Masculinization in common carp (Cyprinus carpio) by androgen immersion: The interaction effect of hormone concentration and immersion time / Masculinização na carpa comum (Cyprinus carpio) por imersão em androgênio: Efeito da interação da concentração hormonal e do tempo de imersão

Synthetic androgens (male hormones) administered to fish nursery are being used in aquaculture to avoid sexual differentiation and unwanted spawning at the eggs or the first feeding fry stage of fish. Present trial was conducted with the aim to produce male common carp (Cyprinus carpio) by egg immersion technique. Through this little insight, the effect of different hormone concentrations (17α-methyltestosterone @ HC:150, 300, 450 and 600 µgl-1) with immersion times (IT: 24, 48 and 72 hrs) and their interaction effect (HC x IT) on the hatching percentage of Cyprinus carpio eggs, percent survival and percent of male's production was evaluated specifically. Results showed that egg hatching percentage decreased with increased IT likewise, survival of treated fry was affected by increasing the IT (P<0.001). The main interaction effect of HC x IT showed that the highest percent of male individuals (95%) was obtained at 450-600 µgl-1 HC for 72 hrs IT, followed by 88-92.50% at 150-300 µgl-1 HC for 72-hrsof IT, 87.50% at 48-hrs of IT for rest of the hormone treatments, and lowest 47.50% was recorded in control (P<0.05). Increased percent male of Cyprinus carpio was obtained with increasing HC across all ITs. It was observed that the immersion treatment at 600µgl-1 for 72 hours was more effective to change the sex ratio of pre hatch Cyprinus carpio. A comparative outlook made from this experimental trial that sex induction of Cyprinus carpio by eggs immersion using synthetic male steroid hormone is an alternative safe technique of fish sex reversal in contrast to oral administration of hormone in fish feed.

Andrógenos sintéticos (hormônios masculinos) administrados ao viveiro de peixes estão sendo usados ​​na aquicultura para evitar a diferenciação sexual e a desova indesejada nos ovos ou no primeiro estágio de alimentação dos peixes. O presente estudo foi conduzido com o objetivo de produzir carpa comum masculina (Cyprinuscarpio) pela técnica de imersão em ovos. Com essa pequena percepção, o efeito de diferentes concentrações hormonais (17α-metiltestosterona @ HC: 150, 300, 450 e 600 µgl-1) com tempos de imersão (IT: 24, 48 e 72 horas) e seu efeito de interação (HC x IT) na porcentagem de eclosão dos ovos de Cyprinuscarpio, a porcentagem de sobrevivência e a porcentagem da produção masculina foram avaliadas especificamente. Os resultados mostraram que a porcentagem de incubação de ovos diminuiu com o aumento da TI da mesma forma, a sobrevivência dos alevinos tratados foi afetada pelo aumento da TI (P <0,001). O principal efeito de interação do HC x IT mostrou que o maior percentual de indivíduos do sexo masculino (95%) foi obtido com 450-600 µgl-1 HC por 72 horas de TI, seguido por 88-92,50% com 150-300 µgl-1 HC para 72 horas de TI, 87,50% às 48 horas de TI para o restante dos tratamentos hormonais, e 47,50% mais baixos foram registrados no controle (P <0,05). A porcentagem aumentada de macho de Cyprinuscarpio foi obtida com o aumento do HC em todas as TIs. Observou-se que o tratamento de imersão a 600µgl-1 por 72 horas foi mais efetivo na alteração da razão sexual do Cyprinuscarpio antes da eclosão. Uma perspectiva comparativa feita a partir deste ensaio experimental de que a indução sexual de Cyprinuscarpio por imersão de ovos usando hormônio esteróide masculino sintético é uma técnica alternativa segura de reversão do sexo em peixes, em contraste com a administração oral de hormônio na alimentação de peixes.

Redox-Sensitive and Hyaluronic Acid-Functionalized Nanoparticles for Improving Breast Cancer Treatment by Cytoplasmic 17α-Methyltestosterone Delivery.

