Testosterone Metabolites Research Articles

Neuroactive steroids, their metabolites, and neuroinflammation

Neuroinflammation represents a common feature of many neurodegenerative diseases implicated both in their onset and progression. Neuroactive steroids act as physiological regulators and protective agents in the nervous system. Therefore, the attention of biomedical research has been recently addressed in evaluating whether neuroactive steroids, such as progestagens, androgens, and estrogens may also affect neuroinflammatory pathways. Observations so far obtained suggest a general anti-inflammatory effect with a beneficial relapse on several neurodegenerative experimental models, thus confirming the potentiality of a neuroprotective strategy based on neuroactive steroids. In this scenario, neuroactive steroid metabolism and the sophisticated machinery involved in their signaling are becoming especially attractive. In particular, because metabolism of neuroactive steroids as well as expression of their receptors is affected during the course of neurodegenerative events, a crucial role of progesterone and testosterone metabolites in modulating neuroinflammation and neurodegeneration may be proposed. In the present review, we will address this issue, providing evidence supporting the hypothesis that the efficacy of neuroactive steroids could be improved through the use of their metabolites.

Patterns of fecal hormones in a fenced population of white‐tailed deer

AbstractAnalysis of voided feces has become a popular method of monitoring physiological parameters of many wildlife species. During the breeding seasons of 2006–2007 and 2007–2008, we collected fecal samples to determine concentrations of fecal glucocorticoid (FG) and fecal testosterone (FT) metabolites in a fenced population of white‐tailed deer (Odocoileus virginianus). The purpose of the study was to determine how concentrations of FG and FT metabolites fluctuated throughout the breeding season, whether there was a difference between males and females, and whether there was a relationship between the two hormones. FG and FT metabolites of males peaked during the rut, while female glucocorticoid levels remained relatively stable throughout the breeding season. We also observed a significant positive correlation between FG and FT metabolites. During the pre‐rut and rut, males that exhibit high testosterone also had elevated glucocorticoid metabolite concentrations, but in the post‐rut there was no relationship between FT and FG concentrations. We hypothesize that the effect of testosterone on glucocorticoid secretion is partially a result of testosterone‐induced behavior, and not strictly a physiological link between the two hormones. © 2012 The Wildlife Society.

Investigations on hydrogen isotope ratios of endogenous urinary steroids: reference‐population‐based thresholds and proof‐of‐concept

Carbon isotope ratio (CIR) analysis has been routinely and successfully used in sports drug testing for many years to uncover the misuse of endogenous steroids. One limitation of the method is the availability of steroid preparations exhibiting CIRs equal to endogenous steroids. To overcome this problem, hydrogen isotope ratios (HIR) of endogenous urinary steroids were investigated as a potential complement; results obtained from a reference population of 67 individuals are presented herein. An established sample preparation method was modified and improved to enable separate measurements of each analyte of interest where possible. From the fraction of glucuronidated steroids; pregnanediol, 16-androstenol, 11-ketoetiocholanolone, androsterone (A), etiocholanolone (E), dehydroepiandrosterone (D), 5α- and 5β-androstanediol, testosterone and epitestosterone were included. In addition, sulfate conjugates of A, E, D, epiandrosterone and 17α- and 17β-androstenediol were considered and analyzed after acidic solvolysis. The obtained results enabled the calculation of the first reference-population-based thresholds for HIR of urinary steroids that can readily be applied to routine doping control samples. Proof-of-concept was accomplished by investigating urine specimens collected after a single oral application of testosterone-undecanoate. The HIR of most testosterone metabolites were found to be significantly influenced by the exogenous steroid beyond the established threshold values. Additionally, one regular doping control sample with an extraordinary testosterone/epitestosterone ratio of 100 without suspicious CIR was subjected to the complementary methodology of HIR analysis. The HIR data eventually provided evidence for the exogenous origin of urinary testosterone metabolites. Despite further investigations on HIR being advisable to corroborate the presented reference-population-based thresholds, the developed method proved to be a new tool supporting modern sports drug testing procedures.

