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The red-sided garter snake exhibits a dissociated reproductive pattern, mating at a time when their gonads are quiescent and circulating sex steroids, initially reported to be low, have been found to be elevated during the breeding season. However, the only cue identified that initiates courtship behavior and mating in the red-sided garter snake is an extended period of low-temperature dormancy (LTD) followed by exposure to warm temperatures. In this study, we examined a possible association between winter dormancy, sex steroid hormone metabolism, and initiation of reproductive behavior in the male red-sided garter snake. Upon emergence from winter dormancy, courtship behavior was assessed using attractive females as stimuli. Animals receiving implants containing 1,4,6-androstatriene-3,17-dione (ATD), an aromatase inhibiter, showed little or no courtship behavior, while animals implanted with ATD + 17β estradiol (E2 ) or blank implants exhibited normal courtship. In addition, neurons in the pathways critical in the control of reproductive behavior were examined using the Golgi method. Examination of brains collected upon emergence revealed animals implanted with ATD had a significantly reduced number of dendritic spines compared to controls and animals treated with ATD + E2 . This study provides the first documentation that courtship behavior and mating in the male red-sided garter snake appears to be controlled not by LTD alone, but in conjunction with sex steroid hormones. Moreover, behavioral data suggest that the aromatization of androgens during LTD appears to be critical for the initiation of reproductive behavior in the male red-sided garter snakes.

In seasonally breeding songbirds such as canaries, singing behavior is predominantly under the control of testosterone and its metabolites. Short daylengths in the fall that break photorefractoriness are followed by increasing daylengths in spring that activate singing via both photoperiodic and hormonal mechanisms. However, we observed in a group of castrated male Fife fancy canaries maintained for a long duration under a short day photoperiod a large proportion of subjects that sang at high rates. This singing rate was not correlated with variation in the low circulating concentrations of testosterone. Treatment of these actively singing castrated male canaries with a combination of an aromatase inhibitor (ATD) and an androgen receptor blocker (flutamide) only marginally decreased this singing activity as compared to control untreated birds and did not affect various measures of song quality. The volumes of HVC and of the medial preoptic nucleus (POM) were also unaffected by these treatments but were relatively large and similar to volumes in testosterone-treated males. In contrast, peripheral androgen-sensitive structures such as the cloacal protuberance and syrinx mass were small, similar to what is observed in castrates. Together these data suggest that after a long-term steroid deprivation singing behavior can be activated by very low concentrations of testosterone. Singing normally depends on the activation by testosterone and its metabolites of multiple downstream neurochemical systems such as catecholamines, nonapeptides or opioids. These transmitter systems might become hypersensitive to steroid action after long term castration as they probably are at the end of winter during the annual cycle in seasonally breeding temperate zone species.

The estrogen-synthesizing enzyme aromatase is abundant at the synapse in the zebra finch hippocampus (HP), and its inhibition impairs spatial memory function. To more fully test the role of local estradiol (E2) synthesis in memory, the HP of adult male zebra finches was exposed to either control pellets or those containing the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD), ATD and E2, ATD and the G protein-coupled estrogen receptor (GPER) agonist G1, or the antagonist G15 alone. Birds were tested for spatial memory acquisition and performance, and HP levels of the postsynaptic protein PSD95 were measured. ATD-treated birds took longer to reach criterion than control birds, whereas acquisition in ATD+E2 and ATD+G1 birds was indistinguishable from control and ATD treatments. Interestingly, all G15 birds failed to acquire the task. Following a retention interval, ATD birds took the longest to reach the (formerly) baited cup and made the most mistakes. ATD+E2 animals displayed the lowest retention latencies and made fewer mistakes than ATD-treated birds, and ATD+G1 birds did not significantly differ from controls in retention latencies. The amount of PSD95 in the HP was lowest in ATD-treated animals compared with birds with silicone-only–implanted craniotomies, ATD+E2, and ATD+G1 birds, who did not differ in this expression. Thus, spatial memory acquisition and performance appear aromatase and E2 dependent, an effect more reliably revealed after consolidation and/or recall compared to acquisition. E2 may exert this effect via GPERs, resulting in an increase in PSD95 levels that may modify receptor activity or intracellular signaling pathways to increase synaptic strength.

Androsta-1,4,6-triene-3,17-dione (ATD) is an irreversible steroidal aromatase inhibitor and is marketed as a supplement. It has been reported to effectively reduce estrogen biosynthesis and significantly increase the levels of endogenous steroids such as dihydrotestosterone and testosterone in human. ATD abuses have been reported in human sports. Its metabolism in human has been studied, and the in vitro metabolic study of ATD in horses has been reported, however, little is known about its biotransformation and elimination in horses. This paper describes the in vitro and in vivo metabolism studies of ATD in horses, with an objective of identifying the target metabolites with the longest detection time for controlling ATD abuse. In vitro metabolism studies of ATD were performed using homogenized horse liver. ATD was found to be extensively metabolized, and its metabolites could not be easily characterized by gas chromatography/mass spectrometry (GC/MS) due to insufficient sensitivity. Liquid chromatography/high resolution mass spectrometry (LC/HRMS) was therefore employed for the identification of in vitro metabolites. The major biotransformations observed were combinations of reduction of the olefin groups and/or the keto group at either C3 or C17 position. In addition, mono-hydroxylation in the D-ring was observed along with reduction of the olefin groups and/or the keto group at C17 position. Fourteen in vitro metabolites, including two epimers of androsta-1,4,6-trien-17-ol-3-one (M1a, M1b), androsta-4,6-diene-3,17-dione (M2), boldione (M3), androsta-4,6-diene-17β-ol-3-one (M4), androsta-4,6-diene-3-ol-17-one (M5), boldenone and epi-boldenone (M6a, M6b), four stereoisomers of hydroxylated androsta-1,4,6-trien-17-ol-3-one (M7a to M7d), and two epimers of androsta-1,4-diene-16α,17-diol (M8a, M8b), were identified. The identities of all metabolites, except M1a, M5, M7a to M7d, were confirmed by matching with authentic reference standards using LC/HRMS. For the in vivo metabolism studies, two thoroughbred geldings were each administered with 800 mg of ATD by stomach tubing. ATD, and twelve out of the fourteen in vitro metabolites, including M1a, M1b, M2, M4, M5, M6, M7a to M7d, M8a and M8b, were detected in post-administration urine. Two additional urinary metabolites, namely stereoisomers of hydroxylated androsta-4,6-dien-17-ol-3-one (M9a, M9b), were tentatively identified by mass spectral interpretation. Elevated level of testosterone was also observed. In post-administration blood samples, only the parent drug, M1b and M2 were identified. This study showed that the detection of ATD administration would be best achieved by either monitoring the metabolites M1b (androsta-1,4,6-trien-17β-ol-3-one) or M4 (both excreted as sulfate conjugates) in urine, which could be detected for up to a maximum of 77 h post-administration. The analyte of choice for plasma is M1b, which could be detected for up to 28 h post administration.

A hyperactive hypothalamo-pituitary-adrenal (HPA) axis is a prominent feature in depression. It has been shown that androgens inhibit HPA activity and that estrogens stimulate it. We have therefore investigated, in human postmortem hypothalamus, whether depression features an increase in aromatase, which is the rate-limiting enzyme for the conversion of androgens to estrogens. In addition, we have tested the effect of an aromatase inhibitor on depression-like symptoms in a frequently used animal model for depression. At first, aromatase immunoreactivity (ir) was quantified in the central part of the hypothalamic paraventricular nucleus (PVN) of 10 major depressive disorder (MDD) patients and 10 well-matched control subjects. Subsequently an animal experimental study was performed using the chronic unpredictable mild stress (CUMS) rats as depression model. The effect of administration of 1,4,6-androstatriene-3,17-dione (ATD), an aromatase inhibitor, was investigated by silastic capsule implantation. In the postmortem study, the amount of PVN aromatase-ir decreased significantly in the MDD group compared to the controls (P=0.029). In the animal study, ATD was found to cause significantly increased testosterone (T) levels, both in plasma and in the hypothalamus. However, ATD administration did not show significant effects on the depression-like behaviors or plasma corticosterone levels in CUMS rats. Based on our observations in human postmortem material and the animal experiment, we have to conclude that alterations in aromatase in adulthood do not seem to play a major role in the pathogenesis of the symptoms of depression.