Novel reduction-responsive hyaluronic acid-chitosan-lipoic acid nanoparticles (HACSLA-NPs) were designed and synthesized for effective treatment of breast cancer by targeting Cluster of Differentiation 44 (CD44)-overexpressing cells and reduction-triggered 17α-Methyltestosterone (MT) release for systemic delivery. The effectiveness of these nanoparticles was investigated by different assays, including release rate, 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT), lactate dehydrogenase (LDH), caspase-3 activity, Rhodamine 123 (RH-123), and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). In vitro experiments revealed that Methyltestosterone/Hyaluronic acid-chitosan-lipoic acid nanoparticles (MT/HACSLA-NPs) illustrated a sustained drug release in the absence of glutathione (GSH), while the presence of GSH led to fast MT release. HACSLA-NPs also showed high cellular internalization via CD44 receptors, quick drug release inside the cells, and amended cytotoxicity against positive CD44 BT-20 breast cancer cell line as opposed to negative CD44, Michigan Cancer Foundation-7 (MCF-7) cell line. These findings supported that these novel reduction-responsive NPs can be promising candidates for efficient targeted delivery of therapeutics in cancer therapy.

Inter- and Intraspecific Variation in Sex Hormone-Induced Sex-Reversal in Medaka, Oryzias latipes and Oryzias sakaizumii.

We compared sex-reversal ratios induced by 17α-methyltestosterone (MT) and 17ß-estradiol (E2) exposure in two inbred medaka strains: Hd-rR derived from Oryzias latipes and HNI-II from O. sakaizumii. All MT exposures (0.2-25 ng mL-1) induced complete XX sex-reversal in HNI-II. Although MT exposure at 0.2 ng mL-1 induced XX sex-reversal at > 95% in Hd-rR, other concentrations tested caused XX sex-reversal at lower frequencies (<50%). MT exposure at 1, 5, and 25 ng mL-1 induced XY sex-reversal in Hd-rR, but not in HNI-II. In Hd-rR, E2 exposure induced XY sex-reversal at > 10 ng mL-1, and in all fish feminization occurred 500 ng mL-1. In HNI-II, E2 induced XY sex-reversal at 50 and 250 ng mL-1, but only at rates below 20%. To clarify whether the strain differences in sex hormone-induced sex-reversal are characteristic of each species, we examined the effects of MT and E2 exposure on sex differentiation in five and two additional strains or wild stocks/populations of O. latipes and O. sakaizumii, respectively. MT exposure induced low XX and high XY sex-reversal rates in O. latipes, except in the Shizuoka population, but the trend was reversed in O. sakaizumii. Furthermore, E2-induced XY sex-reversal rates varied intraspecifically in O. latipes. Our results demonstrated that sensitivity to MT and E2 varied within O. latipes species. To evaluate the ecological impacts of environmental chemicals using medaka, it is important to define not only the species, but the strains, stocks, and populations to obtain accurate results.

The co-administration of estradiol/17α-methyltestosterone leads to male fate in the protogynous orange-spotted grouper, Epinephelus coioides.

Estrogen plays a pivotal role in the sex differentiation of teleosts, whereas the precise function of androgens is more controversial. In this study, orange-spotted grouper (Epinephelus coioides) fry were treated with letrozole (an aromatase inhibitor, AI), 17α-methyltestosterone (MT), or MT and 17ß-estradiol (E2) simultaneously, during the period of gonadal formation and sex differentiation. MT feeding at 50 days after hatching resulted in gonadal dysgenesis, which could be rescued by E2 supplementation. Different doses of AI treatment led to different phenotypes: undifferentiated gonads were maintained in the AI group fed a low dose (5 mg/kg diet), whereas female-to-male sex reversal was observed in the AI group fed a high dose (100 mg/kg diet). MT and MT + E2 treatment could induce female-to-male sex reversal during sex differentiation (90 days after hatching). The expression of female pathway genes was suppressed, while the expression of genes in the male pathway was up-regulated in the MT + E2 group. Consistent with the expression of sex-related genes, the serum 11- ketotestosterone level was also upregulated in MT and MT + E2 group. Finally, we examined the expression of male-specific mark (DMRT1) and proliferating cell nuclear antigen in MT and MT + E2 induced sex reversal, and the result indicated that male germ cells and somatic cells may origin from the gonium and proliferative somatic cells surrounding the efferent duct, respectively. Overall, our data suggested that estrogen acts as a natural inducer of female differentiation, and that the co-administration of estrogen and androgen during sex differentiation leads to a male sex fate in the protogynous orange-spotted grouper.

MT-Feeding-Induced Impermanent Sex Reversal in the Orange-Spotted Grouper during Sex Differentiation.

