Testosterone Metabolites Research Articles

Effects of transdermal administration of testosterone gel on the urinary steroid profile in hypogonadal men: Implications in antidoping analysis

Testosterone is one of the most abused pseudo-endogenous anabolic steroids in sport doping. The current method adopted to detect the abuse of testosterone and other pseudo-endogenous steroids (endogenous steroids when administered exogenously) is first based on the longitudinal monitoring of several urinary biomarkers, which constitute the so called “steroidal module” of the Athlete Biological Passport (ABP): atypical samples undergo a confirmation analysis based on the measurement of the 13C/12C isotopic ratio of selected target compounds, to distinguish their endogenous or exogenous origin. At the same time, testosterone administration can be allowed in athletes diagnosed with hypogonadism, provided they are granted a therapeutic use exemption by the relevant medical authority.In this pilot study we have investigated whether the approach based on the preliminary determination of the urinary steroid profile, in the format considered in the steroidal module of the ABP, also integrated with the inclusion of the sulfo-conjugates and of additional target steroids, can retain its validity also in the case of hypogonadal athletes. We have studied the effects of a single low dose (40 mg) of testosterone gel (T-gel) on the urinary concentration of the markers of steroidal module of the ABP, as well as on some additional steroid markers. The study was based on the analysis of urinary samples from 19 non-hospitalized hypogonadal men, 10 of them with late-onset hypogonadism (LOH), collected before, after 4 h and after 24 h the transdermal self-administration of 40 mg of T-gel. None of the patient had any co-morbidities possibly affecting the urinary excretion of the steroidal markers. The steroidal markers were quantified by gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) after the enzymatic hydrolysis of the respective glucuro-conjugates and the chemical hydrolysis of the respective sulfo-conjugates. Targeted GC-MS/MS analysis was carried out operating in electron impact (EI) ionization mode, with acquisition in multiple reaction monitoring (MRM) mode.Our preliminary results show that, as expected, the treatment with T-gel leads, in all hypogonadal men, to an increase of the urinary concentration of the glucuro-conjugate metabolites of testosterone and its main metabolites, with special relevance to those with 5α-reduction. Furthermore, samples collected from non-LOH hypogonadal men showed an increase also in the levels of epitestosterone glucuronide, testosterone sulfate and epitestosterone sulfate. Apart from their biochemical and pharmacological relevance, these outcomes could be leveraged to refine the analytical strategy currently followed in the antidoping field for the analysis of the urinary steroidal markers, with potential implications also in other forensic and/or clinical investigations.

Exposure of managed red river hogs (Potamochoerus porcus) to urine from males stimulates estrous cycling and modulates fecal sex steroid metabolites in males and females

For unknown reasons, reproductive success varies among zoos in managed red river hogs. In response to urine exposure from novel conspecifics, we hypothesized that males with low libido would exhibit increased concentrations of testosterone metabolites and that acyclic and/or non-breeding females would be induced to cycle or cycle more regularly. Estrous cycle length and progesterone metabolites in same-sex housed females were compared prior to and following exposure to novel red river hog male urine. Male testosterone metabolites and female progesterone metabolites as well as estrous cycle length were compared among: 1) proven-breeder females and males; 2) non-breeding females newly paired with novel males; 3) non-breeding females and males exposed to urine from novel females and males. Fecal samples were collected 3–5 times per week for eight to 12 months, lyophilized, extracted, and assayed for progesterone and testosterone metabolites with validated enzyme immunoassays. Introduction of female urine resulted in an increased number of estrous cycles per female per month, and decreased luteal and increased follicular progesterone metabolites in females. Introduction of male urine resulted in an increase in testosterone metabolites in males. Average progesterone metabolites for pregnant proven-breeder females were more than double that for pregnant females newly paired to novel males. An interaction between season and treatment group, as well as the acyclicity of females from July through November irrespective of treatment group, suggest that season may confound and warrant judicious interpretation of the results. Additionally, females housed with pregnant females were either acyclic or did not carry their pregnancies to term, indicating that reproductive suppression may occur in females. In conclusion, urine may be a cost-effective and efficient means to manipulate estrous cycling in managed red river hogs. Furthermore, careful consideration of the number of females in a managed herd is recommended to avoid reproductive suppression.

The role of steroid hormones and individual traits in food intake in the wood mouse (Apodemus sylvaticus).

