Steroid hormones and the fertilizing capacity of spermatozoa

The effects of eleven different steroid hormones on $\text{in vitro}$ development of fertilizing capacity by hamster sperm were examined. Capacitation of epididymal sperm occurred only in the presence of female genital tract secretions. Fertilizing ability of sperm was poor if estradiol-17β, cortisol, chlormadinone acetate, medroxyprogesterone acetate, or megestrol acetate were present in the incubation medium at 10^?5M, whereas similar concentrations of estradiol-17α, progesterone, norethisterone acetate, ethynodiol diacetate, or norgestrel had little effect. Testosterone was a weak inhibitor of capacitation. Capacitation activity of female uterus and oviduct washings was higher at estrus than diestrus. This activity was reduced by treating intact animals with progesterone, cortisol, or testosterone, but increased by estradiol-17β or HCG. Estradiol-17α has no effect. Activity was low in pregnant or ovariectomized hamsters. Treatment of ovariectomized animals with estradiol-17β increased capacitation activity, but estradiol-17α, HCG or progesterone treatment was ineffective.     

Microbial transformations of steroids

Capacity to acetylate testosterone was demonstrated in the species of yeast and yeast-like organisms which simultaneously ferment lactose (Saccharomyces fragilis, S. lactis, Candida pseudotropicalis andTorulopsis sphaerica). This capacity can thus be made use of as a supplementary diagnostic test for classifying the above microorganisms. Microbial acetylation can also be employed for separating mixtures of steroid 17-hydroxy-epimers, the acetylation taking place only with the 17β-hydroxyderivative while the corresponding 17α-epimer remains intact. No acetylation of the steroid molecule with hydroxy group in the 11α, 11β, 20β and 21 positions takes place under these conditions.     

Repair replication in human cells studied by sodium iodide isopycnic centrifugation of DNA in a fixed-angle rotor

A method for purification of ethynyl steroids from biological fluids has been developed using silver-sulfoethyl cellulose column chromatography. Ethynyl steroids were applied in methanol, and the firm binding allowed the columns to be washed with methanol, ethyl acetate, or diethyl ether to remove endogenous materials. Nonethynylated steroids did not bind to silver-sulfoethyl cellulose. Release of ethynylated steroids was achieved with a saturated NaCl/methanol solution. Dehydration or de-ethynylation of ethynyl estradiol, ethynodiol diacetate, and ethynodiol was not observed. The utility of this technique for purification of ethynyl steroid metabolites from the urine of a beagle metabolizing norethynodrel was demonstrated.     

Estradiol regulates N-cadherin mRNA levels in the mouse ovary

N-cadherin (N-cad) is a calcium-dependent cell adhesion molecule which is present in the granulosa cells of the mouse ovarian follicle. This cell adhesion molecule has been implicated as a key modulator of follicular development. The regulators of N-cad mRNA levels in the ovary have not been identified. We have examined the ability of steroids to influence ovarian N-cad mRNA levels in vivo. Immature mice were injected with either progesterone, testosterone, 17β-estradiol, or 17α-estradiol. Only 17β-estradiol caused a rapid and significant increase in the ovarian N-cad mRNA levels. We speculate that this steroid is a major regulator of N-cad-mediated granulosa cell interactions in vivo. © 1995 Wiley-Liss, Inc.     

A new class of long-acting hormonal steroid preparation: synthesis of dimeric androgens coupled at C3-C3 and C-17-C3 and of an androgen-progestogen combination

3β-Hydroxy-4-androsten-17-one was prepared from 4-androsten-3, 17-dione according to the method of Klimstra and Colton (1) and dimerized by means of esterification with succinic acid. The reduction with lithium-tri-t-butoxyaluminium hydride gave a testosterone derivative coupled between C₃-C₃ which showed after a single Injection of 10 mg a protracted but relatively weak androgenic effect in castrated male rats. The direct esterification of testosterone hemisuccinate with 4-androsten-3β,17β-diol gave the testosterone derivative coupled between C₁₇-C₃ which showed a more even and more protracted time response curve than testosterone enanthate. The testosterone-ethynodiol succinate also coupled between C₁₇-C₃, showed an androgenic depot-effect similar to that of the dimeric C₁₇-C₃ testosterone derivative.     