In this study, we systematically investigated the process of sex reversal induced by 17-methyltestosterone (MT) feeding and MT-feeding withdrawal at the ovary differentiation stage in orange-spotted groupers, Epinephelus coioides. Gonadal histology showed that MT feeding induced a precocious sex reversal from immature ovaries to testes, bypassing the formation of an ovarian cavity, and MT-feeding withdrawal led to an ovarian fate. In both the MT feeding and MT-feeding withdrawal phases, cytochrome P450 family 11 subfamily B (cyp11b) gene expression and serum 11-KT levels were not significantly changed, suggesting that the MT-treated fish did not generate endogenous steroids, even though active spermatogenesis occurred. Finally, by tracing doublesex-expressing and Mab-3-related transcription factor 1 (dmrt1)-expressing cells and TUNEL (terminal deoxynucleotidyl transferase 2-deoxyuridine, 5-triphosphate nick end labeling) assays, we found that the efferent duct formed first, and then, the germ cells and somatic cells of the testicular tissue were generated around the efferent duct during MT-feeding-induced precocious sex reversal. Collectively, our findings provide insights into the molecular and cellular mechanisms underlying sex reversal induced by exogenous hormones during sex differentiation in the protogynous orange-spotted grouper.

17α-Methyltestosterone implants accelerate spermatogenesis in common snook, Centropomus undecimalis, during first sexual maturation.

The common snook, Centropomus undecimalis, is an emerging species for intensive fish culture, however, some reproductive aspects of this species, especially the development of the testes and the action of androgen hormones on spermatogenesis have not been studied. The objective of this study was to evaluate the effects of 17α-methyltestosterone (MT) on spermatogenesis and steroidogenesis during the first sexual maturation of the common snook. The fish, which were reproduced in captivity, had a body weight of 305.80 ± 35.60 g and a total length of 34,11 ± 1,08 cm. We used ethylene-vinyl-acetate (EVAc) implants with four concentrations of the hormone MT: T1 (0.3 mg/kg); T2 (3.0 mg/kg); T3 (15.0 mg/kg) and T4 (30.0 mg/kg), and a control group that did not receive the hormone. The gonads increased (P < 0.05) in relation to the concentrations of MT. Histological analysis revealed a progression of spermatogenesis in the MT treatments, especially in T3 and T4. Sperm release was attained in some fish treated with MT. However, there was a partial suppression of the levels of testosterone (T) and 11-ketotestosterone (11-KT) in plasma in the MT treatments, indicating a negative feedback on steroidogenesis. However, this suppression of T and 11-KT in plasma did not prevent an increase in the gonadosomatic index and the progression of gametogenesis. There was also an increase of estradiol (E2) in plasma in the treatments with the highest MT concentrations. The results suggest that the application of EVAc implants with MT at concentrations of 15 and 30 mg/kg stimulates the development and growth of the testes and accelerates spermatogenesis.

Molecular regulation of sex change induced by methyltestosterone -feeding and methyltestosterone -feeding withdrawal in the protogynous orange-spotted grouper.

The sex identity of fish can be easily manipulated by exogenous hormones. Treatment with 17-methyltestosterone (MT) has been widely used to induce a male fate, but the molecular and cellular processes underlying sex changes induced by MT treatments and the withdrawal of MT are not well studied. In this study, we systematically investigated gonadal histology, gene expression profiles, sex steroid hormone levels, and cellular changes during sex changes induced by MT-feeding and MT-feeding withdrawal in the protogynous orange-spotted grouper, Epinephelus coioides. Based on gonadal histology, we demonstrated that MT-feeding-induced sex reversal can be divided into early and late phases: in the early phase, male and female germ cells coexist, and MT-feeding withdrawal leads to a female fate; in the late phase, only male germ cells are observed, and MT-feeding withdrawal does not reverse the process, leading to a male fate. In both the early and late phases, cytochrome P450 family19 subfamily A member 1 (cyp19a1a) gene expression increased in response to MT-feeding withdrawal. Finally, by tracing doublesex- and Mab-3-related transcription factor 1 (dmrt1)-expressing cells, we found that gonia-like cells in the germinal epithelium might be the major germ cell sources for developing testes during sex reversal. Collectively, our findings provide insights into the molecular and cellular mechanisms underlying sex changes induced by exogenous hormones.

Mechanism-based risk assessment strategy for drug-induced cholestasis using the transcriptional benchmark dose derived by toxicogenomics.

