 8α, 3α,17β-dihydroxy-5α-androstan-11-one 9β, and 3α,17α-dihydroxy-5α-androstan-11-one 9α. The structures of these metabolites (3–9) were elucidated using several spectroscopic techniques. Computer-aided prediction of potential biological activities of the isolated prednisone metabolites (3–9) revealed potential inhibition of prostaglandin E2 9-ketoreductase (PGE2 9-KR). Docking studies applied to PGE2 9-KR allowed recommendation of the metabolites 4, 8β, and 8α for further pharmacological study as PGE2 9-KR inhibitors.     

Serum steroid hormone levels in relation to the reproductive cycle ofSebastes taczanowskii andS. schlegeli

Synopsis Changes in serum steroid hormones were studied during the reproductive cycle of two viviparous rockfishes of the genusSebastes. During the annual reproductive cycle of femaleS. taczanowskii, serum levels of estradiol-17β (E₂) gradually increased from their lowest in August to maximal levels towards the end of vitellogenesis in February, and rapidly returned to low levels in pregnant females in April and May. Serum progesterone (prog) fluctuated at low levels throughout the annual reproductive cycle, while 17α, 20β-dihydroxy-4-pregnen-3-one (17α, 20β-diOHprog) levels were high during gestation in May. Serum levels of these steroids were also measured in femaleS. schlegeli at weekly intervals during late vitellogenesis, gestation and post-parturition. In pregnant females, E₂ was high during vitellogenesis, then decreased and remained low throughout gestation. The 17α, 20β-diOHprog levels were low during vitellogenesis, then rapidly increased and remained high during gestation. On the other hand, prog values remained low, with temporal peaks coinciding with the peak of 17α, 20β-diOHprog during gestation. Based on these results and the literature, it is suggested that E₂ is the most important steroid involved in vitellogenesis and that 17α, 20β-diOHprog may play an important role in final oocyte maturation and subsequent gestation.     

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.     

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     

Comparative analysis of milt quality and steroid levels in blood and seminal fluid of Persian sturgeon males, Acipenser persicus during final maturation induced by hormonal treatment

Steroids both in seminal fluid (SF) and blood serum (BS) as well as the milt quality (sperm motility and sperm production) were investigated during final maturation of Persian sturgeon. The BS levels of testosterone (T), 11-Ketotestosterone (11-KT), progesterone (P), 17α,20β,21-trihydroxy-4-pregnen-3-one (20βS), cortisol (C) and 17α,hydroxyprogesterone (OHP) elevated after pituitary preparation (PP) treatment and then decreased during stripping period for spermiating males. Such elevations did not occur for non-spermiating individuals and steroids remained in basal levels after PP treatment until the end of stripping period. For both groups (spermiating and non-spermiating fish), the BS levels of 17α,20β-dihydroxy-4-pregnen-3-one (DHP) did not show significant changes during experiment. During stripping period, the values of all tested steroids were significantly lower in SF than in BS of spermiating males. SF levels of 20βS and 11-KT showed a decreasing trend and the other steroids were unchanged during this period. Significant positive correlations were found between the values of 20βs and 11-KT in BS with their levels in SF. Also, BS and SF levels of 20βs and 11-KT were positively correlated with sperm motility characteristics (percentage and duration of motility) and sperm production (sperm density and milt volume), respectively. The results showed the probable involvement of 20βs, P, OHP, T, 11-KT and C in final maturation of Persian sturgeon, especially 20βs and 11-KT had good correlations with qualitative parameters of milt. The lower levels of steroids in SF than those in BS might also be essential for viability of Persian sturgeon spermatozoa. Probably, there are mechanisms that stabilize the concentrations of a number of hormones in the SF.     

Olfactory sensory input increases gill ventilation in male round gobies (Neogobius melanostomus) during exposure to steroids

In teleostean fish, ventilation increases have been observed in response to low dissolved oxygen levels, visual stimuli, and gustatory cues. However, olfactory sensory input may also stimulate gill ventilation rate. We investigated whether olfactory sensory input mediates gill ventilation responses, as suggested by the observation that steroidal compounds detected by the olfactory system elicited increases in opercular activity in the perciform teleost, the round goby (Neogobius melanostomus). Close parallels between gill ventilation and olfactory responses, led us to conduct an empirical study that used two different olfactory sensory deprivation techniques to seek a causal relationship between olfactory epithelial activity and hyperventilation. Chemical lesion of olfactory sensory neurons or mechanical occlusion of the nasal cavities inhibited gill ventilation responses of reproductive male round gobies to estrone (1,3,5(10)-estratrien-3-ol-17-one) and to ovarian extracts. This direct evidence demonstrates the role of olfactory sensory input for the gill ventilation response to putative reproductive pheromones and may represent an important regulatory mechanism for odorant sampling during pheromone communication.     

Ovarian steroid sulphate functions as priming pheromone in maleBarilius bendelisis (Ham.)