Profiling of urinary steroids by gas chromatography-mass spectrometry detection and confirmation of androstenedione administration using isotope ratio mass spectrometry

Androstenedione (4-androstene-3,17-dione) is banned by the World Anti-Doping Agency (WADA) as an endogenous steroid. The official method to confirm androstenedione abuse is isotope ratio mass spectrometry (IRMS). According to the guidance published by WADA, atypical steroid profiles are required to trigger IRMS analysis. However, in some situations, steroid profile parameters are not effective enough to suspect the misuse of endogenous steroids. The aim of this study was to investigate the atypical steroid profile induced by androstenedione administration and the detection of androstenedione doping using IRMS. Ingestion of androstenedione resulted in changes in urinary steroid profile, including increased concentrations of androsterone (An), etiocholanolone (Etio), 5α-androstane-3α,17β-diol (5α-diol), and 5β-androstane-3α,17β-diol (5β-diol) in all of the subjects. Nevertheless, the testosterone/epitestosterone (T/E) ratio was elevated only in some of the subjects. The rapid increases in the concentrations of An and Etio, as well as in T/E ratio for some subjects could provide indicators for initiating IRMS analysis only for a short time period, 2-22 h post-administration. However, IRMS could provide positive determinations for up to 55 h post-administration. This study demonstrated that, 5β-diol concentration or Etio/An ratio could be utilized as useful indicators for initiating IRMS analysis during 2-36 h post-administration. Lastly, Etio, with slower clearance, could be more effectively used than An for the confirmation of androstenedione doping using IRMS.     

Development of a GC/C/IRMS method - Confirmation of a novel steroid profiling approach in doping control

In doping control, an athlete can only be convicted with the misuse with endogenous steroids like testosterone (T), if abnormal values of steroid metabolites and steroid ratios are observed and if the subsequent analysis with isotope ratios mass spectrometry (IRMS) confirms the presence of exogenously administered androgens. In this work, we compare the results of a novel steroid profiling approach with the performance an in-house developed IRMS method. The developed IRMS has the advantage over other methods to be relatively short in time and with target compounds androsterone, etiocholanolone, 5β-androstane 3α,17β-diol and 5α-androstane 3α,17β-diol. Pregnanediol was used as an endogenous reference compound (ERC). Reference limits for the IRMS values were established and applied as decision limits for the evaluation of excretion urine from administration with oral T, T-gel, dihydrotestosterone (DHT) - gel and dehydroepiandrosterone (DHEA). Results indicated the importance of both androstanediols as important IRMS markers where relative values compared to an ERC (Δδ(13)C) yielded better detection accuracy than absolute δ(13)C-values. The detection times of all administered endogenous steroids were evaluated using the proposed thresholds. The results of traditional steroid profiling and a new approach based upon minor steroid metabolites monitoring introduced in a longitudinal framework were evaluated with IRMS. With traditional steroid profiling methods, 95% of the atypical samples could be confirmed whereas an additional 74% of IRMS confirmed was provided by a new biomarkers strategy. These results prove that the other steroid profiling strategies can improve the efficiency in detection of misuse with endogenous steroids.     

In vivo MRI evaluation of anabolic steroid precursor growth effects in a guinea pig model

Anabolic steroids are widely used to increase skeletal muscle (SM) mass and improve physical performance. Some dietary supplements also include potent steroid precursors or active steroid analogs such as nandrolone. Our previous study reported the anabolic steroid effects on SM in a castrated guinea pig model with SM measured using a highly quantitative magnetic resonance imaging (MRI) protocol. The aim of the current study was to apply this animal model and in vivo MRI protocol to evaluate the growth effects of four widely used over-the-counter testosterone and nandrolone precursors: 4-androstene-3 17-dione (androstenedione), 4-androstene-3β 17β-diol (4-androsdiol), 19-nor-4-androstene-3β-17β-diol (bolandiol) and 19-nor-4-androstene-3 17-dione (19-norandrostenedione). The results showed that providing precursor to castrated male guinea pigs led to plasma steroid levels sufficient to maintain normal SM growth. The anabolic growth effects of these specific precursors on individual and total muscle volumes, sexual organs, and total adipose tissue over a 10-week treatment period, in comparison with those in the respective positive control testosterone and nandrolone groups, were documented quantitatively by MRI.     

Expression of hepatic 3β-hydroxysteroid dehydrogenase and sulfotransferase 2A1 in entire and castrated male pigs