Cholestasis is one of the major causes of drug-induced liver injury (DILI), which can result in withdrawal of approved drugs from the market. Early identification of cholestatic drugs is difficult due to the complex mechanisms involved. In order to develop a strategy for mechanism-based risk assessment of cholestatic drugs, we analyzed gene expression data obtained from the livers of rats that had been orally administered with 12 known cholestatic compounds repeatedly for 28 days at three dose levels. Qualitative analyses were performed using two statistical approaches (hierarchical clustering and principle component analysis), in addition to pathway analysis. The transcriptional benchmark dose (tBMD) and tBMD 95% lower limit (tBMDL) were used for quantitative analyses, which revealed three compound sub-groups that produced different types of differential gene expression; these groups of genes were mainly involved in inflammation, cholesterol biosynthesis, and oxidative stress. Furthermore, the tBMDL values for each test compound were in good agreement with the relevant no observed adverse effect level. These results indicate that our novel strategy for drug safety evaluation using mechanism-based classification and tBMDL would facilitate the application of toxicogenomics for risk assessment of cholestatic DILI.

Low dose of methyltestosterone in ovariectomised rats improves baroreflex sensitivity without geno- and cytotoxicity.

This study evaluated the effects of the isolated use of a low dose of methyltestosterone (MT) on cardiovascular reflexes and hormonal levels and its geno- and cytotoxic safety in ovariectomized rats. Female Wistar rats were divided into four groups (n = 6), respectively: SHAM (received vehicle methylcellulose 0.5%), SHAM + MT (received MT 0.05 mg/kg), OVX (received vehicle), and OVX + MT (received MT). Twenty-one days after ovariectomy, treatment was given orally daily for 28 days. The Bezold-Jarisch reflex (BJR) was analyzed by measuring the bradycardic and hypotensive responses elicited by phenylbiguanide (PBG) administration. The baroreflex sensitivity (BRS) was evaluated by phenylephrine and sodium nitroprussite. Myocyte hypertrophy was determined by morphometric analysis of H&E stained slides. Biochemical data were analyzed, as well as micronucleus assay. MT improved BRS and increased testosterone values, but did not change estradiol in the OVX group. MT did not promote changes in mean arterial pressure, heart rate, BJR, serum concentrations of troponin I, weight and histopathology of the heart. MT was able to restore the BRS in OVX rats. The geno- and cytotoxic safety of the MT was demonstrated by the absence of an increase in the micronucleus (PCEMN) or change in the ratio between normochromatic erythrocytes and polychromatic erythrocytes (NCE/PCE).

Simultaneous exposure to estrogen and androgen resulted in feminization and endocrine disruption.

Estrogen, which is synthesized earlier in females than androgen in males, is critical for sex determination in non-mammalian vertebrates. However, it remains unknown that what would happen to the gonadal phenotype if estrogen and androgen were administrated simultaneously. In this study, XY and XX tilapia fry were treated with the same dose of 17α-methyltestosterone (MT) and 17ß-estradiol (E2) alone and in combination from 0 to 30 days after hatching. Treatment of XY fish with E2 resulted in male to female sex reversal, while treatment of XX fish with MT resulted in female to male sex reversal. In contrast, simultaneous treatment of XX and XY fish with MT and E2 resulted in female, but with cyp11b2 and cyp19a1a co-expressed in the ovary. Serum 11-ketotestosteron level of the MT and E2 simultaneously treated XX and XY female was similar to that of the XY control, while serum E2 level of these two groups was similar to that of the XX control. Transcriptomic cluster analysis revealed that the MT and E2 treated XX and XY gonads clustered into the same branch with the XX control. However a small fraction of genes, which showed disordered expression, may be associated with stress response. These results demonstrated that estrogen could maintain the female phenotype of XX fish and feminize XY fish even in the presence of androgen. Simultaneous treatment with estrogen and androgen up-regulated the endogenous estrogen and androgen synthesis, and resulted in disordered gene expression and endocrine disruption in tilapia.

Properties of PLA/PCL particles as vehicles for oral delivery of the androgen hormone 17α-methyltestosterone.

The aim of this study was to produce PLA (poly(lactic acid)) and PCL (polycaprolactone) oral carriers through the precipitation of the polymer solutions using supercritical CO2 as an antisolvent for the controlled release of the hydrophobic model drug 17α-methyltestosterone (MT). Such drug is a steroidal hormone used orally to develop and sustain primary and secondary male sex characteristics, e.g. for female Nile tilapia sex reversal in aquaculture. The influence of hormone, PLA and PCL concentrations on particle formation was analyzed, showing that high PCL concentrations produced particles with rougher surfaces and greater mean diameters. The incorporation efficiency of MT ranged from 20 to 51%, and its addition resulted in increases in particle mean diameter from 23 to 54 µm. Aggregation was observed for particles incorporating or not MT and high concentrations of MT led to the formation of more amorphous structures, changing the thermal behavior of the particles. The exposure of the PLA/PCL particles to pH conditions simulating gastrointestinal fish conditions showed that hormone release fraction at acidic pH ranged from 8 to 63% (over 2h), while in the basic pH the proportion released varied from 23 to 60% (over 10h), reaching levels adequate for the desired in vivo activity.