Recent evidence suggests that the aromatization of testosterone to estrogen is important for the organizing effects of neonatal testosterone on neuroendocrine responses to acute challenges. However, the extent to which neonatal inhibition of aromatase alters the stress-induced activation of neural pathways has not been examined. Here we assessed central patterns of c-fos mRNA induced by 30 min of restraint in 65-d-old adult male rats that were implanted with sc capsules of the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD), introduced within 12 h of birth and removed on d 21 of weaning. Neonatal ATD decreased the expression of arginine vasopressin within extrahypothalamic regions in adults, confirming reduced estrogen exposure during development. As adults, ATD-treated animals showed higher corticosterone responses at 30 min of restraint exposure compared with control animals as well as higher c-fos expression levels in the paraventricular nucleus of the hypothalamus. ATD treatment also increased stress-induced c-fos within several limbic regions of the forebrain, in addition to areas involved in somatosensory processing. Based on these results, we propose that the conversion of testosterone to estrogen during the neonatal period exerts marked, system-wide effects to organize adult neuroendocrine responses to homeostatic threat.

A variety of natural and synthetic environmental substances have been shown to disrupt vertebrate reproduction through mimicking or modifying the regulation of the endocrine system. Tests to screen for any such chemicals that directly interact with the steroid hormone receptors are widely available; however, few tests have been developed to identify chemicals that affect endocrine function through non-receptor mediated mechanisms. The aim of this study was, therefore, to develop an assay for the identification of substances that disrupt the activity of enzymes involved in the sex steroid biosynthesis cascade, in particular the aromatase enzyme, CYP19, that catalyses the final conversion of androgens to estrogens. A gonad ex vivo assay was developed using gonad explants harvested from juvenile brown trout and cultured in a modified Leibovitz medium. Effects on sex steroid biosynthesis were quantified through measurement of 17β-estradiol (E2) and testosterone (T) concentrations in the medium after 2 days incubation. Exposure of ovary explants to 100 ng/mL 1,4,6-androstatriene-3,17-dione (ATD), a potent pharmaceutical aromatase inhibitor, reduced E2 concentrations and elevated T concentrations confirming that CYP19 activity could be inhibited in the assay. Exposure of ovary explants to 250 ng/mL prochloraz, an imidazole fungicide, also reduced E2 concentrations but did not affect T levels, consistent with reports that in addition to inhibiting CYP19 activity, prochloraz also inhibits enzymes in the steroidogenic pathway upstream of the CYP19 enzyme. Exposure to a third chemical, tributyltin (TBT), did not affect T or E2 concentrations, further supporting previous evidence that the CYP19 modulating effects of this chemical are not mediated through direct inhibition of CYP19 activity. These results demonstrate that the gonad ex vivo assay developed here can be successfully used to identify substances that disrupt sex steroid biosynthesis and further that it has the potential to inform on their specific mode of action.

Anabolic androgenic steroids and high testosterone doses have been reported to induce impulsive behavior in man and behavioral disinhibition in rats. The purpose of the present study was to investigate whether aromatization of testosterone to estradiol is of importance for the behavioral disinhibiting effect of a high testosterone dose in adult male rats. Testosterone administered via five testosterone-filled silastic capsules implanted subcutaneously (s.c.) to non-castrated, group-housed rats for six days induced behavioral disinhibition in a modified Vogel's drinking conflict model and yielded supraphysiological serum levels of testosterone and increased accessory sex organ weights. Moreover, concurrent administration of the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD; 60 mg/kg/day s.c.) decreased behavioral disinhibition in testosterone-treated rats (without affecting accessory sex organ weights) while behavior was not significantly affected in sham-treated animals. Since some reports indicate that ATD, in addition to inhibit aromatase, also may affect the binding of testosterone to the androgen receptor, the effect of the non-steroidal androgen receptor antagonist flutamide was investigated. Flutamide treatment did not affect disinhibited behavior in testosterone-treated rats. However, in sham-treated animals, flutamide (50 mg/kg/day) produced behavioral disinhibition. These results suggest that estradiol is of importance in the mechanisms underlying behavioral disinhibition in non-castrated rats treated with a high testosterone dose. Speculatively, aromatization may be involved in pro-impulsive effects of high testosterone doses in humans.

The orphan G protein coupled receptor, GPR30, has the characteristics of a high affinity, specific estrogen membrane receptor on Atlantic croaker oocytes and mediates estrogen inhibition of oocyte maturation in this perciform fish. In order to determine the broad applicability of these findings to other teleosts, similar experiments were conducted in a cyprinid fish, zebrafish, in the present study. GPR30 mRNA expression was detected in zebrafish oocytes but not in the ovarian follicular cells. Both spontaneous and 17, 20beta-dihyroxy-4-pregnen-3-one (DHP)-induced maturation of follicle-enclosed zebrafish oocytes was significantly decreased when they were incubated with either estradiol-17beta, or the GPR30 agonists, ICI 182 780 and tamoxifen, or with the GPR30 specific agonist G-1. On the other hand spontaneous oocyte maturation increased two-fold when zebrafish ovarian follicles were incubated with an aromatase inhibitor, ATD. Moreover, the stimulatory effects of ATD on germinal vesicle breakdown (GVBD) were partially reversed by co-treatment with 100 nM of E2 or G-1. These results suggest that endogenous estrogens acting through GPR30 are involved in maintaining meiotic arrest of zebrafish oocytes.

Testosterone secreted by male testes during fetal development is aromatized to oestradiol (E(2)) or reduced to the androgen, dihydrotestosteorne (DHT), within specific tissues. The female brain is assumed to develop in the relative absence of gonadal steroid hormones, as the ovary is steroidogenically quiescent until later in postnatal life. However, the proximity of a female fetus to male littermates in utero can increase her exposure to testosterone, and thereby its metabolites. To date, it is has been difficult to dissociate the effects of male-derived E(2) from those of DHT on the developing female brain. In the present study, anogential distance (AGD) in females was used as an androgen-dependent bioassay, whereas progesterone receptor (PR) expression within the medial preoptic nucleus (MPN) was used as an E-dependent measure. Pregnant dams received the aromatase inhibitor, 1,4,6-androstatriene-3,17-dione (ATD), or vehicle from embryonic day 16 (ED16) to ED21. On ED22, AGD and PR-immunoreactivity (-ir) were measured in females that had zero, one, or two males (0-2M) or females that had three, four, or five males (3-5M) in the uterine horn. AGD was significantly greater in 3-5M females compared to 0-2M females, suggesting that male littermates are the source of androgenic exposure in the female fetus. ATD treatment significantly decreased PR-ir in the MPN, demonstrating E(2) regulation of PR. However, the total number of males in the uterine horn did not effect PR expression. There was no correlation between PR-ir and AGD, suggesting that these measures are influenced independently. Together, these results suggest that although male littermates provide a significant source of androgens to female fetuses, the amount of E(2) aromatized from male-derived testosterone may not be the only biologically relevant source of androgens or E(2). Alternative sources of E(2) may be essential in ensuring the normal development of the female brain.

The effects of administration of the aromatase inhibitor 1,4,6-androstatrien-3.17-dione (ATD) to female rats during the last third of pregnancy on the formation of behavior of offspring of both genders in a novel environment were studied. Animal behavior was assessed in the open field and elevated cross maze tests. Inhibition of testosterone aromatization during the prenatal period of development resulted in increases in anxiety and emotionality in experimental rats at age one month; increases in these measures in adult animals were seen in both males and females exposed to prenatal ATD. Intergender differences between control males and experimental females, in terms of behavioral measures in the novel environment such as motor activity, the duration of the freezing and grooming reactions, as well as well the level of anxiety, disappeared. It is concluded that impairment of testosterone metabolism during the prenatal period of development affects the formation of the behavior of rats in a novel environment as determined by genetic gender.