The present study investigated the effect of surgical (SC) and immunological castration on the steroid metabolizing enzymes 3β-hydroxysteroid dehydrogenase (3β-HSD) and sulfotransferase 2A1 (SULT2A1) in male pigs. Thirty-two male pigs were divided in four groups; in one group the pigs were SC before the age of 7 days, two groups were injected with Improvac(?) a vaccine against gonadotropin releasing hormone (immunological castration), while the pigs in the last group remained entire males (EMs). Immunological castration was in one group performed by vaccine injection at ages 11 and 14 weeks, while the other group received injections at ages 17 and 21 weeks. Plasma, adipose and liver tissue were collected at the time of slaughter. Plasma was analyzed for concentrations of testosterone and oestradiol. The adipose tissue was analyzed for the concentration of androstenone, while the liver tissue was analyzed for mRNA and protein expression of 3β-HSD and SULT2A1. Independent of method, all castrated pigs showed greater mRNA and protein expression of 3β-HSD and lower levels of all steroids in plasma compared with EMs. Moreover, there was a strong correlation between mRNA and protein expression of 3β-HSD and steroid levels. The same was not valid for expression of SULT2A1. It is concluded that steroid levels can increase expression of the steroid metabolizing enzyme 3β-HSD and thereby influence steroid metabolism, e.g. of androstenone.     

Estimation of Steroids and Steroid Metabolism in a Lower Vertebrate (Lampetra planeri Bloch)

Pregnenolone, androstenedione and testosterone were identified by RIA in tissue homogenates of the pronephric region, the opisthonephros, the gonads and in plasma samples from male and female immature and mature adult brook lampreys. Additionally, hydroxysteroid dehydrogenase activity was determined spectrophotometrically in homogenates from the same tissues of mature and spent adult brook lampreys employing pregnenolone, testosterone or 3β,17β-dihydroxy-5β-androstane as substrates. The steroid levels show differences corresponding to developmental stages, tissues and sex. Remarkable quantities of testosterone were measured in the testicular tissue homogenates, in homogenates obtained from the pronephric region and in the serum.     

Effect of 17 beta-estradiol and testosterone on guanosine 3', 5'-cyclic monophosphate in the rat adrenal cortex

The levels of guanosine 3′, 5′-cyclic monophosphate (cGMP) were measured in the rat adrenal cortex after administration of a single dose of either 17β-estradiol or testosterone. Young immature rats received 10 μg 17β-estradiol (females) or 100 μg testosterone (males). After testosterone administration, cGMP levels progressively rose to about 150 per cent of the control values after 4–6 hrs, and remained elevated until at least 9 hr. Administration of 17β-estradiol resulted in a similar increase in cGMP, which began at 2 hr and persisted until 9 hr, reaching levels of about 180 per cent of the controls. Our data are further evidence of general effect of steroid hormones on cGMP in their target tissues.     

The metabolism of androgens in rat preputial glands

The metabolism of testosterone, androstenedione and dehydroepiandrosterone in the rat preputial gland has been studied. A high activity of 5α-reductase is present as shown by the formation of 17β hydroxy-5α-androstan-3-one and 5α-androstan-3, 17-dione as the major products from testosterone and androstenedione respectively. Other enzyme activities are present including 17β-hydroxy steroid dehydrogenase, but the amounts of testosterone and 17β-hydroxy-5α-androstan-3-one formed from androstenedione and dehydroepiandrosterone are low. The main product of dehydroepiandrosterone metabolism was androstenedione indicating a high level of 3β-hydroxy steroid dehydrogenase 4-5 isomerase activity. The metabolism was compared with that in rat skin where it was found that the extent of metabolism was much less. The possible significance of the various products formed and of differences between skin and preputial gland metabolism is discussed. Some differences were noted between the metabolism of androgens by rat skin and preputial gland and the metabolism of androgens by human skin.     

Effect of oxidizing adulterants on human urinary steroid profiles

Steroid profiling is the most versatile and informative technique adapted by doping control laboratories for detection of steroid abuse. The absolute concentrations and ratios of endogenous steroids including testosterone, epitestosterone, androsterone, etiocholanolone, 5α-androstane-3α,17β-diol and 5β-androstane-3α,17β-diol constitute the significant characteristics of a steroid profile. In the present study we report the influence of various oxidizing adulterants on the steroid profile of human urine. Gas chromatography–mass spectrometry analysis was carried out to develop the steroid profile of human male and female urine. Oxidants potassium nitrite, sodium hypochlorite, potassium permanganate, cerium ammonium nitrate, sodium metaperiodate, pyridinium chlorochromate, potassium dichromate and potassium perchlorate were reacted with urine at various concentrations and conditions and the effect of these oxidants on the steroid profile were analyzed. Most of the oxidizing chemicals led to significant changes in endogenous steroid profile parameters which were considered stable under normal conditions. These oxidizing chemicals can cause serious problems regarding the interpretation of steroid profiles and have the potential to act as masking agents that can complicate or prevent the detection of the steroid abuse.     

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.     