Sexual Fate Reprogramming in the Steroid-Induced Bi-Directional Sex Change in the Protogynous Orange-Spotted Grouper, Epinephelus coioides.

Androgen administration has been widely used for masculinization in fish. The mechanism of the sex change in sexual fate regulation is not clear. Oral administration or pellet implantation was applied. We orally applied an aromatase inhibitor (AI, to decrease estrogen levels) and 17α-methyltestosterone (MT, to increase androgen levels) to induce masculinization to clarify the mechanism of the sex change in the protogynous orange-spotted grouper. After 3 mo of AI/MT administration, male characteristics were observed in the female-to-male sex change fish. These male characteristics included increased plasma 11-ketotestosterone (11-KT), decreased estradiol (E2) levels, increased male-related gene (dmrt1, sox9, and cyp11b2) expression, and decreased female-related gene (figla, foxl2, and cyp19a1a) expression. However, the reduced male characteristics and male-to-female sex change occurred after AI/MT-termination in the AI- and MT-induced maleness. Furthermore, the MT-induced oocyte-depleted follicle cells (from MT-implantation) had increased proliferating activity, and the sexual fate in a portion of female gonadal soma cells was altered to male function during the female-to-male sex change. In contrast, the gonadal soma cells were not proliferative during the early process of the male-to-female sex change. Additionally, the male gonadal soma cells did not alter to female function during the male-to-female sex change in the AI/MT-terminated fish. After MT termination in the male-to-female sex-changed fish, the differentiated male germ cells showed increased proliferating activities together with dormancy and did not show characteristics of both sexes in the early germ cells. In conclusion, these findings indicate for the first time in a single species that the mechanism involved in the replacement of soma cells is different between the female-to-male and male-to-female sex change processes in grouper. These results also demonstrate that sexual fate determination (secondary sex determination) is regulated by endogenous sex steroid levels.

Ultimobranchial gland respond in a different way in male and female fresh water teleost Mastacembelus armatus (Lacepede) during reproductive cycle.

The present study was carried out to analyze the differences in the activity of ultimobranchial gland (UBG) between male and female fresh water teleost Mastacembelus armatus during reproductive cycle. Considerable variations in the nuclear diameter of UBG cells and plasma calcitonin (CT) levels during different reproductive phases of testicular and ovarian cycle suggested that the activity of the UBG depends upon the sexual maturity of fishes. A positive correlation was observed between plasma CT and sex steroid levels and the gonadosomatic index in both sexes which further confirmed the involvement of UBG in the processes related to gonadal development in fishes irrespective of the sex. Sudden increase in the level of plasma CT and nuclear diameter of UBG cells after administration of 17 α-methyltestosterone in males and 17 ß-estradiol in females during resting phase of the reproductive cycle clearly showed that UBG becomes hyperactive with increases in the level of sex steroids. Plasma calcium level was also found to be positively correlated with gonadal maturation in females. However no such change in plasma calcium level in relation to testicular cycle was observed. Thus it can be concluded that UBG becomes hyperactive during gonadal maturation but its role differs between male and female fishes. In females it may involved in both gonadal maturation and plasma calcium regulation while in males its involvement in calcium regulation was not justified. Variations in the level of CT during various phases of testicular cycle evidenced its involvement in gonadal maturation only.

Effects of 17α-methyltestosterone on the reproduction of the freshwater snail Biomphalaria glabrata.

17-α-methyltestosterone (MT) is a synthetic hormone used in fish hatcheries to induce male monosex. Snails hold promise as possible test models to assess chemicals acting on the endocrine system. Biomphalaria glabrata is an aquatic gastropod mollusk (Pulmonata, Planorbidae) that can be easily maintained in aquaria, predisposing the species for use in ecotoxicological testing. This study evaluated the reproductive effects of MT on B. glabrata by examining histological changes and its reproductive performance. Ten snails per group were exposed for 4 weeks to different concentrations of MT (0.01, 0.1, and 1.0 mg/L). The total number of laid eggs, egg mass per group, size of type V oocytes, and production of spermatozoids were determined. Reproduction of B. glabrata was affected by MT. At the lowest concentration (0.01 mg/L), MT caused a statistically significant increase in the number of egg mass per snail compared with controls unexposed to MT. Histopathology analyses showed an increase in the sperm production at the higher MT concentrations of 0.1 and 1.0 mg/L. Chromatographic analyses of water samples showed that MT concentrations rapidly declined within a 96-h period. These results highlight the importance of giving more support to regulatory authorities, since MT is not registered for use on fish hatcheries in many countries around the world. Wastewater from fish farms discharged into aquatic ecosystems should be monitored for MT residues, since its presence could compromise the reproduction of other native snail species.