Genetic Variation in SLCO2B1 is Associated with Serum Levels of Testosterone and its Metabolites Prior to and Two Days after Testosterone Administration

Organic anion transporting polypeptides (OATP/SLCO) 2B1 and 1B3 mediate the cellular influx of androgens. Very few studies have specifically evaluated genetic variation and androgen transport. The aim of this study was to evaluate if human genetic variation in SLCO2B1 and SLCO1B3 genes are associated with the disposition of testosterone and five of its metabolites before and two days after the administration of a single intramuscular dose (500 mg) of testosterone enanthate in healthy volunteers (N=43). Serum concentration of testosterone and its metabolites were determined by GCMS/LCMS. Two functional polymorphisms in SLCO2B1 (rs12422149) and SLCO1B3 (rs4149117) genes were characterized by allelic discrimination. These SNPs have been hitherto shown to markedly affect the cellular uptake of hormones and hence, they were selected. Individuals homozygous for the SLCO2B1 G-allele displayed lower levels of total testosterone prior to (5.09 ng/ml) and two days after (13.58 ng/ml) testosterone injection, as compared to individuals expressing the A-allele (6.01 ng/ml and 21.88 ng/ml, respectively) (p=0.05 and p=0.002, respectively). Moreover, the concentration of the testosterone metabolites (dihydrotestosterone (DHT), androsterone, 5α-androstane- 3α,17β-diol-17-glucuronide) were significantly lower in subjects with GG genotype at 48 hours post-dose. Individuals homozygous for the SLCO2B1 G allele had a smaller increase in serum testosterone and DHT levels (0.03-fold/kg and 0.02-fold/kg) compared to carriers of the A allele (0.05-fold/kg and 0.03-fold/kg) (p=0.04 and p=0.02, respectively). The SLCO1B3 polymorphism did not show any significant association with serum testosterone levels or the other androgens metabolites investigated. In conclusion, here we report, for the first time to the best of our knowledge, that genetic variation in SLCO2B1 gene impacts the systemic concentrations of testosterone and some of its metabolites, after administration of a single-dose testosterone enanthate. These data also serve as a foundation to examine in the future the role of human genetic variation in SLCO2B1 in relation to testosterone pharmacokinetics, particularly in the context of sports medicine and doping testing or clinical use as a replacement therapy. Keywords: Diagnostics for sports medicine, Doping and genetic testing, genetic variation, personalized medicine, SLCO2B1, testosterone.

Sex, stress and social status: Patterns in fecal testosterone and glucocorticoid metabolites in male Ethiopian wolves

Ethiopian wolves, Canis simensis, live in large multi-male family packs, where males are philopatric and do not disperse. Within a pack, mating and breeding is largely monopolized by the dominant male and female, although extra-pack copulations are common, and subordinate males may sire pups in neighboring packs. Regardless of paternity, all males in a pack help rear the pups. We non-invasively studied patterns in fecal testosterone and glucocorticoid metabolite concentrations using radioimmunoassays of fecal samples collected from nine wild male Ethiopian wolves between August 2007 and February 2008. We tested the predictions of the Challenge Hypothesis, namely that fecal testosterone metabolite concentrations would be higher during the annual mating season, which is the portion of the reproductive cycle when mating and increased aggression typically occur, and lower when there were pups in the pack for which to care. Contrary to the predictions of the Challenge Hypothesis, we did not detect patterns in fecal testosterone metabolite concentrations associated with reproductive stage during our study period. Similarly, we found no patterns associated with reproductive stage in male fecal glucocorticoid metabolite concentrations. Dominant males had higher average fecal testosterone and glucocorticoid metabolite concentrations than did subordinates, which may be related to higher rates of aggression and mate guarding in dominant males of group-living canids, a pattern also reported in African wild dogs, Lycaon pictus.

Testosterone effect on the expression of genes that mediate testosterone metabolism and genes that mediate the effect of those metabolites on the prostate