Availability of food resources affects animal survival and reproduction. Thus, coping with changes in food availability is one of the most crucial behavioural and physiological processes in wildlife. Food intake is a key concept in animal ecology that is directly conditioned by food quality and abundance or diet choice, but may also vary according to individual-related factors (e.g. foraging behaviours, social rank or energy-demanding periods) and the influence of the endocrine system on energy metabolism. Here, we studied food intake in relation to individual characteristics (sex, breeding condition and age) and whether steroid hormones (testosterone and corticosterone metabolites) mediate food intake in wild wood mouse (Apodemus sylvaticus). Field work was carried out in February-March 2014 in Monte de Valdelatas (Madrid, Spain). Wood mice were live-trapped for 10 consecutive days in four independent plots. Traps were baited with 4g of toasted corn and food intake was calculated by subtracting the remaining bait found inside traps. Fresh faecal samples from 130 different wood mice were collected and faecal testosterone and corticosterone metabolites (FTM and FCM, respectively) were analysed by enzyme immunoassays. Food intake was higher in females than males, probably due to greater energy requirements. Non-breeders and young individuals also showed a higher food intake. These individuals usually hold a lower social rank which is associated to limited food resources because of dominants; thus, increased food intake may be a result of freely exploit food bait inside traps while avoiding risky competition. In addition, food intake negatively correlated with FTM levels and positively with FCM levels indicating that both hormones have an active role mediating food intake in the wood mouse. Our data suggest that food intake is a function of both individual traits and the endocrine system that accordingly respond throughout different energy-demanding periods.

Design and development of letrozole nanoemulsion: A comparative evaluation of brain targeted nanoemulsion with free letrozole against status epilepticus and neurodegeneration in mice.

The target of the current study is to formulate letrozole loaded nanoemulsion (LET-NE) for the direct nose to brain delivery to reduce peripheral effects of letrozole (LET). LET-NE is compared against intraperitoneally administered free LET in kainic acid (KA) induced status epilepticus (SE) in mice. LET loaded nanoemulsion (LET-NE) was prepared by aqueous microtitration method using Triacetin, Tween 80 and PEG-400 as the oil phase, surfactant, and co-surfactant. Nanoemulsion was studied for droplet size, polydispersity index (PDI), zeta potential, percentage transmittance, drug content, surface morphology. TEM images of developed formulation demonstrated spherical droplets with a mean diameter of 95.59 ± 2.34 nm, PDI of 0.162 ± 0.012 and zeta potential of -7.12 ± 0.12 mV respectively. In in-vitro and ex-vivo drug release, LET-NE showed prolonged drug release profile as compared to suspension. SE was induced by KA (10 mg/kg, i.p.) in Swiss albino mice. Behavioral seizure monitoring, biochemical estimations, and histopathological examination were performed. The onset time of SE was significantly enhanced and % incidence of SE was reduced by intranasal administration of LET-NE as compared to KA and LET administered intraperitoneally. Biochemical estimations revealed that LET-NE effectively decreased levels of 17-β estradiol while the levels of 5α-Dihydrotestosterone (5α-DHT) and 3α-androstanediol (3α-Diol) were significantly increased in the hippocampus. In cresyl violet staining LET-NE showed better protection of the hippocampus from neurotoxicity induced by KA as compared to LET. Also, in gamma scintigraphy of mouse brain, intranasal administration of nanoemulsion exhibited the presence of high concentration of LET. The study demonstrates the anticonvulsant and neuroprotective effect of LET-NE probably by inhibition of aromatization of testosterone into 17-β estradiol, proconvulsant, and diverting the pathway into the synthesis of testosterone metabolites, 3α-Diol with known anticonvulsant and neuroprotective action. Brain targeting of LET-NE showed better anticonvulsant and neuroprotective action than LET.

Major glucuronide metabolites of testosterone are primarily transported by MRP2 and MRP3 in human liver, intestine and kidney

Testosterone glucuronide (TG), androsterone glucuronide (AG), etiocholanolone glucuronide (EtioG) and dihydrotestosterone glucuronide (DHTG) are the major metabolites of testosterone (T), which are excreted in urine and bile. Glucuronides can be deconjugated to active androgen in gut lumen after biliary excretion, which in turn can affect physiological levels of androgens. The goal of this study was to quantitatively characterize the mechanisms by which TG, AG, EtioG and DHTG are eliminated from liver, intestine, and kidney utilizing relative expression factor (REF) approach. Using vesicular transport assay with recombinant human MRP2, MRP3, MRP4, MDR1 and BCRP, we first identified that TG, AG, EtioG, and DHTG were primarily substrates of MRP2 and MRP3, although lower levels of transport were also observed with MDR1 and BCRP vesicles. The transport kinetic analyses revealed higher intrinsic clearances of TG by MRP2 and MRP3 as compared to that of DHTG, AG, and EtioG. MRP3 exhibited higher affinity for the transport of the studied glucuronides than MRP2. We next quantified the protein abundances of these efflux transporters in vesicles and compared the same with pooled total membrane fractions isolated from human tissues by quantitative LC–MS/MS proteomics. The fractional contribution of individual transporters (ft) was estimated by proteomics-based physiological scaling factors, i.e., transporter abundance in whole tissue versus vesicles, and corrected for inside-out vesicles (determined by 5′-nucleotidase assay). The glucuronides of inactive androgens, AG and EtioG were preferentially transported by MRP3, whereas the glucuronides of active androgens, TG and DHTG were mainly transported by MRP2 in liver. Efflux by bile canalicular transport may indicate the potential role of enterohepatic recirculation in regulating the circulating active androgens after deconjugation in the gut. In intestine, MRP3 possibly contributes most to the efflux of these glucuronides. In kidney, all studied glucuronides seemed to be preferentially effluxed by MRP2 and MDR1 (for EtioG). These REF based analysis need to be confirmed with in vivo findings. Overall, characterization of the efflux mechanisms of T glucuronide metabolites is important for predicting the androgen disposition and interindividual variability, including drug-androgen interaction in humans. The mechanistic data can be extrapolated to other androgen relevant organs (e.g. prostate, testis and placenta) by integrating these data with quantitative tissue proteomics data.