Somatic growth effects of intramuscular injection of growth hormone in androgen-treated juvenile Nile tilapia, Oreochromis niloticus (Perciformes: Cichlidae).

Little is known about the effects of the interaction of growth hormone (GH) with 17 alpha-methyltestosterone (17-MT) during fish growth. We evaluated this in the present study to assess the effect on fish growth. Fish in two batches of juvenile tilapia (Oreochromis niloticus) (approximately 5.0cm in length) were randomly assigned in triplicate to three treatments and a control group, distributed among 12 fiberglass tanks of 1 000L capacity (50 fish per tank) in an experiment covering a period of six weeks. The experimental groups were: a) fish treated with 17-MT and GH in mineral oil (RGH); b) fish treated with 17-MT and mineral oil without the addition of GH (R); c) fish treated with GH in mineral oil but not 17-MT (NGH); and d) fish of the control group, which were treated with mineral oil but not 17-MT or GH (N). The GH was injected into the fish at a rate of 0.625mg/g body weight. Morphometric data were recorded at the beginning of the experiment (T0) and at 15, 30 and 45 days (T15, T30 and T45), and various indicators of growth were assessed: condition factor (K); survival percentage (S), feed conversion rate (FCR), percentage weight gain (WG) and (v) daily weight gain. The optimum dietary level was calculated assuming 5% food conversion to total weight in each group. During the experiment, the fish were provided with a commercial food containing 45% protein. The data showed that GH injection resulted in a greater weight gain in fish treated with 17-MT (the RGH treatment group), being particularly significant increase in weight during T15 and T30 (p<0.05). High values of K were found in the R and RGH treatments during the initial days of the experiment, which may have been a consequence of the better nutritional status affecting both weight gain and growth in body length, as a result of the additive effects of 17-MT and GH. The fish in groups not treated with 17-MT and treated with 17-MT and added GH showed greater increases in WG per day, higher K values and lower FCRs than fish in the other groups, which suggests that greater feed efficiency occurred in the hormone-treated fish. Fish in the RGH treatment showed the most growth, suggesting a possible interaction between 17-MT and injected GH.

Somatic growth effects of intramuscular injection of growth hormone in androgen-treated juvenile Nile tilapia, Oreochromis niloticus (Perciformes: Cichlidae)

Little is known about the effects of the interaction of growth hormone (GH) with 17 a-methyltestosterone (17-MT) during fish growth. We evaluated this in the present study to assess the effect on fish growth. Fish in two batches of juvenile tilapia (Oreochromis niloticus) (approximately 5.0cm in length) were randomly assigned in triplicate to three treatments and a control group, distributed among 12 fiberglass tanks of 1 000L capacity (50 fish per tank) in an experiment covering a period of six weeks. The experimental groups were: a) fish treated with 17-MT and GH in mineral oil (RGH); b) fish treated with 17-MT and mineral oil without the addition of GH (R); c) fish treated with GH in mineral oil but not 17-MT (NGH); and d) fish of the control group, which were treated with mineral oil but not 17-MT or GH (N). The GH was injected into the fish at a rate of 0.625mg/g body weight. Morphometric data were recorded at the beginning of the experiment (T) and at 15, 30 and 45 days (T, T and T), and various indicators of growth were assessed: condition factor (K); survival percentage (S), feed conversion rate (FCR), percentage weight gain (WG) and (v) daily weight gain. The optimum dietary level was calculated assuming 5% food conversion to total weight in each group. During the experiment, the fish were provided with a commercial food containing 45% protein. The data showed that GH injection resulted in a greater weight gain in fish treated with 17-MT (the RGH treatment group), being particularly significant increase in weight during T and T (p<0.05). High values of K were found in the R and RGH treatments during the initial days of the experiment, which may have been a consequence of the better nutritional status affecting both weight gain and growth in body length, as a result of the additive effects of 17-MT and GH. The fish in groups not treated with 17-MT and treated with 17-MT and added GH showed greater increases in WG per day, higher K values and lower FCRs than fish in the other groups, which suggests that greater feed efficiency occurred in the hormone-treated fish. Fish in the RGH treatment showed the most growth, suggesting a possible interaction between 17-MT and injected GH.