AimsThe aim of this study was to investigate the effect of testosterone treatment on the proliferation index and the mRNA expression levels of 5α-reductase, CYP7B1, androgen receptor (AR), and estrogen receptor β (ΕRβ) in the canine prostate. Main methodsImmature dogs were treated with testosterone for one month, after which prostate gland growth was assessed by comparing the proliferation index in prostates from testosterone-treated dogs with that of untreated control dogs. The relative mRNA expression levels of the aforementioned genes in the prostate glands of testosterone-treated and untreated dogs were determined by real time PCR. Key findingsTestosterone treatment induced a highly significant reduction in proliferation index in prostate gland. This inhibition of prostate gland growth was associated with differential mRNA expression of 5α-reductase, CYP7B1, AR, and ΕRβ by the prostate gland of testosterone-treated dogs, as compared to that of untreated dogs. While the expression levels of 5α-reductase and CYP7B1 mRNA were significantly down-regulated by testosterone treatment, the expression level of ER-β mRNA was highly up-regulated. In contrast, AR mRNA expression was not significantly altered. SignificanceProstate gland proliferation appeared to be associated with the expression levels of genes that encode proteins that control intra-prostatic levels of testosterone metabolites and their respective receptors. Testosterone treatment may regulate gene expression in the prostate to generate a phenotype that suppresses growth-promoting signaling through AR and enhances anti-proliferative signaling through ERβ. Therefore, targeting disturbances of this genetic machinery in benign prostate hyperplasia and prostate cancer is of a therapeutic potential.

Differential effects of testosterone metabolites oestradiol and dihydrotestosterone on oxidative stress and carotenoid-dependent colour expression in a bird

Despite extensive research, the potential costs that keep secondary sexual traits honest and evolutionary stable remain somewhat elusive. Many carotenoid-based signals are regulated by testosterone (T), which has been suggested to impose a cost to the signaller by suppression of the immune system or an increase in oxidative stress. Results are, however, inconsistent, which may be due to the fact that T can be metabolised to both 5α-dihydrotestosterone (DHT, a potent androgen) and oestradiol (E2, a potent oestrogen). To evaluate for the first time the independent effect of these testosterone metabolites on oxidative status, circulating carotenoids and a carotenoid-dependent sexual signal, we administered DHT and E2 to captive non-breeding adult kestrels Falco tinnunculus of both sexes. E2 increased oxidative damage and downregulated the antioxidant barrier without affecting colouration or circulating carotenoids. In contrast, DHT did not affect oxidative status, but increased skin redness, again without affecting circulating carotenoids. No sex-specific effects were found. These results suggest that the pro-oxidant activity of T could be induced indirectly by its metabolite, E2, whereas the other metabolite, DHT, stimulates signal expression. Finally, the study shows that changes in oxidative damage or antioxidant status of plasma were not correlated with either skin redness or circulating carotenoids.

Influence of Testosterone Metabolites on Song-Control System Neuroplasticity during Photostimulation in Adult European Starlings (Sturnus vulgaris)

The song-control system is a network of discrete nuclei in the songbird brain that controls the production and learning of birdsong and exhibits some of the best-studied neuroplasticity found in the adult brain. Photoperiodic growth of the song-control system during the breeding season is driven, at least in part, by the gonadal steroid testosterone. When acting on neural tissue, however, testosterone can be metabolized into 5α-dihydrotestosterone (DHT) or 17β-estradiol (E2), which activate different hormonal signaling pathways. By treating adult starlings with both testosterone metabolites and metabolite antagonists, we attempted to isolate the effects of androgen and estrogen treatment on neuroplasticity during photostimulation in male and female European starlings (Sturnus vulgaris). Photostimulation resulted in a large HVC volume typical of the breeding season in all treatments independent of hormone treatment. E2 had additional effects on HVC growth by reducing neuron density and enhancing early survival of new neurons recruited to HVC in females but did not significantly affect HVC volume. Conversely, DHT reduced the migration of new neurons, assessed by the expression of doublecortin, to HVC. DHT also increased syrinx mass and maintained RA (robust nucleus of the arcopallium) cytoarchitecture in the presence of aromatase inhibitors. In addition, we document the first evidence of sex-specific neuroplastic responses of the song-control system to androgens and estrogens. These findings suggest that the contributions of DHT and E2 signaling in songbird neuroplasticity may be regulated by photoperiod and that future studies should account for species and sex differences in the brain.