The chemoselectivity of rigid cyclic alpha,beta-unsaturated carbonyl group on the reducing agents was influenced by the ring size and steric factor. Cholesterol (cholest-5-en-3beta-ol) and dehydroepiandrosterone (DHEA) were oxidized with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone to form 1,4,6-cholestatrien-3-one and 1,4,6-androstatriene-3,17-dione. They were reduced with NaBH(4), lithium tri-sec-butylborohydride (l-Selectride), LiAlH(4), 9-borabicyclo[3.3.1]nonane (9-BBN), lithium triethylborohydride (Super-hydride), and BH(3) x (CH(3))(2)S in various conditions, respectively. Reduction of 1,4,6-cholestatrien-3-one and 1,4,6-androstatriene-3,17-dione by NaBH(4) (4 equiv.) produced 4,6-cholestadien-3beta-ol and 4,6-androstadiene-3beta,17beta-diol, respectively. Reduction by l-Selectride (12 equiv.) afforded 4,6-cholestadien-3alpha-ol and 4,6-androstadiene-3alpha,17beta-diol, chemoselectively. Reaction with Super-hydride (12 equiv.) produced 4,6-cholestadien-3-one and 3-oxo-4,6-androstadien-17beta-ol. Reduction of 1,4,6-cholestatrien-3-one by 9-BBN (14 equiv.) produced 1,4,6-cholestatrien-3alpha-ol, but 1,4,6-androstatriene-3,17-dione was not reacted with 9-BBN in the reaction conditions. Reaction of LiAlH(4) (6 equiv.) formed 4,6-cholestadien-3beta-ol and 3-oxo-1,4,6-androstatrien-17beta-ol. Reduction of 1,4,6-cholestatrien-3-one by BH(3) x (CH(3))(2)S (11 equiv.) gave cholestane as major compound and unlike reactivity of cholesterol, 1,4,6-androstatriene-3,17-dione by 8 equiv. of BH(3) x (CH(3))(2)S formed 3-oxo-1,4,6-androstatrien-17beta-ol. LiAlH(4) and BH(3) x (CH(3))(2)S showed relatively low chemoselectivity.

There is some evidence for an unusual body odour in schizophrenia that has been linked to a hexenoic acid derivative (trans-3-methyl-2-hexenoic acid; MHA). Poor body odour has been linked to increased negative symptoms and reduced olfactory identification ability. However, the relationship between these findings and MHA, including olfactory sensitivity for MHA, has not been examined. Olfactory sensitivity thresholds were assessed for MHA and n-butyl-alcohol (NBA), in normal controls (CTL; n=24), patients with chronic schizophrenia (CHR; n=32) and a first-episode psychosis cohort (FE; n=31). In addition, forced choice detection of the pheromonal steroids 5-alpha-androst-16-en-3-one, androsterone-sulphate and estrone-3-sulphate was performed along with a measure of olfactory identification. CHR patients had significantly reduced sensitivity to MHA, but not NBA, compared to FE and CTL subjects. While sensitivity to pheromones was not different between the groups, CHR patients who could not detect them also showed poorer sensitivity to MHA. Further, the CHR group showed a significant association between reduced MHA sensitivity and greater levels of disorganised and negative symptoms. No relationships between identification and sensitivity for any substance were found. Our findings are the first to report reduced sensitivity for MHA in chronic schizophrenia patients, in the absence of similar impairment for more traditionally used substances. This may be linked to olfactory habituation effects, abnormal chemical processing or a genetic predisposition.

The use of anabolic androgenic steroids (AAS) is prohibited in both human and equine sports. The conventional approach in doping control testing for AAS (as well as other prohibited substances) is accomplished by the direct detection of target AAS or their characteristic metabolites in biological samples using hyphenated techniques such as gas chromatography or liquid chromatography coupled with mass spectrometry. Such an approach, however, falls short when dealing with unknown designer steroids where reference materials and their pharmacokinetics are not available. In addition, AASs with fast elimination times render the direct detection approach ineffective as the detection window is short. A targeted metabolomics approach is a plausible alternative to the conventional direct detection approach for controlling the misuse of AAS in sports. Because the administration of AAS of the same class may trigger similar physiological responses or effects in the body, it may be possible to detect such administrations by monitoring changes in the endogenous steroidal expression profile. This study attempts to evaluate the viability of using the targeted metabolomics approach to detect the administration of steroidal aromatase inhibitors, namely androst-4-ene-3,6,17-trione (6-OXO) and androsta-1,4,6-triene-3,17-dione (ATD), in horses. Total (free and conjugated) urinary concentrations of 31 endogenous steroids were determined by gas chromatography-tandem mass spectrometry for a group of 2 resting and 2 in-training thoroughbred geldings treated with either 6-OXO or ATD. Similar data were also obtained from a control (untreated) group of in-training thoroughbred geldings (n = 28). Statistical processing and chemometric procedures using principle component analysis and orthogonal projection to latent structures-discriminant analysis (OPLS-DA) have highlighted 7 potential biomarkers that could be used to differentiate urine samples obtained from the control and the treated groups. On the basis of this targeted metabolomic approach, the administration of 6-OXO and ATD could be detected for much longer relative to that of the conventional direct detection approach.

Aromatase, the enzyme that aromatizes androstenedione (A) to estrone and testosterone (T) to estradiol (E), affects androgen control of male sex behavior in many vertebrates. In male monkeys, rats and quail, E mimics the ability of T to promote mating, and aromatase inhibitors block mating induced by T but not E. Aromatase inhibitors include androgens with different A-rings than T and A, e.g., 1,4,6-androstatriene-3,17-dione (ATD), azoles, e.g., fadrozole, and androgens alpha-halogenated at carbon 6, e.g., 6 alpha-bromoA, 6 alpha-fluoroA and 6 alpha-fluoroT. 6 alpha-FluoroT is the only 6 alpha-halogenated androgen studied in regard to mating. It promotes mating in male rats and quail and was studied, before it was known to inhibit aromatase, because it cannot be aromatized yet has the same A-ring as T. 6 alpha-FluoroT might promote mating by binding estrogen receptors (ER) directly, i.e., unassisted, or by metabolism to an androgen that binds ER. Since neither process would require aromatase, this study tested both hypotheses by determining how mating induced in castrated male rats by 6 alpha-fluoroT is affected by ATD and fadrozole. Both aromatase inhibitors inhibited the effects of 6 alpha-fluoroT on mating. Thus, 6 alpha-fluoroT does not promote mating by direct ER binding or metabolism to another androgen. Since aromatase underlies a process in which 6 alpha-fluoroT, unlike most nonaromatizable androgens, mimics T effects on male sex behavior, the process must involve a feature that 6 alpha-fluoroT shares with T but not other nonaromatizable androgens. A-ring structure is a candidate. A hypothesis is also offered for how aromatase may participate without aromatizing the androgen. (C) 2015 Elsevier Inc. All rights reserved.

In order to assess the efficacy of selected aromatase inhibitors on Atlantic salmon ( Salmo salar ) ovarian and brain tissue, in vitro systems were developed for measuring 17β-estradiol (E 2 ) production by these tissues. Isolated vitellogenic follicles, or homogenised whole brains were incubated at 10 °C in complete Cortlands solution for 18 or 42 h respectively, and E 2 levels in the medium were determined by RIA. The addition of testosterone to the medium increased E 2 production in all preparations. E 2 production by whole brain homogenate was reduced by co-incubation with the aromatase inhibitors 1,4,6-androstatriene-3,17-dione (ATD), 4-androstene-4-ol-3,17-dione (OHA), aminoglutethimide, fadrozole or miconazole. Fadrozole, ATD, and OHA reduced E 2 production by vitellogenic follicles at a medium concentration of 0.1 μg mL − 1 , whereas miconazole was only effective at 10 μg mL − 1 . This study demonstrates a simple and rapid screening method for assessing the efficacy of aromatase inhibitors on fish tissues, and that the aromatase inhibitors ATD, OHA and fadrozole are potent inhibitors of both brain and gonadal aromatase in vitro , in Atlantic salmon.