Regulation of gonadotrophin-releasing hormone secretion by testosterone in male sheep

In males, including the ram, testosterone, acting via its primary metabolites oestradiol and dihydrotestosterone (DHT), suppresses circulating LH concentrations. This effect is due primarily, although not totally, to decreased frequency of gonadotrophin-releasing hormone (GnRH) pulses. The arcuate-ventromedial region (ARC-VMR) of the mediobasal hypothalamus and possibly the medial preoptic area (mPOA) are sites at which oestradiol acts to suppress GnRH, but the site of DHT action is not known. Given that native GnRH neurones appear to contain few or no oestrogen or androgen receptors, the effects of testosterone metabolites probably are exerted by modulating activity of inhibitory interneurone systems such as beta-endorphin, dopamine, and gamma-aminobutyric acid (GABA). Although beta-endorphin clearly inhibits GnRH secretion, the observation that testosterone treatment during a long-day photoperiod reduced proopiomelanocortin (POMC) mRNA in the arcuate nucleus while coincidentally suppressing GnRH release indicates that beta-endorphin does not mediate the inhibitory effect of testosterone on GnRH. Activation of GABAA receptors in either the mPOA or ARC-VMR suppressed LH, whereas activation of GABAB receptors in the ARC-VMR increased LH pulse amplitude. Therefore, it is suggested that GABA acts in both regions to regulate LH. Whereas testosterone affects GABA metabolism in the rat hypothalamus, its effect in the ram hypothalamus is yet to be determined. Testosterone treatment activated dopaminergic cells in the retrochiasmatic A15 area in the same animals in which it suppressed POMC mRNA in the arcuate nucleus. This dopaminergic system may partially mediate the negative feedback effect of testosterone in the ram analogous to its role in partially mediating the negative effect of oestrogen in the ewe. Future studies must concentrate on determining how these and other putative inhibitory neuronal systems interact and how they in turn are regulated by environmental factors such as photoperiod.

Evaluation of fecal hormones for noninvasive research on reproduction and stress in humpback whales (Megaptera novaeangliae)

Fecal hormone analysis shows high potential for noninvasive assessment of population-level patterns in stress and reproduction of marine mammals. However, the marine environment presents unique challenges for fecal sample collection. Data are still lacking on collection methodology and assay validations for most species, particularly for those mysticete whales that have variable diets. In this study we tested collection techniques for fecal samples of free-swimming humpback whales (Megaptera novaeangliae), and validated immunoassays for five steroid and thyroid hormones. Resulting data were used for preliminary physiological validations, i.e., comparisons to independently confirmed sex and reproductive state. Pregnant females had significantly higher fecal progestins and glucocorticoids than did other demographic categories of whales. Two possible cases of previously undetected pregnancies were noted. Males had significantly higher fecal testosterone metabolites than nonpregnant females. Fecal glucocorticoids were significantly elevated in pregnant females and mature males compared to nonpregnant females. Calf fecal samples had elevated concentrations of all fecal hormones. Fecal thyroid hormones showed a significant seasonal decline from spring to summer. Though sample sizes were small, and sampling was necessarily opportunistic, these patterns indicate that noninvasive fecal hormone analysis may facilitate studies of reproduction, stress and potentially energetics in humpback whales.