Sociodemographic Correlates of Fecal Androgen Levels in Wild Male White-Handed Gibbons (Hylobates lar)

Despite the central role of testosterone in influencing many aspects of the male life cycle, information on the influence of social and behavioral factors associated with androgen output is available for only a few primate species, mainly those living in multimale–multifemale societies. We collected 322 fecal samples and measured fecal testosterone metabolite levels in 19 adult male white-handed gibbons (Hylobates lar) from 13 groups living in the Khao Yai National Park, Thailand, to examine the extent to which androgen output is related to sociodemographic variables (social organization, social status, presence of infants, age) in a socially flexible species, i.e., pair-living/multimale groups. We predicted that androgen levels would be higher in males 1) that live in potentially more unstable single-male/single-female units; 2) that are primary (dominant) vs. secondary; 3) that live in groups with dependent infants; and 4) that are of prime vs. senior age. We found marked differences in androgen levels among males and a significant effect of time of the year. Males living in pairs exhibited significantly higher androgen concentrations vs. males living in multimale groups, and males residing in groups with infants showed a significant increase in androgen levels throughout the sampling year. However, we found no relationship with male age or social status. The increased androgen output found in both pair-living males and those residing with infants might be a physiological mechanism that facilitates male aggression to cope with potential threats posed by social challenges and the demand for infant/group defense, respectively. More generally, our data indicate that in socially flexible species male androgen output is less strongly linked to key life-history variables than in species that live permanently in multimale–multifemale groups.

Selective Cleavage of Human Sex Hormone-Binding Globulin by Kallikrein-Related Peptidases and Effects on Androgen Action in LNCaP Prostate Cancer Cells

Stimulation of the androgen receptor via bioavailable androgens, including testosterone and testosterone metabolites, is a key driver of prostate development and the early stages of prostate cancer. Androgens are hydrophobic and as such require carrier proteins, including sex hormone-binding globulin (SHBG), to enable efficient distribution from sites of biosynthesis to target tissues. The similarly hydrophobic corticosteroids also require a carrier protein whose affinity for steroid is modulated by proteolysis. However, proteolytic mechanisms regulating the SHBG/androgen complex have not been reported. Here, we show that the cancer-associated serine proteases, kallikrein-related peptidase (KLK)4 and KLK14, bind strongly to SHBG in glutathione S-transferase interaction analyses. Further, we demonstrate that active KLK4 and KLK14 cleave human SHBG at unique sites and in an androgen-dependent manner. KLK4 separated androgen-free SHBG into its two laminin G-like (LG) domains that were subsequently proteolytically stable even after prolonged digestion, whereas a catalytically equivalent amount of KLK14 reduced SHBG to small peptide fragments over the same period. Conversely, proteolysis of 5α-dihydrotestosterone (DHT)-bound SHBG was similar for both KLKs and left the steroid binding LG4 domain intact. Characterization of this proteolysis fragment by [(3)H]-labeled DHT binding assays revealed that it retained identical affinity for androgen compared with full-length SHBG (dissociation constant = 1.92 nM). Consistent with this, both full-length SHBG and SHBG-LG4 significantly increased DHT-mediated transcriptional activity of the androgen receptor compared with DHT delivered without carrier protein. Collectively, these data provide the first evidence that SHBG is a target for proteolysis and demonstrate that a stable fragment derived from proteolysis of steroid-bound SHBG retains binding function in vitro.

Certification of steroid carbon isotope ratios in a freeze‐dried human urine reference material

An accurate method for the measurement of carbon isotope ratios of steroids in human urine has been developed at the National Measurement Institute, Australia (NMIA) for the certification of a freeze-dried human urine reference material (CRM NMIA MX005). The method measures δ(13)C values by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) analysis following hydrolysis, solvent extraction and high performance liquid chromatography (HPLC) purification. Reference δ(13)C values for testosterone metabolites etiocholanolone, androsterone, and endogenous reference compounds (ERCs) 11β-hydroxyandrosterone and pregnanediol were determined, as well as information δ(13)C values for testosterone, epitestosterone, 11-oxoetiocholanolone, and a range of differences (Δ(13)C) between testosterone metabolites and ERCs. The measurement uncertainty was rigorously evaluated with expanded uncertainties for the reference δ(13)C values between 1.1 and 1.6 ‰ at the 95% coverage level.