It has been shown previously that female mice homozygous for an alpha-fetoprotein (AFP) null allele are sterile as a result of anovulation, probably due to a defect in the hypothalamic-pituitary axis. Here we show that these female mice exhibit specific anomalies in the expression of numerous genes in the pituitary, including genes involved in the gonadotropin-releasing hormone pathway, which are underexpressed. In the hypothalamus, the gonadotropin-releasing hormone gene, Gnrh1, was also found to be down-regulated. However, pituitary gene expression could be normalized and fertility could be rescued by blocking prenatal estrogen synthesis using an aromatase inhibitor. These results show that AFP protects the developing female brain from the adverse effects of prenatal estrogen exposure and clarify a long-running debate on the role of this fetal protein in brain sexual differentiation.

Many chemicals in the aquatic environment are able to adversely affect in vitro brain and ovarian aromatase expression/activity. However, it remains to be determined if these substances elicit in vivo effect in fish. With the view to further understanding possible effects of endocrine disrupting chemicals (EDCs) on aromatase function, we first developed methods to measure brain and ovarian aromatase expression/activity in a model species, the zebrafish, and assessed the effect of estradiol (E2) and androstatrienedione (ATD), a steroidal aromatase inhibitor. We showed that CYP19b gene was predominantly expressed in the brain whereas in the ovary CYP19a mRNA level was predominant. Moreover, aromatase activities (AA) were higher in brain than in ovary. In adult zebrafish, E2 treatment had no effect on aromatase expression/activity in brain, whereas at larval stage, E2 strongly triggered CYP19b expression. In the ovaries, E2 led to a complete inhibition of both CYP19a expression and AA. Exposure to ATD led to a total inhibition of both brain and ovarian AA but had no effect on CYP19 transcripts abundance. Together, these results provide relevant knowledge concerning the characterization of aromatase in the zebrafish, and reinforce the idea that brain and ovarian aromatase are promising markers of EDCs in fish and deserve further in vivo studies.

Gonadal steroids, most importantly testosterone (T), are considered to be a major factor in the expression of adult song behavior in temperate-zone songbirds. The action of T within specific brain regions involved in the regulation of song may occur either directly, or through its androgenic or estrogenic metabolites. In the present study, we tested steroid-dependence of great tit dawn song by blocking both known pathways of T action by simultaneous implantation of flutamide, an anti-androgen, and ATD, an aromatase inhibitor. By our knowledge, this is the first study investigating the effects of androgen inhibitors on dawn song in free-living birds. Male great tits were implanted during their mate's egg laying stage, being the time of maximal male song activity at dawn. Treatment with ATD and flutamide significantly increased plasma T levels, probably because feedback mechanisms on T secretion were inhibited. The treatment decreased the likelihood of showing dawn song, which is in line with the hypothesis that sex steroids are involved in the endocrine control of song behavior. In males that did show dawn song, we found no evidence for a treatment effect on song quality. Although the implants were present for the larger part of the breeding season, males were able to maintain control of a territory and mate and to complete their brood cycle as successful as control males.

Organizational effects of testosterone during a critical period of neonatal life have major irreversible effects on adult sexual behavior. We have investigated whether perinatal androgen changes also affect another major sexually differentiated system, the hypothalamo-pituitary-adrenal axis. This was assessed in male rats who had been exposed to perinatal flutamide or 1,4,6-androstatriene-3,17-dione (ATD). Once the animals reached adulthood, an automated sampling system was used to collect blood from freely moving animals at 10-min intervals over 24 h, followed by a noise stress and then the administration of lipopolysaccharide (LPS). Perinatal flutamide- and ATD-treated rats not only had higher mean corticosterone levels and increased frequency and amplitude of corticosterone pulses over the 24 h compared with vehicle-injected controls, but they also showed markedly increased corticosterone responses to both noise and LPS. All parameters of increased hypothalamo-pituitary-adrenal activity resembled the normal physiological state of the intact adult female rather than that of the intact adult male rat. Furthermore, 3 h after LPS administration, both flutamide- and ATD-treated animals had markedly higher levels of corticotropin-releasing factor mRNA in the parvocellular paraventricular nucleus (PVN) and proopiomelanocortin mRNA in the adenohypophysis. Flutamide-treated rats also had a greater level of PVN arginine vasopressin mRNA. PVN glucocorticoid receptor mRNA levels were significantly lower in both the flutamide- and the ATD-treated male rats. These data highlight the importance of perinatal exposure to both testosterone and estrogen(s) on the development of a masculinized circadian corticosterone profile and stress-induced hypothalamo-pituitary-adrenal axis activity in the adult male rat.

Four steroids that share the 17-hydroxy-3-oxopregna-4,6-diene structure – cyproterone acetate, chlormadinone acetate, megestrol acetate, and potassium canrenoate – have been shown previously to behave with different potency as liver-specific genotoxic agents, the response being markedly higher in female than in male rats, but similar in humans of both genders. In this study, performed to better define the relationship between chemical structure and genotoxicity, dydrogesterone (DGT) with double bonds C4 C5 and C6 C7, dienogest (DNG) with double bonds C4 C5 and C9 C10, and 1,4,6-androstatriene-17β-ol-3-one acetate (ADT) with double bonds C1 C2, C4 C5 and C6 C7, were compared with cyproterone acetate (CPA) for their ability to induce DNA fragmentation and DNA repair synthesis in primary cultures of hepatocytes from three rats of each sex. At subtoxic concentrations, ranging from 10 to 90 μM, all four steroids consistently induced a dose-dependent increase of DNA fragmentation, which in all cases was higher in females than in males; their DNA damaging potency decreased in the order CPA > DNG > ADT > DGT. Under the same experimental conditions, the responses provided by the DNA repair-synthesis assay were positive or inconclusive in hepatocytes from female rats and consistently negative in hepatocytes from male rats. In the induction of apoptotic cells, examined in primary hepatocytes from female rats, CPA was more active than ADT and DGT, and DNG was inactive. Considered as a whole these findings suggest that a liver-specific genotoxic effect more marked in female than in male rats might be a common property of steroids with two or three double bonds.

In mid- to high-latitude songbirds, seasonal reproduction is stimulated by increasing day length accompanied by elevated plasma sex steroid levels, increased singing, and growth of the song control nuclei (SCN). Plasticity of the SCN and song behavior are primarily mediated by testosterone (T) and its metabolites in most species studied thus far. However, the majority of bird species are tropical and have less pronounced seasonal reproductive cycles. We have previously documented that equatorial rufous-collared sparrows (Zonotrichia capensis) exhibit seasonal neuroplasticity in the SCN. Manipulating T in these birds, however, did not alter singing behavior. In the current study, we investigated whether T mediates plasticity of the SCN in a similar manner to temperate songbirds. In the first experiment, we treated captive male birds with T or blank implants during the nonbreeding season. In a second experiment, we treated captive male birds with either blank implants, T-filled implants, T with flutamide (FLU; an androgen receptor antagonist) or T with FLU and 1,4,6-androstatriene-3,17-dione (ATD; an estrogen synthesis inhibitor) during the breeding season. In both experiments, the volumes of the brain areas high vocal center (HVC), Area X, and robust nucleus of the arcopallium (RA) were measured along with singing behavior. In summary, T stimulated growth of HVC and RA, and the combined effect of FLU and ATD reversed this effect in HVC. Area X was not affected by T treatment in either experiment. Neither T-treated birds nor controls sang in captivity during either experiment. Together, these data indicate that T mediates seasonal changes in the HVC and RA of both tropical and higher- latitude bird species even if the environmental signals differ. However, unlike most higher-latitude songbirds, we found no evidence that motivation to sing or growth of Area X are stimulated by T under captive conditions.

Using quantitative immunocytochemical procedures, the total number of estrogen and androgen receptors was estimated in a large number of hypothalamic and limbic nuclei of male rats, in which brain estrogen formation was inhibited neonatally by treatment with the aromatase inhibitor 1,4,6-androstatriene-3,17-dione. The highest densities of estrogen receptor immunoreactivity were observed in the periventricular preoptic area and the medial preoptic area. Neonatally estrogen-deprived males showed a higher estrogen receptor immunoreactivity than control males in the periventricular preoptic area and the ventrolateral portion of the ventromedial nucleus of the hypothalamus, i.e. those brain areas in which sex differences have been reported, with female rats showing a greater estrogen binding capacity than male rats. The highest densities of androgen receptor immunoreactivity were found in the septohypothalamic nucleus, the medial preoptic area, the posterior division of the bed nucleus of the stria terminalis and the posterodorsal division of the medial amygdaloid nucleus. No significant differences in distribution or total numbers of androgen receptors were found between neonatally estrogen-deprived males and control males. These findings suggest that neonatal estrogens, derived from the neural aromatization of testosterone, are involved in the sexual differentiation of the estrogen receptor system in the periventricular preoptic area and the ventromedial hypothalamus. The role of neonatal estrogens in the development of the forebrain androgen receptor system is less clear.