Sex differences in diurnal cortisol, testosterone and DHEA functioning in incarcerated adolescents

This study evaluated the effect of androsterone (AND), a metabolite of testosterone, on the ability of selected classical and novel antiepileptic drugs to prevent seizures caused by maximal electroshock (MES), which may serve as an experimental model of human generalized tonic-clonic seizures in mice. Single intraperitoneal (i.p.) administration of AND (80 mg kg−1) significantly raised the threshold for convulsions in the MES seizure threshold test. Lower doses of AND (5, 10, 20, and 40 mg kg−1) failed to change the threshold. AND at a subthreshold dose of 40 mg kg−1 significantly enhanced the protective activity of carbamazepine, gabapentin, and phenobarbital against MES-induced seizures decreasing their median effective doses (ED50) values ± SEM from 8.59 ± 0.76 to 6.05 ± 0.81 mg kg−1 (p = 0.0308) for carbamazepine, from 419.9 ± 120.6 to 111.5 ± 41.1 mg kg−1 (p = 0.0405) for gabapentin, and from 20.86 ± 1.64 to 10.0 ± 1.21 mg kg−1 (p = 0.0007) for phenobarbital. There were no significant changes in total brain concentrations of carbamazepine, gabapentin, and phenobarbital following AND administration. This suggests that the enhancing effects of AND on the protective activity of these antiepileptic drugs are not related to pharmacokinetic factors. A lower dose of AND (20 mg kg−1) had no effect on the protective activity of carbamazepine, gabapentin, and phenobarbital. AND administered at a dose of 40 mg kg−1 failed to change the anticonvulsant activity of lamotrigine, oxcarbazepine, phenytoin, topiramate, and valproate in the MES test. In the chimney test, AND given at a dose enhancing the protective activity of carbamazepine, gabapentin, and phenobarbital (which alone was without effect on motor performance of mice) did not affect impairment of motor coordination produced by the antiepileptics. Our findings recommend further preclinical and clinical research on AND in respect of its use as adjuvant therapy in the management of epilepsy in men with deficiency of androgens.

Associations between biological variables involved in estrogen signaling in pre- and post- menopausal women – Findings from the women 40+ healthy aging study

This study evaluated the effect of androsterone (AND), a metabolite of testosterone, on the ability of selected classical and novel antiepileptic drugs to prevent seizures caused by maximal electroshock (MES), which may serve as an experimental model of human generalized tonic-clonic seizures in mice. Single intraperitoneal (i.p.) administration of AND (80 mg kg−1) significantly raised the threshold for convulsions in the MES seizure threshold test. Lower doses of AND (5, 10, 20, and 40 mg kg−1) failed to change the threshold. AND at a subthreshold dose of 40 mg kg−1 significantly enhanced the protective activity of carbamazepine, gabapentin, and phenobarbital against MES-induced seizures decreasing their median effective doses (ED50) values ± SEM from 8.59 ± 0.76 to 6.05 ± 0.81 mg kg−1 (p = 0.0308) for carbamazepine, from 419.9 ± 120.6 to 111.5 ± 41.1 mg kg−1 (p = 0.0405) for gabapentin, and from 20.86 ± 1.64 to 10.0 ± 1.21 mg kg−1 (p = 0.0007) for phenobarbital. There were no significant changes in total brain concentrations of carbamazepine, gabapentin, and phenobarbital following AND administration. This suggests that the enhancing effects of AND on the protective activity of these antiepileptic drugs are not related to pharmacokinetic factors. A lower dose of AND (20 mg kg−1) had no effect on the protective activity of carbamazepine, gabapentin, and phenobarbital. AND administered at a dose of 40 mg kg−1 failed to change the anticonvulsant activity of lamotrigine, oxcarbazepine, phenytoin, topiramate, and valproate in the MES test. In the chimney test, AND given at a dose enhancing the protective activity of carbamazepine, gabapentin, and phenobarbital (which alone was without effect on motor performance of mice) did not affect impairment of motor coordination produced by the antiepileptics. Our findings recommend further preclinical and clinical research on AND in respect of its use as adjuvant therapy in the management of epilepsy in men with deficiency of androgens.

Does the 2nd and 4th digit ratio reflect prenatal androgen exposure?

The aim of our study was to describe the effect of prenatal testosterone exposure on 2D:4Din both sexes, and todeterminewhetherthis effectismediated via the androgen receptor. In addition, the sex differences in lengths of 2D, 4D, and 2D:4D ratio were analyzed. Clinical studies suggest a negative correlation between prenatal testosterone exposure and ratio of thelengths ofthe second and fourth digits (2D:4D). However,less is known about the underlying molecular mechanisms. Pregnant rats were treated with olive oil, testosterone, flutamide or testosterone with flutamide daily from the fourteenth day of pregnancy until delivery. The finger lengths of adult offspring were measured using both, digital scanning of the paws and μCT analysis of the phalanges. None of the aforementioned methods revealed any effect of testosterone on 2D:4D.μCT measurements showed that prenatal hyperandrogenism in both sexes leads to shorter 2D compared to controls. Moreover, the testosterone treatment in males resulted in the shortening of 4Dwhen compared to controls. Prenatal hyperandrogenism leads to shorter lengths of 2D and 4D;however, itdoes not affect2D:4D ratio. Whether other steroid hormones and/or testosterone metabolites affect the 2D:4D ratio requires further investigation (Tab. 5, Fig. 3, Ref. 32).