Testosterone metabolite nonessential for androgen effects

Testosterone metabolite nonessential for androgen effects

Comparative GC-MS Based In vitro Assays of 5α-Reductase Activity Using Rat Liver S9 Fraction

α-Dihydrotestosterone (DHT) is the primary active metabolite of testosterone, catalyzed by 5α-reductase (5αR) in the skin, prostate, and liver. In this study, the 5αR activity in rat liver S9 fraction in the presence of a NADPH-generating system was evaluated and compared by gas chromatography-mass spectrometry (GC-MS)-based in vitro assays. Testosterone and a 5αR inhibitor, finasteride, were added to the S9 fractions and incubated at 37 o C for 1 h. Both testosterone and DHT were quanti- tatively measured and compared with two different GC-MS-based steroid profiling techniques. DHT was not detected by con- ventional GC-MS analysis in the absence of finasteride when the concentration of testosterone in the S9 fraction was less than 0.2 µM, whereas the isotope-dilution GC-MS (GC-IDMS) system was able to evaluate the 5αR activity. Because the S9 fraction contains more reactive enzymes and is easier to collect from tissues compared with a microsomal solution, the combination of the S9 fraction and GC-IDMS technique may be a promising assay for evaluating the 5αR activity in large-scale clinical studies.

Hyperinsulinemia Amplifies GnRH Agonist Stimulated Ovarian Steroid Secretion in Women with Polycystic Ovary Syndrome

In vitro data show that insulin may enhance basal and LH-stimulated ovarian androgen secretion, particularly in theca cells from women with polycystic ovary syndrome (PCOS). However, in vivo studies gave inconsistent results. The objective of the study was to assess whether hyperinsulinemia affects in vivo ovarian steroid secretion and steroid metabolism. This was a controlled cross-sectional study, conducted in a tertiary care academic center. Nine young PCOS women participated in the study. Participants were submitted, in two separate days, to a GnRH agonist stimulation (buserelin 100 μg, s.c.), during a 17-h hyperinsulinemic (80 mU/m(2) · min) euglycemic clamp and, as a control, during saline infusion. Adrenal steroid secretion was suppressed by dexamethasone. During both protocols, before and after GnRH agonist stimulation, serum insulin, gonadotropins, cortisol, progesterone, 17-hydroxyprogesterone, androstenedione, testosterone, estradiol, and urinary androgen metabolites were measured. Insulin increased from 25.1 ± 13.3 to 341.5 ± 102.6 mU/liter during the clamp, whereas it did not significantly change during saline infusion. Baseline steroids and gonadotropins were similar in the two protocols. During hyperinsulinemia, GnRH agonist-stimulated serum progesterone and androstenedione were significantly higher than during saline infusion, and 17-hydroxyprogesterone was of borderline significance. Moreover, 24 h after GnRH agonist stimulation, testosterone was higher after hyperinsulinemia. Serum gonadotropins and estradiol response did not differ between the protocols. Urinary androgen metabolites excretion significantly increased after GnRH agonist stimulation, but the increase was similar during insulin and saline infusions. These in vivo data show that sustained hyperinsulinemia potentiates gonadotropin-stimulated ovarian androgen steroidogenesis. Insulin-induced increase in ovarian hormone secretion is not accompanied by an increased steroid metabolism.

The differences between aromatizable and non-aromatizable androgens in relation to body composition and metabolic syndrome risk factors in men

The relationships between the parameters of metabolic syndrome and non-aromatizable metabolites of testosterone have been discussed in literature. Some papers describe these metabolites as one of the possible causes of male-type obesity. On the contrary, other studies show a protective influence of dihydrotestosterone on visceral obesity.The aim of this study to analyse the relationship between anthropometric parameters, lipid spectrum, glycemia and the level of endogenous testosterone and dihydrotestosterone, and to compare the effects of these androgens. Our population-based study involved 232 healthy men ranging from 20 to 78 years with BMI 18 to 39kg/m2. Serum testosterone, dihydrotestosterone and sex hormone binding globulin SHBG levels, lipid spectrum, glucose metabolism parameters were measured and the oral glucose tolerance test was carried out in all subjects. Their anthropometric parameters (weight, height, waist, hips, waist-to-hip ratio, 14 skin folds) and body composition parameters were determined and calculated by the Antropo program. Multiple regression analysis showed a correlation between hormonal levels, esp. of testosterone and dihydrotestosterone, and the anthropometric data, lipid spectrum and parameters of glucose regulation. Low testosterone and/or dihydrotestosterone was correlated to a higher body-mass index, fat content, waist diameter, total-, HDL-, LDL-cholesterol and triglycerides, fasting glucose, insulin resistance and lower muscle and bone mass. In addition, statistical analysis using multivariate regression with reduction in dimensionality did not discover any striking difference between aromatizable and non-aromatizable androgens in their association to lipid and glucose metabolism parameters in healthy, normosthenic men. In conclusion, the association of endogenous testosterone and dihydrotestosterone to anthropometric data, lipid spectrum and insulin sensitivity are of the same quality; however, the effect of the circulating levels of dihydrotestosterone is quantitatively smaller.