The study reveals that pre-ovulatory females of the fishBarilius bendelisis (Ham.) release sex steroids and their conjugates into the water and that a steroid sulphate of these compounds functions as a potent sex pheromone which stimulates milt production in conspecific males prior to spawning. Since males exposed to the purified sub-fraction III of the steroid sulphate fraction have increased milt volume and more spermatozoa with greater motility, the function of this priming pheromone appears to be to enhance male spawning success. High turbulence and faster water currents render the hillstream ecosystem extremely challenging for chemical communication. Therefore, ovulatory female fish secrete highly water soluble steroid sulphates for rapid pheromonal action in males. Inhibited milt volume in olfactory tract lesioned (OTL) males exposed to the steroid sulphate fraction and 17α,20β-dihydroxy-4-pregnen-3-one supports the concept that the pheromonally induced priming effect in male fish is mediated through olfactory pathways.     

Metabolism of 17β-hydroxy-2-hydroxymethylene-5α-androstan-3-one in the rabbit

An acidic metabolite, 2α-carboxy-5α-androstane-3α, 16α, 17αtriol and two neutral metabolites, 2α-hydroxymethyl-5α-androstane-3α, 17α-diol, and 2α-hydroxymethyl-5α-androstane-3α, 16α, 17α-triol have been identified in the urine of rabbits orally dosed with 17β-hydroxy-2-hydroxymethylene-5α-androstan-3-one. 2α-Hydroxymethyl-5α-androstane-3α, 16α, 17α-triol was previously obtained from the urine of rabbits dosed with 17β-hydroxy-2α-methyl-5α-androstan-3-one. The acidic metabolite was the major urinary excretion product.     

Steroid isotopic standards for gas chromatography-combustion isotope ratio mass spectrometry (GCC-IRMS)

Carbon isotope ratio (CIR) analysis of urinary steroids using gas chromatography-combustion isotope ratio mass spectrometry (GCC-IRMS) is a recognized test to detect illicit doping with synthetic testosterone. There are currently no universally used steroid isotopic standards (SIS). We adapted a protocol to prepare isotopically uniform steroids for use as a calibrant in GCC-IRMS that can be analyzed under the same conditions as used for steroids extracted from urine. Two separate SIS containing a mixture of steroids were created and coded CU/USADA 33-1 and CU/USADA 34-1, containing acetates and native steroids, respectively. CU/USADA 33-1 contains 5α-androstan-3β-ol acetate (5α-A-AC), 5α-androstan-3α-ol-17-one acetate (androsterone acetate, A-AC), 5β-androstan-3α-ol-11, 17-dione acetate (11-ketoetiocholanolone acetate, 11k-AC) and 5α-cholestane (Cne). CU/USADA 34-1 contains 5β-androstan-3α-ol-17-one (etiocholanolone, E), 5α-androstan-3α-ol-17-one (androsterone, A), and 5β-pregnane-3α, 20α-diol (5βP). Each mixture was prepared and dispensed into a set of about 100 ampoules using a protocol carefully designed to minimize isotopic fractionation and contamination. A natural gas reference material, NIST RM 8559, traceable to the international standard Vienna PeeDee Belemnite (VPDB) was used to calibrate the SIS. Absolute δ¹³C_VPDB and Δδ¹³C_VPDB values from randomly selected ampoules from both SIS indicate uniformity of steroid isotopic composition within measurement reproducibility, SD(δ¹³C) < 0.2‰. This procedure for creation of isotopic steroid mixtures results in consistent standards with isotope ratios traceable to the relevant international reference material.     

Biotransformation of methyl ent-17-hydroxy-16β-kauran-19-oate by Rhizopus stolonifer

Methyl ent-17-hydroxy-16β-kauran-19-oate was fed to a 2-day-old culture of the fungus Rhizopus stolonifer, fermenting at room temperature (25 °C) in an orbital shaker (2 l). After 11 days, both broth and mycelia were extracted with ethyl acetate. Two novel compounds were isolated from this experiment: methyl ent-9α,17-dihydroxy-16β-kauran-19-oate and methyl ent-7α,17-dihydroxy-16β-kauran-19-oate. Their structures were fully confirmed by spectroscopic methods. Received: 22 July 1999 / Received revision: 2 November 1999 / Accepted: 12 November 1999     

Androgen metabolism by rat epididymis. 1. Metabolic conversion of 3 H-testosterone in vivo

The epididymis of adult rats metabolize ³H-testosterone by experiments $\text{in vivo}$. Thirty minutes after the injection of 100 μCi ³H-testosterone, some 10 per cent of the total radioactivity of the epididymis was found in the water-soluble fraction, whereas 90 per cent was found in the ether soluble fraction (free steroids). The free steroids were examined further and the following androgenic metabolites identified: $\text{testosterone}$ (17β-hydroxy-4-androsten-3-one) 8, 9%, $\text{androstendipne}$ (4-androstene-3, 17-dione, 2,7%,$\text{5α-A-dione}$ (5α-androstane-3, 17-dione) 6,5%, $\text{DHT}$ (17β-hydroxy-5α-androstan-3-one) 47, 2%, $\text{3β-diol}$ (5α-androstane-3β, 17β-diol) 4, 4%, $\text{3α-diol}$ (5α-androstane-3α,17β-diol) 20, 8% and $\text{androsterone}$ (3α-hydroxy-5α-androstan-3-one) 3,4%. The relative amount of each metabolite is given in per cent of total radioactivity in the ether soluble fraction.     