The synthesis and biological evaluation of 4-thiosubstituted derivatives of 1,4-androstadienedione, 4,6-androstadienedione, and 1,4,6-androstatrienedione as inhibitors of aromatase are described. Inhibitory activity of synthesized compounds was assessed using a human placental microsomal preparation as the enzyme source and [1 beta-3H]androstenedione as substrate. Under initial velocity assay conditions of low product formation, the inhibitors demonstrated potent inhibition of aromatase, with apparent Kis ranging from 9.8 to 137 nM and with Km for androstenedione being 38 nM. However, unlike other 1,4-androstadienediones and 1,4,6-androstatrienediones in which time-dependent inactivation was observed, the 4-thiosubstituted analogs were found to be competitive inhibitors and did not produce any time-dependent inactivation of aromatase.

In an earlier article an enlarged subpopulation of vasopressin containing neurons was found in the suprachiasmatic nucleus (SCN) of homosexual men as compared to heterosexuals. The present study investigates the possibility that the number of vasopressin neurons in the SCN and sexual partner preference behavior in male rats are both influenced by sex hormones during brain development. For this purpose, we studied groups of adult male rats that had been treated either prenatally or preand postnatally with the aromatase inhibitor ATD (1,4,6-androstatriene-3,17-dione) which blocks the aromatization of testosterone to estradiol. Rats treated with ATD in both preand postnatal periods showed ‘bisexual’ partner preference behavior and appeared to have 59% more vasopressin-expressing neurons in the SCN than the controls. The prenatally treated rats did not differ from the controls. This observation supports the hypothesis that the increased number of vasopressin neurons found earlier in the SCN of adult homosexual men might reflect differences that took place in the interaction between sex hormones and the brain early in development.

Male rats were neonatally treated with cholesterol or a substance that bloccks the aromatization of testosterone to estradiol (1,4,6-androstatriene-3,17-dione: ATD). At weaning (21 days) they were either housed alone or in small groups (2–3 animals) and tested for partner preference behavior (PPB) in adulthood. Choice was between an estrous female and an active male (Part I) and between an estrous female and an ATD-male (Part II). Tests were carried out in a 3-compartment box. Social isolation did not have major effects on PPB except when sexual interaction with the stimulus animals was prevented (Part I). In this case, isolates (ATD and control) showed higher preference scores (PS) for the estrous female and spent less time in the empty middle compartment. When the choice was between an estrous female and an ATD-male, partner PS decreased in all males, most clearly in ATD-males. The latter animals spent more time with the stimulus ATD-male than they had done in previous PPB tests with the normal stimulus male. In contrast to partner preference behaviors, sexual behavior was clearly affected by social isolation. Isolates (ATD and control) displayed lower frequencies of mounts and intromissions. These effects persisted over consecutive tests. Ejaculation was not affected. In conclusion, the present results confirm earlier findings about the significance of neonatal endocrine conditions for the organization of adult PPB in male rats. The presence or absence of social conspecifics after weaning apepars to have little influence on adult PPB.

A semiautomated three-compartment box (3CB) for studying partner preference behavior of rats is described. This apparatus automatically records the rat's time spent in each compartment, as well as the locomotor activity (i.e., the number of visits an animal pays to each compartment). Software was developed for calculating partner preference scores. Behavioral testing in the semiautomated 3CB, which is a modification of an earlier version, is less time consuming and less laborious. Three 3CBs can be observed simultaneously by two trained observers, and the behavioral interactions of three experimental animals with the stimulus animals can be observed and scored by hand. The use of the new apparatus was validated by studying adult partner preference behavior of neonatally ATD-treated male rats. The collected data fully corroborate previous results, obtained in the earlier version of the 3CB, again revealing the behavioral bisexual nature of these ATD males. A new finding was the much higher locomotor activity of the ATD males compared to controls.

A sensitive and selective high-performance liquid chromatographic method for the determination of FCE 24928 (4-amino-androsta-1,4,6-triene-3, 17-dione) in human plasma is reported. The drug was extracted from buffered (pH = 8) plasma samples with methylene chloride-isooctane, then analysed by reversed-phase liquid chromatography. Quantitation was achieved by ultraviolet detection of the eluate at 238 nm. Blank plasma samples from humans, dog and rat assayed as described showed no significant peak at the retention time of the compound of interest. The suitability of the method for in vivo samples was tested by measuring the plasma levels of FCE 24928 in rats that received oral doses of the test compound.

A range of chemicals found in the aquatic environment have the potential to influence endocrine function and affect sexual development by mimicking or antagonizing the effects of hormones, or by altering the synthesis and metabolism of hormones. The aim of this study was to evaluate whether the effects of chemicals interfering with sex hormone synthesis may affect the regulation of early ovarian development via the modulation of sex steroid and insulin-like growth factor (IGF) systems. To this end, ex vivo ovary cultures of juvenile brown trout (Salmo trutta fario) were exposed for 2 days to either 1,4,6-androstatriene-3,17-dione (ATD, a specific aromatase inhibitor), prochloraz (an imidazole fungicide), or tributyltin (TBT, a persistent organic pollutant). Further, juvenile female brown trout were exposed in vivo for 2 days to prochloraz or TBT. The ex vivo and in vivo ovarian gene expression of the aromatase (CYP19), responsible for estrogen production, and of IGF1 and 2 were compared. Moreover, 17β-estradiol (E2) and testosterone (T) production from ex vivo ovary cultures was assessed. Ex vivo exposure to ATD inhibited ovarian E2 synthesis, while T levels accumulated. However, ATD did not affect ex vivo expression of cyp19, igf1, or igf2. Ex vivo exposure to prochloraz inhibited ovarian E2 production, but did not affect T levels. Further prochloraz up-regulated igf1 expression in both ex vivo and in vivo exposures. TBT exposure did not modify ex vivo synthesis of either E2 or T. However, in vivo exposure to TBT down-regulated igf2 expression. The results indicate that ovarian inhibition of E2 production in juvenile brown trout might not directly affect cyp19 and igf gene expression. Thus, we suggest that the test chemicals may interfere with both sex steroid and IGF systems in an independent manner, and based on published literature, potentially lead to endocrine dysfunction and altered sexual development.

Aromatase activity is abundant in limbic and nonlimbic neural structures in zebra finches, especially in the telencephalon near neural circuits that control singing behavior. Also, male songbirds can have high estrogen levels in blood. Because it is difficult to detect aromatase activity in other male tissues, we have postulated that the brain itself is the source of the estrogen present in the blood of males. Previously, we developed methods to measure estrogen synthesis in vivo by injecting [3H]androgen into the systemic circulation or directly into tissues and then determining the quantity of [3H]estrogen entering or leaving the brain. Our results support our hypothesis that the brain is the primary site of estrogen synthesis in males of this species. Here, we confirm that [3H]estrogen emanates from the brain after systemic [3H]androgen injection by showing that the presumptive estrogen in jugular plasma is significantly reduced by treatment with an aromatase inhibitor. In females, but not males, estrogens can be found in large amounts in carotid plasma after systemic androgen injection, presumably due to ovarian estrogen synthesis. However, carotid estrogen levels are variable in females, so it is difficult to determine if the female brain contributes estrogen to blood as is seen in males. The data presented here suggest that the adrenal of males is not an important site of aromatization, because [3H]estrogens are undetected in the adrenals of castrated or sham-castrated males after adrenal injections of [3H] androgen. Nevertheless, at least 2.8- to 17.3-fold more [3H]estrogen was present in the jugular than in the carotid of castrated males after systemic injection of [3H]androgen. Because androgen levels were in the physiological range, we conclude that the brain routinely enriches the estrogen content of blood in normal adult males of this species.