The testosterone metabolite 3α-androstanediol inhibits oxidative stress-induced ERK phosphorylation and neurotoxicity in SH-SY5Y cells through an MKP3/DUSP6-dependent mechanism

Testosterone exerts neuroprotective effects on the brain, but the mechanisms by which these effects are exerted appear to be different in males and females. While in females they involve local conversion to estradiol, in males they may be androgen receptor-dependent, or mediated through metabolism to neurosteroids such as 5α-androstane-3α,17β-diol (3α-diol), which acts through different mechanisms than testosterone itself. Recently, we demonstrated that 3α-diol can protect neurons and neuronal-like cells against oxidative stress-induced neurotoxicity associated with prolonged phosphorylation of the extracellular signal-regulated kinase (ERK). The mechanism(s) responsible for these effects remain unknown. In the present study, we sought to determine whether the ERK-specific phosphatase, mitogen-activated protein kinase phosphatase 3/dual specificity phosphatase 6 (MKP3/DUSP6), is involved in the cytoprotective effects of 3α-diol in SH-SY5Y human female neuroblastoma cells. 3α-diol inhibited ERK phosphorylation and ameliorated cell death induced by the oxidative stressor hydrogen peroxide (H2O2). These protective effects were significantly reduced by pre-treatment with the MKP3/DUSP6 inhibitor BCI. In addition, H2O2 decreased expression of MKP3/DUSP6, and this was prevented by co-treatment with 3α-diol. These findings suggest that the protective effects of 3α-diol are mediated through regulation of ERK phosphorylation in neurotoxic conditions and indicate that these effects may be exerted through modulation of MKP3/DUSP6. Targeting the regulation of MKP3/DUSP6 may be beneficial in reducing toxicity under conditions of oxidative stress.

108 Fecal metabolite monitoring as a tool to assess sexual maturation in polar bears

Polar bears (Ursus maritimus) in managed care generally are paired for breeding starting around the age of 5 or 6 years; however, DNA analyses of the wild population indicate that males as young as 2 or 3 may sire offspring and females as young as 4 can produce cubs. There are no reports describing longitudinal reproductive hormone parameters in juvenile polar bears. The objective of the current study was to determine if seasonal shifts in testosterone and progesterone (P4) metabolites are detectable in feces of polar bears 2-3 years old as part of a greater effort to characterise reproductive metabolites in a large cohort of juveniles throughout sexual maturation. Subjects were 2-year-old male (n=3) and female (n=3) polar bears residing at 5 zoological institutions in the USA. Individuals were monitored for 1 (1.1) or 2 (2.2) years. Fecal samples were collected noninvasively 3-4 times/week and hormone metabolites were extracted as previously described. Testosterone was evaluated as an indicator of gonadal activity in both sexes, whereas P4 was measured in samples collected from females only. Metabolites were quantified in duplicate using established enzyme immunoassay techniques. Student’s and paired t-tests were used to compare mean metabolite concentrations between seasons [breeding (BS; January-June) and nonbreeding (NBS; July-December)] by sex and within individual, respectively. All values are reported as mean concentration (ng metabolite/g dried feces±standard error of the means) and P-values less than 0.05 indicated statistical significance. Mean testosterone concentration of the 2-year-old males was 153.1±112.5. Overall, testosterone concentrations were higher in samples collected from 2-year-old males during BS versus NBS; however, when examined within individual, this held true for only 1 of 3 males. Mean testosterone of the 3-year-old male was 170.2±19.6 and values were significantly higher in BS (282.1±27.2) versus NBS (74.6±7.3). The overall mean testosterone and P4 concentrations of the 2-year-old females were 56.0±21.7 and 57.4±7.5, respectively. Two of the 3 females exhibited significantly higher testosterone concentrations during BS compared to NBS and all 3 exhibited higher P4 in the breeding versus the NBS. The 3-year-old female had significantly higher testosterone in BS (63.7±4.1) versus NBS (40.5±2.4) and showed evidence of regular ovarian cycles during BS. Despite no detectable differences in mean P4 between seasons (56.3±7.9 and 55.1±3.7), this female exhibited a distinct increase in P4 from October to November compared to the rest of NBS (85.7±8 v. 43.5±2.8), suggestive of pseudopregnancy. These results indicate that fecal reproductive hormone metabolite monitoring can be used to detect changes in metabolite excretion patterns associated with sexual maturation in polar bears and that males and females as young as 2 years old may exhibit seasonal variations in reproductive hormones. These data suggest bears in zoological institutions may be achieving sexual maturation earlier than believed previously and should be considered when managing bears in human care.