Hydroxylation of steroids by <Emphasis Type="Italic">Curvularia lunata</Emphasis> mycelium in the presence of methyl-β-cyclodextrine

Transformation of 16 Δ⁵-3β-hydroxy- and Δ⁴-3-ketosteroids of androstane and pregnane classes was carried out using Curvularia lunata mycelium suspended in phosphate buffer with methyl-β-cyclodextrine (MCD). As the result, 20 monohydroxy- and dihydroxy-metabolites, whose structure was determined using spectra of proton magnetic resonance and mass-spectra, have been isolated. Hydroxylation of Δ⁵-3β-hydroxy-steroids occurred mostly in the C-7α position whereas hydroxylation of Δ⁴-3-ketosteroids was in the C-11β position. Only androst-4-en-3,17-dione, 9α-hydroxy-androstenedione, and androsta-1,4-diene-3,17-dione were hydroxylated at C-14α position. Besides main 11β-derivatives, the 6β- and 7β-hydroxy-derivatives with yield 10 and 30%, respectively, were isolated during transformation of progesterone and hydroxymethyl pregnadienone. The ratio of MCD to transforming steroid was 1: 1 (mol/mol). Hydrocortisone and 7α-hydroxyandrostenolone with the yield 55 and 77%, respectively, were obtained at the maximal concentrations of cortexolone 20 g/l and androstenolone acetate 10 g/l in the presence of MCD. Absorption of steroids on mycelium, lower speed of their transformation, low concentrations of modifying substrates, and low yield of hydroxyderivatives have been observed in the absence of MCD.     

Sertoli cells from immature rats : in vitro stimulation of steroid metabolism by FSH.

Sertoli cells from 10 day old rats convert androstenedione to testosterone and 5α-androstane-3α,17β-diol, testosterone to 17β-hydroxy-5α-androstan-3-one and 5α-androstane-3α,17β-diol, and 17β-hydroxy-5α-androstan-3-one to 5α-andro-stane-3α,17β-diol after 72 hours $\text{in vitro}$. Conversions of androstenedione to testosterone and 5α-androstane-3α,17β-diol, and testosterone to 5α-androstane-3α,17β-diol were 2 to 3 times greater in FSH treated cultures. Steroid conversion was not stimulated significantly by LH or TSH. The results are interpreted as evidence that in young rats Sertoli steroid metabolism is stimulated by FSH, that Sertoli cells are an androgen target and that FSH may induce or facilitate Sertoli androgen responsiveness.     

Polar steroids from <Emphasis Type="Italic">Solaster endeca</Emphasis> starfish and the physiological activity of polar steroids from three starfish species

Four polyhydroxylated steroids, new (20R)-5α-cholestan-3β,6α,8,15α,24,26-hexaol (I) and known (20R,25S)-5α-cholestan-3β,6α,8,15β,16β,26-hexaol, (20R,25S)-5α-cholestan-3β,6α,15β,16β,26-pentaol, and marthasterone sulfate were isolated from the Solaster endeca starfish inhabiting the Sea of Okhotsk and characterized. Steroid (I) contains a 24,26-dihydroxylated side chain, which is unique for starfish polyols. The isolated steroids and related metabolites from two starfish species of the Evasterias genus (in total, 15 compounds) were weakly cytotoxic in a human HeLa cell culture and some of them were inhibitors of non-specific esterase from mouse Ehrlich carcinoma. The effects of these compounds on the p53 protein activity were studied in a yeast two-hybrid test system and both inhibitors and stimulators of this activity were found among them.     

Non-erythroid beta spectrin interacting proteins and their effects on spectrin tetramerization

With yeast two-hybrid methods, we used a C-terminal fragment (residues 1697–2145) of non-erythroid beta spectrin (βII-C), including the region involved in the association with alpha spectrin to form tetramers, as the bait to screen a human brain cDNA library to identify proteins interacting with βII-C. We applied stringent selection steps to eliminate false positives and identified 17 proteins that interacted with βII-C (IP_βII-C s). The proteins include a fragment (residues 38–284) of “THAP domain containing, apoptosis associated protein 3, isoform CRA g”, “glioma tumor suppressor candidate region gene 2” (residues 1-478), a fragment (residues 74–442) of septin 8 isoform c, a fragment (residues 704–953) of “coatomer protein complex, subunit beta 1, a fragment (residues 146–614) of zinc-finger protein 251, and a fragment (residues 284–435) of syntaxin binding protein 1. We used yeast three-hybrid system to determine the effects of these βII-C interacting proteins as well as of 7 proteins previously identified to interact with the tetramerization region of non-erythroid alpha spectrin (IP_αII-N s) [1] on spectrin tetramer formation. The results showed that 3 IP_βII-C s were able to bind βII-C even in the presence of αII-N, and 4 IP_αII-N s were able to bind αII-N in the presence of βII-C. We also found that the syntaxin binding protein 1 fragment abolished αII-N and βII-C interaction, suggesting that this protein may inhibit or regulate non-erythroid spectrin tetramer formation.