Actualmente, durante el crecimiento de los peces existe poco conocimiento sobre los efectos de la interacción de la hormona del crecimiento (HC) con 17 α-metiltestosterona (17-MT). En el presente estudio los peces en dos lotes de tilapia (Oreochromis niloticus) (5.0cm de longitud), fueron asignados al azar por triplicado a tres tratamientos y un grupo control, distribuidos en 12 tanques de fibra de vidrio de 1 000 litros (50 peces por tanque), en un período de seis semanas. Los tratamientos fueron: a) peces tratados con 17-MT+HC en aceite mineral (RGH), b) peces tratados con 17-MT+aceite mineral sin la adición de HC (R), c) los peces que no fueron tratados con 17-MT-tratado+HC en aceite mineral (NGH), y d) los peces que no fueron tratados con 17-MT+aceite mineral (N). La hormona de crecimiento humano recombinante (Humatrope, Eli Lilly & Co., Windlesham, Inglaterra), se inyectó en el pez con una dosis de 0.625mg por gramo de peso corporal. Los datos morfométricos se registraron al comienzo del experimento (T) y en los días 15, 30 y 45 (T, T y T), Se registraron diversos indicadores de crecimiento: factor de condición (K), porcentaje de supervivencia (S), la tasa de conversión alimenticia (FCR), porcentaje de ganancia de peso (GP) y el aumento de peso al día. El nivel óptimo dietético fue calculado suponiendo 5% de conversión de alimentos al peso total de cada grupo. Durante el experimento fue usada una dieta comercial con el 45% de proteína. De los resultados presentados, es evidente que la inyección de HC dio lugar a una mayor ganancia de peso en el 17-MT-los peces tratados (el grupo de tratamiento RGH), y la diferencia fue significativa, tanto en T y T (p<0.05) para ambas comparaciones. De manera similar, los altos valores de K se presentaron en los tratamientos R y RGH durante los primeros días de cultivo. Esto puede haber sido asociado con un mejor estado nutricional que afectó tanto el desarrollo de peso y la longitud del cuerpo del pez, como resultado del efecto aditivo de 17-MT y GH. Los tratamientos no andrógenos y los grupos tratados con andrógenos y con HC mostraron un mayor incremento en la ganancia de peso por día, los mayores valores de K y menores tasas de conversión del alimento, lo que sugiere una mayor eficiencia de la alimentación en los peces tratados con hormonas. Peces en el tratamiento RGH mostraron el mayor crecimiento, lo que sugiere una posible interacción entre el 17 de α-metiltestosterona (17-MT) y hormona de crecimiento inyectada.

Endocrine characterization of the designer steroid methyl-1-testosterone: investigations on tissue-specific anabolic-androgenic potency, side effects, and metabolism.

Various products containing rarely characterized anabolic steroids are nowadays marketed as dietary supplements. Herein, the designer steroid methyl-1-testosterone (M1T) (17ß-hydroxy-17α-methyl-5α-androst-1-en-3-one) was identified, and its biological activity, potential adverse effects, and metabolism were investigated. The affinity of M1T toward the androgen receptor (AR) was tested in vitro using a yeast AR transactivation assay. Its tissue-specific androgenic and anabolic potency and potential adverse effects were studied in a Hershberger assay (sc or oral), and tissue weights and selected molecular markers were investigated. Determination of M1T and its metabolites was performed by gas chromatography mass spectrometry. In the yeast AR transactivation assay, M1T was characterized as potent androgen. In rats, M1T dose-dependently stimulated prostate and levator ani muscle weight after sc administration. Oral administration had no effect but stimulated proliferation in the prostate and modulated IGF-I and AR expression in the gastrocnemius muscle in a dose-dependent manner. Analysis of tyrosine aminotransferase expression provided evidence for a strong activity of M1T in the liver (much higher after oral administration). In rat urine, 17α-methyl-5α-androstane-3α,17ß-diol, M1T, and a hydroxylated metabolite were identified. In humans, M1T was confirmed in urine in addition to its main metabolites 17α-methyl-5α-androst-1-ene-3α,17ß-diol and 17α-methyl-5α-androstane-3α,17ß-diol. Additionally, the corresponding 17-epimers as well as 17ß-hydroxymethyl-17α-methyl-18-nor-5α-androsta-1,13-dien-3-one and its 17-epimer were detected, and their elimination kinetics was monitored. It was demonstrated that M1T is a potent androgenic and anabolic steroid after oral and sc administration. Obviously, this substance shows no selective AR modulator characteristics and might exhibit liver toxicity, especially after oral administration.