Effects of androsterone on the protective action of various antiepileptic drugs against maximal electroshock-induced seizures in mice

This study evaluated the effect of androsterone (AND), a metabolite of testosterone, on the ability of selected classical and novel antiepileptic drugs to prevent seizures caused by maximal electroshock (MES), which may serve as an experimental model of human generalized tonic-clonic seizures in mice. Single intraperitoneal (i.p.) administration of AND (80 mg kg−1) significantly raised the threshold for convulsions in the MES seizure threshold test. Lower doses of AND (5, 10, 20, and 40 mg kg−1) failed to change the threshold. AND at a subthreshold dose of 40 mg kg−1 significantly enhanced the protective activity of carbamazepine, gabapentin, and phenobarbital against MES-induced seizures decreasing their median effective doses (ED50) values ± SEM from 8.59 ± 0.76 to 6.05 ± 0.81 mg kg−1 (p = 0.0308) for carbamazepine, from 419.9 ± 120.6 to 111.5 ± 41.1 mg kg−1 (p = 0.0405) for gabapentin, and from 20.86 ± 1.64 to 10.0 ± 1.21 mg kg−1 (p = 0.0007) for phenobarbital. There were no significant changes in total brain concentrations of carbamazepine, gabapentin, and phenobarbital following AND administration. This suggests that the enhancing effects of AND on the protective activity of these antiepileptic drugs are not related to pharmacokinetic factors. A lower dose of AND (20 mg kg−1) had no effect on the protective activity of carbamazepine, gabapentin, and phenobarbital. AND administered at a dose of 40 mg kg−1 failed to change the anticonvulsant activity of lamotrigine, oxcarbazepine, phenytoin, topiramate, and valproate in the MES test. In the chimney test, AND given at a dose enhancing the protective activity of carbamazepine, gabapentin, and phenobarbital (which alone was without effect on motor performance of mice) did not affect impairment of motor coordination produced by the antiepileptics. Our findings recommend further preclinical and clinical research on AND in respect of its use as adjuvant therapy in the management of epilepsy in men with deficiency of androgens.

Age, seasonality, and correlates of aggression in female Apennine chamois

Intrinsic and environmental stressors, such as age and seasonality, may influence social behavior and endocrine levels in gregarious foragers, but little is known about how season and age affect both behavioral and physiological responses. We evaluated seasonal/age variation of aggression and vigilance, and seasonal/age variation of endocrine levels (fecal cortisol and testosterone metabolites), in a gregarious herbivore, the Apennine chamois Rupicapra pyrenaica ornata. We examined a period of decreasing resource abundance and maternal care from July to October, a key period for nursing, weaning, and early growth of offspring. Aggression rate, vigilance, and cortisol levels decreased throughout July–October, while aggression intensity showed the reverse. Aggression intensity peaked when chamois were on the most nutritious vegetation patches. Dominance increased with age, and prime-aged females (4–8 years old) showed higher cortisol and testosterone levels and were involved in aggressive interactions more often than subadult or older females. Our findings suggest that, in early summer, when nursing effort is the greatest, selection of nutritious food patches leads to frequent encounters between female chamois, enhancing aggression rate, vigilance, and endogenous stress response. The progressive decrease in food abundance throughout July–October triggers competition for scarce resources and increased intensity of aggression. Most likely, the energetic demands of lactation and offspring guarding were key determinants of behavioral and physiological stress of female chamois. Our results suggest a multi-factorial compromise between reproductive state and stress levels, in a group-living species. The cost of motherhood: in female chamois, aggression, vigilance, and stress peak during lactation, despite abundant food resources. In females, aggression and dominance increase with age. A multi-factorial compromise between reproductive stage and stress may be necessary in a group-living animal. We suggest that motherhood is a stressful event for female chamois.

Abstract 041: Brain Cytochrome P450 1B1-Testosterone Generated Metabolite 6Beta-Hydroxytestosterone Contributes to Angiotensin II-Induced Increased Sympathetic Outflow and Blood Pressure