Local modulation of follicular and gametogenic functions by ovarian androgens and estrogens in mammalian species has been proposed. This study examined the effects of elevated androgen/estrogen ratios during follicular maturation in vivo by inhibiting aromatase activity in rhesus monkeys. To obviate steroid feedback effects, gonadotropin-treated animals were used. Beginning at menses (day 1), animals received human (h) FSH (60 IU/day, im) on days 1-6, followed by hFSH plus hLH (60 IU/day, im) on days 7-9 to promote the growth of multiple follicles. Ovulatory maturation was induced by hCG (1000 IU, im) on day 10. On days 8-10, four animals received an aromatase inhibitor, 1,4,6-androstatrien-3,17-dione (ATD; 1-1.25 g, orally, twice/day), while five served as controls and received no further treatment. Within 8 h of ATD treatment, a 63% reduction in serum estradiol levels relative to control values was evident, which reached maximal suppression (84%) by day 10. A marked elevation (17-fold) in serum androstenedione and a lesser increase (2.6-fold) in serum testosterone occurred with aromatase inhibition, yielding androstenedione/estradiol (18.0) and testosterone/estradiol (1.9) ratios greater than those in controls (0.6 and 0.3, respectively). ATD treatment did not alter follicular diameters or the total number of follicles per animal (20 +/- 3) relative to control values (16 +/- 3). Of the total cohort classified, the proportion of oocytes collected at prophase I was greater (P0.05) after ATD treatment (31%) than in controls (11%). Completion of oocyte meiosis to metaphase II was retarded (P0.05) in ATD-treated (4%) compared to control (26%) animals. Furthermore, the in vitro fertilization rate of metaphase II oocytes from ATD-treated animals (9%) was reduced (P0.05) relative to that in controls (25%). While basal progesterone production by luteinizing granulosa cells in vitro was similar between groups, the addition of hCG in vitro enhanced progesterone secretion by cells from ATD-treated animals (3.1 +/- 0.3-fold over basal) to a greater extent (P = 0.05) than in controls (1.5 +/- 0.3-fold). Progesterone receptor was detected by immunocytochemistry in nuclei of luteinizing granulosa cells from ATD-treated animals as well as controls. Serum progesterone profiles and the length of the luteal phase were similar between groups. Thus, acute elevation of serum androgen/estrogen ratios in vivo during follicular maturation was detrimental to the gametogenic functions of the primate follicle, but did not alter follicular growth, events of early luteinization, or subsequent luteal function.(ABSTRACT TRUNCATED AT 400 WORDS)

Steroidal sex hormones play an important role in the neural control of breathing. Previous studies in our laboratory have shown that gonadectomy in young male rats (3 months) eliminates a form of respiratory plasticity induced by intermittent hypoxia, known as long term facilitation (LTF). Testosterone replenishment restores LTF in gonadectomized male rats, and this is dependent on the conversion of testosterone to oestradiol by aromatase. By middle age (12 months), male rats no longer exhibit LTF of hypoglossal motor output; phrenic LTF is significantly reduced, and this persists into old age. We tested the hypothesis that LTF can be restored in old male rats by administration of testosterone. Intact Fischer 344 rats (>20 months) were implanted with Silastic tubing containing testosterone (T), T plus an aromatase inhibitor (T+ADT), or 5α-dihydrotestosterone (DHT), a form of testosterone not converted to oestradiol. One week post-surgery, LTF of hypoglossal and phrenic motor output was measured. By comparison with control rats, hypoglossal LTF was increased in testosterone-treated rats, with levels approaching that of normal young rats. LTF was not restored in T+ADT or DHT-treated rats. Aromatase levels in hypoglossal and phrenic nuclei did not change with age. As serum testosterone levels did not decline with age, local bioavailability of testosterone in old rats may be a limiting factor in the expression of this form of respiratory plasticity. Our findings suggest that testosterone supplementation could potentially be used to enhance upper airway control in the elderly.

Partner preference behavior can be viewed as the outcome of a set of hierarchical choices made by an individual in anticipation of mating. The first choice involves approaching a conspecific verses an individual of another species. As a rule, a conspecific is picked as a mating partner, but early life experiences can alter that outcome. Within a species, an animal then has the choice between a member of the same sex or the opposite sex. The final choice is for a specific individual. This review will focus on the middle choice, the decision to mate with either a male or a female. Available data from rats, mice, and ferrets point to the importance of perinatal exposure to steroid hormones in the development of partner preferences, as well as the importance of activational effects in adulthood. However, the particular effects of this hormone exposure show species differences in both the specific steroid hormone responsible for the organization of behavior and the developmental period when it has its effect. Where these hormones have an effect in the brain is mostly unknown, but regions involved in olfaction and sexual behavior, as well as sexually dimorphic regions, seem to play a role. One limitation of the literature base is that many mate or ‘partner preference studies’ rely on preference for a specific stimulus (usually olfaction) but do not include an analysis of the relation, if any, that stimulus has to the choice of a particular sexual partner. A second limitation has been the almost total lack of attention to the type of behavior that is shown by the choosing animal once a ‘partner’ has been chosen, specifically, if the individual plays a mating role typical of its own sex or the opposite sex. Additional paradigms that address these questions are needed for better understanding of partner preferences in rodents.

Sex steroid hormones during development permanently alter, or organize, the brain and behavior, while during adulthood they act to reversibly modulate, or activate, physiology and behavior. Testosterone exerts both organizational and activational effects on the magnitude of the hypothalamic-pituitary-adrenal (HPA) axis response to acute stress. What has never been approached is how testosterone can organize habituation of the HPA axis, in which stress induced elevations in ACTH and corticosterone release decline over repeated exposures to the same stimulus. In the current study we examined HPA responses to repeated psychogenic stress in 65-day-old, adult male rats that received subcutaneous capsules containing the antiandrogen flutamide or the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD), introduced within 12h of birth and removed on day 21 of weaning. An additional group of castrated, adult male rats were used to differentiate organizational from activational effects of testosterone. All treatment groups displayed smaller declines in ACTH in response to repeated restraint compared to control animals. Remarkably, the normal decline in corticosterone failed to occur in flutamide- and ATD-treated animals. By contrast, males that were castrated as adults showed a significant reduction in corticosterone after repeated stress. Taken together, these findings underscore an organizing influence of both androgen receptors and estrogen conversion on HPA habituation to repeated psychogenic stress, which appears to occur independent of the activational effects of testosterone.

In order to clarify the effect of the prenatal (PN) treatment of the drug 1,4,6-androstatriene-3,17-dione (ATD) which blocks the conversion of testosterone into estradiol on male sexual behavior of the rats offsprings, from the effect of the mild stress induced by the PN administration of the Propylene glycol (PG), the vehicle used to dissolve ATD.Pregnant Wistar rats were divided into three groups. The CON group did not receive any kind of treatment. The other two groups (PG and ATD) were injected i.p. during gestation (days 11-22) with 0 and 5 mg of ATD, dissolved in 0.1 ml of PG, respectively, doses reported by other authors. Sexual performance of the male pups was analyzed three months later in four successive tests.In the first sexual test of these naive rats, the percentage of males mounting, intromitting, ejaculating and the ejaculation frequency of the ATD group decreased significantly in comparison with the CON group. Also in the first and 4th tests, mounting, intromission and ejaculation latencies, as in the post-ejaculatory refractory period, ATD group, was significantly longer in comparison with the CON group. PG males showed a male sexual behavior (MSB) similar to that observed in the ATD group, but the differences did not reach statistical significance when they were compared with the CON group.We considered that the PN stress induced by the daily administration of PG and ATD, results in a slower execution of the MSB in both groups and avoid distinguish the effect of the ATD. Then chronic PN injections, as a route of administration, could act as mild stressor and may have additive effects on drugs affecting brain sexual differentiation.