Chronic exposure to anabolic androgenic steroids alters activity and synaptic function in neuroendocrine control regions of the female mouse.

Disruption of reproductive function is a hallmark of abuse of anabolic androgenic steroids (AAS) in female subjects. To understand the central actions of AAS, patch clamp recordings were made in estrous, diestrous and AAS-treated mice from gonadotropin releasing hormone (GnRH) neurons, neurons in the medial preoptic area (mPOA) and neurons in the anteroventroperiventricular nucleus (AVPV); regions known to provide GABAergic and kisspeptin inputs to the GnRH cells. Action potential (AP) frequency was significantly higher in GnRH neurons of estrous mice than in AAS-treated or diestrous animals. No significant differences in AAS-treated, estrous or diestrous mice were evident in the amplitude or kinetics of spontaneous postsynaptic currents (sPSCs), miniature PSCs or tonic currents mediated by GABA(A) receptors or in GABA(A) receptor subunit expression in GnRH neurons. In contrast, the frequency of GABA(A) receptor-mediated sPSCs in GnRH neurons showed an inverse correlation with AP frequency across the three hormonal states. Surprisingly, AP activity in the medial preoptic area (mPOA), a likely source of GABAergic afferents to GnRH cells, did not vary in concert with the sPSCs in the GnRH neurons. Furthermore, pharmacological blockade of GABA(A) receptors did not alter the pattern in which there was lower AP frequency in GnRH neurons of AAS-treated and diestrous versus estrous mice. These data suggest that AAS do not impose their effects either directly on GnRH neurons or on putative GABAergic afferents in the mPOA. AP activity recorded from neurons in kisspeptin-rich regions of the AVPV and the expression of kisspeptin mRNA and peptide did vary coordinately with AP activity in GnRH neurons. Our data demonstrate that AAS treatment imposes a "diestrous-like" pattern of activity in GnRH neurons and suggest that this effect may arise from suppression of presynaptic kisspeptin-mediated excitatory drive arising from the AVPV. The actions of AAS on neuroendocrine regulatory circuits may contribute the disruption of reproductive function observed in steroid abuse.

Safety and efficacy of low-dose esterified estrogens and methyltestosterone, alone or combined, for the treatment of hot flashes in menopausal women: a randomized, double-blind, placebo-controlled study.

This study evaluated safety and efficacy of esterified estrogens and methyltestosterone administered alone or in combination for the treatment of hot flashes in menopausal women. The 0.30-mg esterified estrogens and 0.30-mg methyltestosterone combination was the lowest effective dose, and our results are consistent with the known safety profile of estrogen and androgen combination products.

Sex steroid levels in XY males and sex-reversed XX males, of rainbow trout (Oncorhynchus mykiss), during the reproductive cycle.

In this study, the annual cycle of the gonadal steroids testosterone (T), 11-ketotestosterone (11-KT), 17ß-oestradiol (E2) and 17α, 20ß-dihydroxy-4-pregnen-3-one (DHP) was determined using radioimmunoassay and then compared, for XY males (n=35) and sex-reversed XX males (n=27) rainbow trout, to establish possible endocrinology differences. Both in XY males and sex-reversed XX males, significant correlation was shown between body weight and T (r=0.5046 and 0.34078, respectively; p<0.0001) or KT (r=0.52494 and 0.43545, respectively; p<0.0001) concentrations. Plasma androgen levels in XY and sex-reversed XX males were similar and showed an intense seasonal variation. The highest levels for T and 11-KT were detected from December to April with a peak in January (51.67 ± 5.11 and 61.95 ± 4.25 ng/ml, for XY males and 57.1 ± 5.82 and 59.27 ± 4.84 ng/ml, respectively, for XX males). In addition, there was a positive correlation (p<0.0001) between T and 11-KT levels for XY males (r=0.7533) and sex-reversed XX males (r=0.6019). Concentrations of DHP in XY males also showed seasonal variation with a peak in February (25.18 ± 12.99 ng/ml). However, DHP levels in sex-reversed XX males were undetectable (<0.1 ng/ml) over the year. Levels of E2 were undetectable through the year in both groups of trout. In conclusion, the androgenic and oestrogenic profiles of sex-reversed XX males were similar to those observed in XY males. The only difference in the annual gonadal steroid cycle between XY and sex-reversed XX males was in the DHP profile.