Cytochrome P450 1B1 (CYP1B1)-generated testosterone (T) metabolite 6β-hydroxytestosterone (6β-OHT) contributes to angiotensin (Ang) II-induced hypertension in the male mice. However, the site of action of CYP1B1-testosterone metabolite 6β-OHT in Ang II-induced hypertension is unknown. The demonstration that Ang II increases blood pressure by its action in the subfornical organ and the presence of CYP1B1 in the brain led us to hypothesize that T contributes to Ang II-induced hypertension via its metabolism to 6β-OHT in the brain. To test this hypothesis, we examined the effect of intracerebroventricularly (ICV) administered T in castrated (Cas) wild-type ( Cyp1b1 +/+ ) and Cas Cyp1b1 -/- mice on the action of systemic Ang II (700 ng/kg/min) for 14 days. T (3 μg/2μL/injection/every 2nd day) or its vehicle (2-Hydroxypropyl-β-cyclodextrin dissolved in artificial CSF) was injected via a cannula implanted in the brain. Mean arterial blood pressure (MAP, mmHg) was measured by radiotelemetry (n=4-5). Ang II increased MAP in T but not its vehicle-injected Cas Cyp1b1 +/+ mice on Day 12 (159±2 vs. 98±2, P<0.05). In Cas Cyp1b1 -/- mice Ang II increased MAP in ICV injected 6β-OHT (1.5 μg/2μL/injection every 2nd day) but not T (151±3 vs. 123±5, P<0.05). Power spectral analysis of the data on day 12 showed that Ang II increased the low to high-frequency ratio of heart rate variability, index of sympathetic outflow modulation in ICV T injected Cas Cyp1b1 +/+ but not Cas Cyp1b1 -/- mice (3.8±0.0.4 vs. 1.9±0.0); whereas ICV 6β-OHT in the later mice increased this ratio (4.7±0.6). Ganglionic blocker hexamethonium (30 mg/Kg, ip) on day 14 of Ang II infusion resulted in greater reduction in MAP (P<0.05) in centrally 6β-OHT injected Cas Cyp1b1 -/- , and T-injected Cas Cyp1b1 +/+ (Δ81±11 and Δ87±10) than in T injected Cas Cyp1b1 -/- mice (Δ50±8). Furthermore, in intact Cyp1b1 -/- , but not Cas Cyp1b1 -/- mice transduction with adenovirus (Ad) of CYP1B1-DNA (ICV 2μL of 1.0 X 10 12 particles/mL) increased Ang II-induced systolic blood pressure (P<0.05, n=5/group) measured by tail-cuff on Day12 (176±11 vs. 120±2, mm Hg). These data suggest that T contributes to Ang II-induced hypertension most likely via generation by the brain CYP1B1 to 6β-OHT, which increases sympathetic outflow in male mice.

Characterization of the behavior and reproductive endocrinology of giant river otters (Pteronura brasiliensis) in managed care.

Propagation of giant river otters (GRO) in zoos is inconsistent: some pairs never reproduce while others are prolific in producing young but can be hindered by low cub survival. Developing effective breeding programs requires understanding normal reproductive parameters and behavior. Fecal samples were collected for 6-16 months from five breeding pairs, two individual females, and one female pair at seven zoos, and analyzed for fecal progesterone, estrogen, testosterone, and glucocorticoid (FGM) metabolites via enzyme immunoassay. Enclosure characteristics and management routines were recorded at six facilities where behavior was assessed over 1 week. Median fecal progestogens during pregnancy and pseudopregnancy were ∼2.5-3.8× greater than basal concentrations. Gestation lasted 66.5 ± 3.5 days (62-70 days); pseudopregnancies lasted 58 ± 11.6 days (41-69 days). Elevated progestogens indicate ovulation but cannot distinguish pregnancy from pseudopregnancy. Periodically sustained, elevated progestogens observed in two females housed without a male indicated spontaneous ovulation. Elevations in fecal estrogens were not associated with estrus, and seasonality in male testosterone was not observed. Wavering scream and contact call vocalizations among reproductively successful males and females, respectively, suggested the importance of social communication. Most facilities housing successful pairs had larger enclosures with more water than land area, vegetation, and limited public exposure. Baseline FGM were negatively correlated with enclosure size and percentage of water area (p < 0.05), and lower baseline FGM were associated with reproductive success (p < 0.05). These results suggest that housing GRO in spacious enclosures with open water and some insulation from disturbance might promote appropriate behavior, lower FGM, and reproduction.

On the Role of Testosterone in Anxiety-Like Behavior Across Life in Experimental Rodents.

Testosterone affects brain functions and might explain some of the observed behavioral sex differences. Animal models may help in elucidating the possible involvement of sex hormones in these sex differences. The effects of testosterone have been intensively investigated, especially in anxiety models. Numerous experiments have brought inconsistent results with either anxiolytic or anxiogenic effects. Besides methodological variations, contradictory findings might be explained by the divergent metabolism of testosterone and its recognition by neurons during prenatal and postnatal development. Gonadectomy and subsequent supplementation have been used to study the role of sex hormones. However, the variable duration of hypogonadism might affect the outcomes and the effect of long-term androgen deficiency is understudied. Testosterone can be metabolized to dihydrotestosterone strengthening the androgen signaling, but also to estradiol converting the androgen to estrogen activity. Moreover, some metabolites of testosterone can modulate γ-aminobutyric acid and serotonergic neurotransmission. Here we review the currently available experimental data in experimental rodents on the effects of testosterone on anxiety during development. Based on the experimental results, females are generally less anxious than males from puberty to middle-age. The anxiety-like behavior of females and males is likely influenced by early organizational effects, but might be modified by activational effects of testosterone and its metabolites. The effects of sex hormones leading to anxiogenesis or anxiolysis depend on factors affecting hormonal status including age. The biological and several technical issues make the study of effects of testosterone on anxiety very complex and should be taken into account when interpreting experimental results.