The thymus can be regenerated in aging rats by surgical or chemical castration and regeneration is inhibited by testosterone, which may exert this effect, at least in part, through its conversion to estradiol. An attempt has been made to regenerate the thymus in intact aging rats using inhibitors of the aromatase system, in the hope that this maneuver could lead to the use of such chemical intervention in the treatment of immunodeficiency syndromes. Young adult and aging (18-month-old) male rats were orchidectomized under ether anesthesia and 7 days later given s.c. implants of testosterone in silicone elastomer (SILASTIC) tubing. Some rats received testosterone together with a five-fold excess of the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD). One group of young intact rats received implants containing 25 mg ATD and a group of 18-month-old intact rats received 125 mg ATD or 25 mg of another, more powerful aromatase inhibitor 4-hydroxyandrostenedione (4-OH). On the 28th day after implanting, rats were killed and the thymus, spleen, prostate gland and seminal vesicles removed for weighing and histology. In addition, estrogen receptors were measured in the thymus. The thymus was enlarged after orchidectomy and greatly restored in aging rats. In aging rats, both aromatase inhibitors restored the thymus, which appeared normal histologically. In addition, ATD enlarged the thymus in young intact animals. Doses of testosterone which restored the accessory sex organs to weights measured in intact rats prevented the effects of orchidectomy on the thymus, and in old rats the effects of testosterone were blocked by ATD in both thymus and spleen. Available cytosolic estrogen receptors were reduced in thymus of testosterone-treated orchidectomized rats, and this effect blocked by ATD, which itself was apparently able to induce estrogen receptors. Receptors could not be detected in thymus from aging rats, but were measureable in cytosols from thymus of orchidectomized or ATD-treated old rats. It is therefore possible to restore the thymus in intact aging rats without recourse to surgical or chemical castration, and such a maneuver may possibly be of use to enhance an immune system weakened by aging or disease.

Most sexual dimorphisms in reproductive behavior are hormonally organized in the guinea pig. This study sought to determine whether the sexually dimorphic requirement for the aromatization of testosterone in the activation of mounting is organized by testosterone prenatally and whether aromatization of testosterone contributes to the organization of mounting behavior. Pregnant females were treated with testosterone, the aromatase inhibitor ATD, or vehicle from days 28–65 of gestation. The offspring were gonadectomized and tested as adults for lordosis and androgen-activated mounting behavior. Prenatal testosterone treatments altered the hormonal requirements for androgen-activated mounting in females such that they resembled normal males, and did not require aromatization as adults. Prenatal inhibition of aromatase activity decreased mounting activity in females but not in males. This treatment had no influence on lordosis in either sex. The results support the hypothesis that the same hormones that activate mounting behavior in the adult guinea pig are responsible for the organization of mounting behavior.

Sex steroids play a significant role in organizing male social behavior, which is associated with low levels of pro-social behavior and high levels of aggression. However, the role of steroids in organizing behavior in highly social males is unclear. The authors tested the hypothesis that low levels of sex steroids facilitate the expression of pro-social behavior in male prairie voles (Microtus ochragaster), predicting that inhibition of testosterone and estradiol would reduce spontaneous-alloparental behavior. Treatment with the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD) or the androgen receptor blocker flutamide, days 8-14, significantly reduced the expression of alloparental behavior in 21-day old males. While both treatments reduced alloparental behavior and increased pup-directed aggression, there were differential treatment effects. Flutamide altered initial response, increasing latency to enter the pup cage and the likelihood of retreat from initial contact. ATD-treated males that were alloparental showed increases in sniffing and latencies to lick and huddle. Results indicate that endogenous steroids play a role in the development of male pro-social behavior and the effects of estrogens and androgens differ.

The protandrous black porgy, Acanthopagrus schlegeli, has a striking life cycle with sex differentiation at the juvenile stage, mono-male development, and male-to-female sex change (with vitellogenic oocytes) at age 3 yr. In the present study, we investigated the possible roles of wnt4 in gonadal development in a nonmammalian model organism (protandrous black porgy), especially in relation to sex differentiation, ovarian growth, and sex change. Fish of various ages were treated with estradiol (E2) or aromatase inhibitor (AI) to determine whether manipulation of the hormonal environment had an effect on these processes. Furthermore, a natural sex change (or =2-yr-old fish) and a nonchemical method to induce an early sex change (or =1-yr-old fish) via the removal of testicular tissue were examined in this study. We present herein an integrative immunohistochemical, cellular, and molecular data set describing these phenomena. During gonadal sex differentiation, no increase in wnt4 expression was detected. A profile of increased wnt4 expression and decreased cyp19a1a expression was associated with ovarian growth (proliferation of oogonia and development of ovarian lamellae) inor =1-yr-old fish. Both E2 and AI induced an increase in wnt4 transcripts and resulted in ovarian development inor =0-yr-old andor =1-yr-old fish. Increased wnt4 transcripts were found in ovarian tissue undergoing development from primary oocytes to vitellogenic oocytes during the natural sex change inor =2-yr-old fish. Removal of testicular tissue inor =1-yr-old fish resulted in successful early sex change (with vitellogenic oocytes) 6 mo after the excision. During the process of the early sex change (3 mo after testis excision), the fish ovary became active and had increased diameter of the primary oocytes; this was in accord with increased ovarian wnt4 expression but not sf1, foxl2, and genes in the steroidogenic pathway, including cyp19a1a. Wnt4 staining further confirmed the profile of wnt4 expression associated with ovarian development. The results of the present study suggest that wnt4 has important roles in late ovarian growth (e.g., oogonia proliferation and structure of ovarian lamellae) and the natural sex change (vitellogenic oocytes) in the protandrous black porgy.

Estradiol impacts a wide variety of brain processes, including sex differentiation, mood, and learning. Here we show that estradiol regulates auditory processing of acoustic signals in the vertebrate brain, more specifically in the caudomedial nidopallium (NCM), the songbird analog of the mammalian auditory association cortex. Multielectrode recordings coupled with local pharmacological manipulations in awake animals reveal that both exogenous and locally generated estradiol increase auditory-evoked activity in NCM. This enhancement in neuronal responses is mediated by suppression of local inhibitory transmission. Surprisingly, we also found that estradiol is both necessary and sufficient for the induction of multiple mitogen-activated protein kinase (MAPK)-dependent genes thought to be required for synaptic plasticity and memorization of birdsong. Specifically, we show that local blockade of estrogen receptors or aromatase activity in awake birds decrease song-induced MAPK-dependent gene expression. Infusions of estradiol in acoustically isolated birds induce transcriptional activation of these genes to levels comparable with song-stimulated animals. Our results reveal acute and rapid nongenomic functions for estradiol in central auditory physiology and suggest that such roles may be ubiquitously expressed across sensory systems.

1. The intensity of the transformation of T into E2 and DHT in the hypothalamus of 21-day-old male rats is greater than in adults. 2. The aromatase inhibitor ATD intensifies the formation of DHT in the hypothalamus of sexually mature male rats. 3. The LH level of LH and T in the blood plasma increases significantly under the influence of ATD in male rats. The reaction of LH is markedly more intense in prepubertal animals as compared with sexually mature animals.

7-Phenyl-1,4,6-androstatriene-3,17-dione (4), 7-benzyl-1,4,6-androstatriene-3,17-dione (5) and 7-phenethyl-1,4,6-androstatriene-3,17-dione (6) were synthesized and evaluated in vitro in human placental microsomes as enzyme-activated irreversible inhibitors of aromatase. The compounds were synthesized from appropriate 7-substituted 4,6-androstadiene-3, 17-diones by reaction with DDQ under neutral conditions. All the compounds produced a first order inactivation of aromatase in the presence of NADPH but not in the absence of NADPH. Substrate 4-androstene-3, 17-dione protected the enzyme from inactivation by the inhibitors. Furthermore, cysteine failed to protect aromatase from inactivation by compounds 5 and 6. In contrast, cysteine partially protected aromatase from inactivation by compound 4. Irreversibility studies illustrated the covalent nature of the inactivation by 4, 5 and 6. The above experimental evidence demonstrated that compounds 5 and 6 are effective enzyme-activated irreversible inhibitors of aromatase.