Steroid hormone signalling system and fungi

Three components of the steroid hormone signalling system, 17β-hydroxysteroid dehydrogenase, androgen binding proteins and steroid hormone signalling molecule testosterone were determined in the filamentous fungus Cochliobolus lunatus for the first time in a fungus. Their possible role in C. lunatus is discussed in comparison with their role in mammalian steroid hormone signalling system. The results are in accordance with the hypothesis, that the elements of the primordial signal transduction system should exist in present day eukaryotic microorganisms.     

Biotransformations of steroid compounds by Chaetomium sp. KCH 6651

Biotransformations of steroid compounds: androstenedione, testosterone, progesterone, pregnenolone and DHEA using Chaetomium sp. 1 KCH 6651 strain as a biocatalyst were investigated. The microorganism proved capable of selective hydroxylation of the steroid substrates. Androstenedione was converted to 14α-hydroxyandrost-4-en-3,17-dione (in over 75% yield) and 6β-hydroxyandrost-4-en-3,17-dione (in low yield), while testosterone underwent regioselective hydroxylation at 6β position. Progesterone was transformed to a single product—6β,14α-dihydroxypregnan-4-en-3,20-dione in high yield, whereas biotransformation of DHEA resulted in the formation of 7α-hydroxy derivative, which was subsequently converted to 7α-hydroxyandrost-4-en-3,17-dione.     

State of steroid secretory cells and concentration of gonadal steroid hormones in the blood plasma of Siberian sturgeon <Emphasis Type="Italic">Acipenser baerii</Emphasis> and sterlet <Emphasis Type="Italic">A. ruthenus</Emphasis> (Acipenseridae) during sex differentiation

Results of studying steroid secretory cells in gonads and the level of estradiol-17β and testosterone in the blood plasma of young Siberian sturgeon Acipenser baerii and sterlet A. ruthenus, aged 8.5 and 9.5 months, respectively, is studied. During sex differentiation, the steroid secretory activity in females is intensified first in the stroma of gonads and then, after the formation of oocytes during the previtellogenesis period, it is shifted to the follicle theca. The development of oocytes is accompanied by a decrease in the concentration of testosterone and an increase in the concentration of estradiol-17β. In males, the intensification of the activity of steroid secretory cells, which was indirectly determined by the state of organoids, is accompanied by an increase in the concentration of testosterone in blood plasma.     

Metabolism of anabolic steroids by recombinant human cytochrome P450 enzymes: Gas chromatographic–mass spectrometric determination of metabolites

Metabolism of steroid hormones with anabolic properties was studied in vitro using human recombinant CYP3A4, CYP2C9 and 2B6 enzymes. The enzyme formats used for CYP3A4 and CYP2C9 were insect cell microsomes expressing human CYP enzymes and purified recombinant human CYP enzymes in a reconstituted system. CYP3A4 enzyme formats incubated with anabolic steroids, testosterone, 17α-methyltestosterone, metandienone, boldenone and 4-chloro-1,2-dehydro-17α-methyltestosterone, produced 6β-hydroxyl metabolites identified as trimethylsilyl (TMS)-ethers by a gas chromatography–mass spectrometry (GC–MS) method. When the same formats of CYP2C9 were incubated with the anabolic steroids, no 6β-hydroxyl metabolites were formed. Human lymphoblast cell microsomes expressing human CYP2B6 incubated with the steroids investigated produced traces of 6β-hydroxyl metabolites with testosterone and 17α-methyltestosterone only. We suggest that the electronic effects of the 3-keto-4-ene structural moiety contribute to the selectivity within the active site of CYP3A4 enzyme resulting in selective 6β-hydroxylation.     

Conversion of 4-androstene-3,17-dione to testosterone by Pisum sativum

4-Androstene-3,17-dione-[4-¹⁴C] was applied to the leaves of growing pea plants, Pisum sativum. Within a week, 28% of the administered steroid was specifically reduced to testosterone. Part of the testosterone was present in esterified form, and 5α-androstane-3β,17β-diol was also identified as a metabolite, but neither epitestosterone nor estrogens were detected.     

Steroid metabolism by human breast and rat mammary carcinomata

The metabolism of dehydroepiandrosterone (DHA) and testosterone by both human breast carcinomata and dimethylbenzanthracene (DMBA)-induced rat mammary carcinomata has been investigated.The rat and human carcinomata converted DHA to testosterone and both DHA and testosterone to 5α-dihydrotestosterone, 5α-androstanediol and 16α-hydroxytestosterone. Tentative evidence is also presented to indicate that some rat adenocarcinomata can convert androgen precursors into estradiol-17β.Although quantitative differences between incubations occurred, the spectrum of steroid transformations was similar in both human and rat tumours. The DMBA-induced rat tumour may therefore prove to be a valuable experimental model for human carcinoma tissue with regard to further steroidogenic studies.