Seasonal dynamics of agonistic behavior and hormones in an ex situ all-male colony of large flying foxes.

Large flying foxes (Pteropus vampyrus) are a socially complex species. In situ colonies typically comprise thousands of individuals in small harems of one male to many females. In ex situ environments, all-male colonies are becoming more common due to a surplus of males in the population. There is limited information describing the hormonal and behavioral patterns of all-male colonies during the breeding season. We assessed seasonal changes in hormones and behavior in an all-male colony of 12 large flying foxes at Disney's Animal Kingdom® . We validated hormone assays using morning urine and fecal samples to assess seasonal changes in excreted immunoreactive testosterone and glucocorticoid metabolites. We collected behavior data using an all-occurrence method, recording agonistic behaviors related to territorial defense (hooking, biting, wing flexing, vocalizing, and wrestling), and sexual behavior (mounting and frontal grabbing). Results indicated that (i) we could reliably measure testosterone and glucocorticoid metabolites concentrations from fecal and urine samples collected from individual bats; (ii) there were distinct relationships between changes in levels of agonism and hormone concentrations throughout the year; and (iii) three agonistic behaviors (chasing, wrestling, and open-mouth threat) peaked prior to the increase in testosterone and glucocorticoid hormones measured during the breeding season. These three behaviors could potentially be used as early indicators to signal the onset of the breeding season and allow time to implement ex situ management changes to reduce the incidence of agonism between individuals.

Effect of Dose and 5α-Reductase Inhibition on the Circulating Testosterone Metabolite Profile of Men Administered Oral Testosterone.

Development of an oral testosterone therapy has proven extremely challenging because of extensive and variable first‐pass metabolism. We investigated the in vivo metabolism of testosterone with increasing oral doses of testosterone, both alone and with the co‐administration of dutasteride (5α‐reductase inhibitor) by liquid‐chromatography tandem mass spectrometry (LC‐MS/MS). In eugonadal men prior to dosing, the circulating concentration of testosterone, androstenedione, etiocholanolone‐glucuronide, and androsterone‐glucuronide was 8.6, 20.9, 9.1, and 55.3%, respectively, of the total testosterone‐related species, whereas testosterone‐glucuronide was ∼1%. When testosterone was dosed orally to men with experimental hypogonadism, a proportion of testosterone‐glucuronide increased to 13%. Dutasteride treatment significantly decreased levels of androsterone and its metabolites. This work reveals extensive metabolism of orally dosed testosterone to androsterone glucuronide via androstenedione, with testosterone‐glucuronide appearing to be the second most important metabolite. This information is of importance in the development of an effective oral testosterone therapy and may have implications for testosterone doping research.

Pregnancy-Induced Perturbation of Urinary Androgenic Steroid Disposition.

ObjectiveTo investigate the excretion and conjugation profile of testosterone (T), Epitestosterone (EpiT), and other androgen metabolites in different phases of pregnancy and postpregnancy as a reflection of the “androgenic exposure.”DesignConsecutive recruitment of pregnant women.SettingMaternity outpatient low-risk pregnancy clinic.PatientsSeventy-seven pregnant women.InterventionsCollection of urine for analyses of sulfate (S) and glucuronide (G) conjugates and metabolic ratios of androgens and androgen metabolites using liquid chromatography-tandem mass spectrometry.Main Outcome MeasuresExcretion profiles and metabolic ratios of G and S conjugates of T, EpiT, dehydroepiandrosterone (DHEA), androsterone (A), etiocholanolone (Etio), and dihydrotestosterone in relation to trimester and postpartum, body mass index, fetal sex, and ethnicity.ResultsT-S excretion increased significantly between the second and third trimester, whereas excretion of T-G did not change. In contrast, both conjugates of EpiT increased markedly, more so for the S-(17-fold) than the G-conjugate (1.6-fold). The preference for S over G conjugation was conspicuous for EpiT and DHEA (S/G ratio 2.1 and 4.7, respectively, in the third trimester), whereas the reverse was true for T, A, and Etio (S/G 0.6, 0.13, and 0.11, respectively).ConclusionsPregnancy influences the androgen excretion profile, with the most profound change being an increase in EpiT excretion throughout the trimesters. EpiT may modulate the effect of T, but its exact role during pregnancy is not known. There were marked differences in the S/G conjugate ratios between androgens upstream and downstream from T in the metabolic network. These results are interesting to compare with the androgen disposition in women with endocrine disorders or abuse of steroids.