Testosterone and tusks: maturation and seasonal reproductive patterns of live, free-ranging male dugongs (Dugong dugon) in a subtropical population

Knowledge of male reproductive status and activity in free-ranging animals is vital to understanding reproductive patterns and population dynamics. Until now, almost all information regarding reproductive behavior of the dugong, a cryptic marine mammal, has relied on post-mortem examination. We examined the relationships between body length, tusk eruption (secondary sexual characteristic), seasonality, and group association on fecal testosterone metabolite concentrations in 322 free-ranging dugongs (159 males, 163 females) in subtropical Moreton Bay, Australia. Fecal testosterone concentrations demonstrated biologically meaningful differences in testicular activity between sexes and across reproductive/age classes, and were correlated with circulating concentrations in serum. Male dugongs have a pre-reproductive period that persists until a body length of 240 cm is achieved. Puberty apparently occurs between 240 and 260 cm body length when fecal testosterone levels increase fourfold (>500 ng/g) over juvenile levels, and is associated with tusk eruption. However, social maturity may be delayed until male dugongs are larger than 260 cm with well-developed tusks. In mature males, the lowest (<500 ng/g) fecal testosterone concentrations occur in the austral autumn months with maximal concentrations in September-October, coincident with the onset of a spring mating season. During spring, solitary mature males had fecal testosterone concentrations double those of mature males sampled within groups, potentially suggesting a mating strategy involving roving of reproductively active males. This study demonstrates that single-point physiological data from individuals across a population have value as indicators of reproductive processes. Our approach provides an efficacious non-lethal method for the census of reproductive status and seasonality in live male dugongs.

Concerns About Serum Androgens Monitoring During Testosterone Replacement Treatments in Hypogonadal Male Athletes: A Pilot Study

A well-tailored testosterone replacement treatment (TRT) in male hypogonadal athletes plays a pivotal role to restore physiological performances, to reduce health risks, and to guarantee the ethic of competition. Few studies evaluated individual androgens profiles during TRT in trained individuals. The aim of this article was to verify the efficacy in restoring eugonadal serum and urinary androgens profiles after testosterone enanthate (TE) and gel (TG) administration. Ten male Caucasian-trained volunteers affected by severe hypotestosteronemia (<8 nmol/L) were included. Serum androgens and urinary testosterone metabolites were evaluated, in the same subjects, before and weekly for 5 weeks after both a single intramuscular TE injection (250 mg) and during a daily administration of TG (50 mg/die of testosterone), respectively. The main outcome measures of this article were serum total testosterone (TT), dihydrotestosterone (DHT), calculated free and bioavailable testosterone (cFT, cBioT), 17-β-estradiol, and urinary glucuronide testosterone metabolites. Supraphysiological TT concentrations were observed in 50% of our volunteers until 7 days after TE and in the 4% of total samples after TG. Serum DHT was high both after TE (all volunteers on day 7 and 50% on day 14) and during TG (32% of total samples). A relatively low number of samples showed normal cFT and cBioT both after TE and TG (20-44%, respectively). Urinary metabolites were related to the type of treatment and to serum androgens profile and resulted in the normal ranges from 15% to 60% of total samples. Besides well-known variations of mean serum TT, we showed a high percentage of serum and urinary samples with abnormal androgens, being TG safer than TE. We conclude that monitoring TRT with TT only may be inaccurate because of abnormal fluctuations of other circulating androgens. Further studies to identify the appropriate markers of eugonadism during TRT are highly warranted both in athletes and in non-athletes.