According to a proposed aromatisation mechanism by which estrogens are biosynthesized from androgens, the novel steroid androsta-4,6,8(9)-triene-3,17-dione (FCE 24918) should behave as a suicide substrate for aromatase. The synthesis of this triene steroid has been accomplished starting from androsta-4,7-diene-3,17-dione (4) by the acid-catalysed cleavage of the corresponding 7,8α-epoxide, 5, and it was obtained together with androsta-4,6,8(14)-triene-3,17-dione (FCE 24917) as a side product. The time-dependent inactivation of placental aromatase by the two isomers was studied comparatively and showed that the 4,6,8(9)-triene moiety acts as a latent alkylating group.

A series of 4 experiments was designed to study the relationships between the activity of the aromatase (AA) in the preoptic area (POA) and the activation by testosterone (T) of copulatory behavior in gonadectomized male and female Japanese quail. The induction of AA by T in the POA is dose- and time-dependent. Levels of AA seen in sexually mature males are restored in castrated birds by a treatment with 20 to 40 mm silastic T capsules which produce physiological levels of steroid in the plasma. The minimal dose of T (10 mm implant) which reliably restores copulatory behavior approximately doubles the AA in the POA. The induction of AA is significantly larger in males than in females. A significant increase in AA is observed within 16 hours after the start of the treatment with T and the induction is maximal after 48 hours. Activation of copulatory behavior follows a similar time course but occurs with a delay of 24-48 hours. These results thus suggest that, in male quail, the activity of the aromatase in the POA is a limiting factor in the activation of copulatory behavior. This idea is confirmed by direct experimentation using an aromatase inhibitor, androstatrienedione (ATD). If T-treated birds receive at the same time silastic implants filled with ATD, the activation of behavior is suppressed for at least one week. This behavioral inhibition is, as expected, accompanied and very probably caused by the inhibition of the aromatase activity in the preoptic area and anterior hypothalamus. No increase of enzyme activity over the level seen in castrates was actually detected during the first 8 days of exposure to T. A moderate increase in AA was seen on day 16 and is probably responsible for the behavioral activation which was observed at the end of the experiment.

Copulatory behaviors in most rodents are highly sexually dimorphic, even when circulating hormones are equated between the sexes. Prairie voles (Microtus ochrogaster) are monomorphic in their display of some social behaviors, including partner preferences and parenting, but differences between the sexes in their masculine and feminine copulatory behavior potentials have not been studied in detail. Furthermore, the role of neonatal aromatization of testosterone to estradiol on the development of prairie vole sexual behavior potentials or their brain is unknown. To address these issues, prairie vole pups were injected daily for the first week after birth with 0.5 mg of the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD) or oil. Masculine and feminine copulatory behaviors in response to testosterone or estradiol were later examined in both sexes. Males and females showed high mounting and thrusting in response to testosterone, but only males reliably showed ejaculatory behavior. Conversely, males never showed feminine copulatory behaviors in response to estradiol. Sex differences in these behaviors were not affected by neonatal ATD, but ATD-treated females received fewer mounts and thrusts than controls, possibly indicating reduced attractiveness to males. In other groups of subjects, neonatal ATD demasculinized males' tyrosine hydroxylase expression in the anteroventral periventricular preoptic area, and estrogen receptor alpha expression in the medial preoptic area. Thus, although sexual behavior in both sexes of prairie voles is highly masculinized, aromatase during neonatal life is necessary only for females' femininity. Furthermore, copulatory behavior potentials and at least some aspects of brain development in male prairie voles are dissociable by their requirement for neonatal aromatase.

Zebra finches, like many other animals, have close social relationships mainly with the family at young ages but begin to express interest in opposite-sex extra-family animals as they enter the late juvenile period and sexual maturity. This experiment tested a set of hypotheses that sex steroids are involved in this developmental transition. At 25-30 days, subjects were implanted subcutaneously with Silastic tubes that were empty (controls), filled with testosterone propionate, filled with estradiol benzoate, or filled with a combination of ATD (an aromatization inhibitor) and flutamide (an anti-androgen). Once a week between ages 30 and 90 days, they were given three-choice tests where the three stimulus types were the family members, unpaired males, or unpaired females. The preferred category was defined as the one adjacent to the proximity zone in which the subject spent the most time. Control males were more likely to prefer females and less likely to prefer the family as they got older, and control females were increasingly likely to prefer males. Males treated with testosterone or estradiol showed a premature increase in preferences for females. Females treated with ATD plus flutamide failed to show the normal increase in preferences for males shown by controls. These results indicate an involvement of sex steroids in the maturation of sexual preferences in a socially monogamous species that relies on visual and auditory, rather than olfactory, cues for sexual or other social behavior.

This study tested the hypothesis that aromatization of testosterone to estradiol is necessary for sexual differentiation of the sheep brain. Pregnant ewes (n = 10) were treated with the aromatase inhibitor 1,4,6- androstatriene-3,17-dione (ATD) during the period of gestation when the sheep brain is maximally sensitive to the behavior-modifying effects of exogenous testosterone (embryonic d 50-80; 147 d is term). Control (n = 10) ewes received vehicle injections. Fifteen control lambs (7 males and 8 females) and 17 ATD-exposed lambs (7 males and 10 females) were evaluated for sexually dimorphic behavioral and neuroendocrine traits as adults. Prenatal ATD exposure had no significant effect on serum concentrations of androgen at birth, growth rates, expression of juvenile play behaviors, or the onset of puberty in male and female lambs. Rams exposed to ATD prenatally exhibited a modest, but significant, decrease in mounting behavior at 18 mo of age. However, prenatal ATD exposure did not interfere with defeminization of adult sexual partner preferences, receptive behavior, or the LH surge mechanism. In summary, our results indicate that aromatization is necessary for complete behavioral masculinization in sheep. However, before we can conclude that aromatization does not play a role in defeminization of the sheep brain, it will be necessary to evaluate whether intrauterine exposure of male fetuses to higher doses of ATD for a more extended period of time can disrupt normal neuroendocrine and behavioral development.

The effects of maternal administration of the aromatase inhibitor, 1,4,6-androstatrien-3,17-dione (ATD), during the last week of gestation on stress reaction of the hypothalamic-pituitary-adrenal axis (HPA) and behavior in a novel environment (open field) and the anxiety level in the elevated plusmaze, were studied in male and female adult offspring. The results showed that parental inhibition of brain testosterone metabolism decreases the basic level of corticosterone in male rats and prolongs hormonal stress reaction of the HPA axis in both sexes. Prenatally treated rats demonstrated significant elevation of the anxiety level and emotionality. There was no sexual dimorphism in behavioral response to a novel environment such as locomotor activity, the time of immobilization, the total duration of grooming reaction, and the anxiety level, between control male and treated female rats. These data suggest a prenatal inhibition of the brain testosterone metabolism after the stress reaction of HPA axis and formation of sexual dimorphism in the anxiety and behavioral response to a novel environment in adulthood.

The purpose of this study was to test whether sex steroid actions are necessary for courtship and pairing in socially monogamous birds. We examined the effects of an aromatase inhibitor, 1,4,6-androstatriene-3,17-dione (ATD), combined with an anti-androgen, flutamide (F), on the behavior and pairing status of initially unpaired male and female zebra finches (Taeniopygia guttata). In the first experiment, 24 adult males were implanted with either a combination of ATD and flutamide or empty implants. Two weeks after implantation, birds were housed in aviaries containing 3 ATD + F males, 3 control males, and 3 females and allowed 2 weeks to pair, with observations 7 times during the 2-week period. A second experiment tested the effects of these same treatments in females. During the first 4 days of testing, ATD + F males were less likely to attack conspecifics than were control males. ATD + F males were also less likely to "greet," or approach, females than were control males, but other courtship behaviors, including directed singing, were unaffected. ATD + F females did not differ from control females on any courtship behavior measured. Furthermore, these treatments did not affect pairing behaviors (time spent clumping or in a nest box together) or the likelihood of pairing with a partner of the opposite sex. ATD + F treatments in females did, however, increase the likelihood of same-sex pairing. This suggests that, although sex steroids may regulate some courtship behaviors in males, they do not regulate pairing behaviors and have little effect on the likelihood that a male or female will be chosen as a mate by a bird of the opposite sex.