Aromatase and 5α-reductase type 2 mRNA in the green anole forebrain: An investigation of the effects of sex, season and testosterone manipulation

Aromatase and 5α-reductase (5αR) catalyze the synthesis of testosterone (T) metabolites: estradiol and 5α-dihydrotestosterone, respectively. These enzymes are important in controlling sexual behaviors in male and female vertebrates. To investigate factors contributing to their regulation in reptiles, male and female green anole lizards were gonadectomized during the breeding and non-breeding seasons and treated with a T-filled or blank capsule. In situ hybridization was used to examine main effects of and interactions among sex, season, and T on expression of aromatase and one isozyme of 5αR (5αR2) in three brain regions that control reproductive behaviors: the preoptic area, ventromedial nucleus of the amygdala and ventromedial hypothalamus (VMH). Patterns of mRNA generally paralleled previous evaluations of intact animals. Although no main effects of T were detected, interactions were present in the VMH. Specifically, the density of 5αR2 expressing cells was greater in T-treated than control females in this region, regardless of season. Among breeding males, blank-treated males had a denser population of 5αR2 positive cells than T-treated males. Overall, T appears to have less of a role in the regulation of these enzymes than in other vertebrate groups, which is consistent with the primary role of T (rather than its metabolites) in regulation of reproductive behaviors in lizards. However, further investigation of protein and enzyme activity levels are needed before specific conclusions can be drawn.

Regional differences in the vasorelaxing effects of testosterone and its 5-reduced metabolites in the canine vasculature

Although the vasorelaxing effects of testosterone (T) and various androgen metabolites have been observed in a variety of blood vessels and species, previous studies have not systematically compared the vasorelaxing effects of androgen metabolites in different vascular beds within the same species. Therefore, we studied the vasorelaxing effects of T and its 5-reduced metabolites (5α- and 5β-DHT) on KCl-induced contractions of the canine left coronary artery, femoral artery and saphenous vein, using standard isometric recordings. KCl contractions were inhibited by each androgen in a concentration-dependent manner from 1.8 to 310μM. Vascular sensitivity and efficacy were expressed as inhibitory concentration 50 (IC50) and maximal relaxation (Rmax), respectively. The coronary artery was significantly more sensitive to androgen-induced vasorelaxation than the saphenous vein or femoral artery. These vasorelaxing responses were unaffected by an antiandrogen (Flutamide) or the sulfhydryl reagent, N-ethylmaleimide, suggesting a nongenomic mechanism independent of signaling mediated by the androgen receptor or G proteins. Concentration–response curves were unchanged in endothelium-denuded preparations; thus, the endothelium appears to have no role in androgen-induced vasorelaxation. 5β-DHT was the most potent androgen in both coronary and femoral artery, but all three androgens were equipotent in the saphenous vein. It is concluded that: 1) significant regional differences exist in vasorelaxing effects of androgen metabolites in the canine vasculature; 2) structural differences in these androgens determine their vasorelaxing efficacy; and 3) regional differences in androgen-induced vasorelaxation may account for some of the conflicting findings reported on the vasorelaxing effects of the androgens.

5α-Reductase, an enzyme regulating glucocorticoid action in the testis of Rhinella arenarum (Amphibia: Anura)

The reduction of A-ring of glucocorticoids to produce 5α-dihydro-derivatives by 5α-reductases has been considered as a pathway of irreversible inactivation. However, 5α-reduced metabolites of corticosterone and testosterone have significant biological activity. In this paper, we investigated whether toad testicular 5α-reductase (5α-Red) is able to transform corticosterone into 5α-dihydrocorticosterone. Furthermore, we studied the role of 5α-reduced metabolite of corticosterone as a glucocorticoid receptor (GR) agonist. The activity of 5α-Red was assayed in subcellular fractions with [3H]corticosterone or [3H]testosterone as substrate. The enzyme localizes in microsomes and its optimal pH is between 7 and 8. The activity is not inhibited by finasteride. These results support the conclusion that toad 5α-Red resembles mammalian type 1 isoenzyme. Kinetic studies indicate that neither Km nor Vmax for both corticosterone and testosterone were significantly different among reproductive periods. The Km value for testosterone was significantly higher than that for corticosterone, indicating that the C-21 steroid is the preferred substrate for the enzyme. Studies of the binding capacity of 5α-dihydrocorticosterone (5α-DHB) to the testicular GR show that 5α-DHB is able to displace the binding of [3H]dexamethasone to testicular cytosol with a similar potency than corticosterone. The inhibition constant (Ki) values for corticosterone and 5α-DHB were similar, 31.33±2.9nM and 35.24±2.3nM, respectively. In vitro experiments suggest that 5α-DHB is an agonist of toad testicular GR, decreasing the activity of the key enzyme for androgen synthesis, the cytochrome P450 17-hydroxylase, C17,20-lyase.