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Steroids Feb 2023Δ6-Methyltestosterone was reported as the main active ingredient of the purported "dietary supplement" Jungle Warfare. This compound is structurally similar to...
Δ6-Methyltestosterone was reported as the main active ingredient of the purported "dietary supplement" Jungle Warfare. This compound is structurally similar to 17α-methyltestosterone, containing an additional Δ6 double bond, and is reported to possess notable androgenic activity, raising concerns over the potential for abuse of Jungle Warfare in sport. The in vivo metabolism of Δ6-methyltestosterone in greyhounds was investigated. Urinary phase I (unconjugated) and phase II (glucuronide) metabolites were detected following oral administration using liquid chromatography-mass spectrometry. No phase II sulfate metabolites were detected. The major phase I metabolite was confirmed as 16α,17β-dihydroxy-17α-methylandrosta-4,6-dien-3-one by comparison with a synthetically-derived reference material. Minor amounts of the parent drug were also confirmed. Glucuronide conjugated metabolites were also observed, but were found to be resistant to hydrolysis using the Escherichia coli β-glucuronidase enzyme. Qualitative excretion profiles, limits of detection, and extraction recoveries were determined for the parent drug and the major phase I metabolite. These results provide a method for the detection of Jungle Warfare abuse in greyhounds suitable for incorporation into routine screening methods conducted by anti-doping laboratories.
Topics: Animals; Dogs; Methyltestosterone; Gas Chromatography-Mass Spectrometry; Glucuronides; Androgens; Mass Spectrometry; Doping in Sports; Anabolic Agents; Substance Abuse Detection
PubMed: 36511323
DOI: 10.1016/j.steroids.2022.109150 -
Regulatory mechanism of LncRNAs in gonadal differentiation of hermaphroditic fish, Monopterus albus.Biology of Sex Differences Oct 2023Monopterus albus is a hermaphroditic fish with sex reversal from ovaries to testes via the ovotestes in the process of gonadal development, but the molecular mechanism...
BACKGROUND
Monopterus albus is a hermaphroditic fish with sex reversal from ovaries to testes via the ovotestes in the process of gonadal development, but the molecular mechanism of the sex reversal was unknown.
METHODS
We produced transcriptomes containing mRNAs and lncRNAs in the crucial stages of the gonad, including the ovary, ovotestis and testis. The expression of the crucial lncRNAs and their target genes was detected using qRT‒PCR and in situ hybridization. The methylation level and activity of the lncRNA promoter were analysed by applying bisulfite sequencing PCR and dual-luciferase reporter assays, respectively.
RESULTS
This effort revealed that gonadal development was a dynamic expression change. Regulatory networks of lncRNAs and their target genes were constructed through integrated analysis of lncRNA and mRNA data. The expression and DNA methylation of the lncRNAs MSTRG.38036 and MSTRG.12998 and their target genes Psmβ8 and Ptk2β were detected in developing gonads and sex reversal gonads. The results showed that lncRNAs and their target genes exhibited consistent expression profiles and that the DNA methylation levels were negatively regulated lncRNA expression. Furthermore, we found that Ptk2β probably regulates cyp19a1 expression via the Ptk2β/EGFR/STAT3 pathway to reprogram sex differentiation.
CONCLUSIONS
This study provides novel insight from lncRNA to explore the potential molecular mechanism by which DNA methylation regulates lncRNA expression to facilitate target gene transcription to reprogram sex differentiation in M. albus, which will also enrich the sex differentiation mechanism of teleosts.
Topics: Male; Animals; Female; RNA, Long Noncoding; Gonads; Ovary; Testis; Sex Differentiation; Smegmamorpha
PubMed: 37880697
DOI: 10.1186/s13293-023-00559-y -
BMC Genomics Jul 2017Sex hormones play important roles in teleost ovarian and testicular development. In zebrafish, ovarian differentiation appears to be dictated by an oocyte-derived signal...
BACKGROUND
Sex hormones play important roles in teleost ovarian and testicular development. In zebrafish, ovarian differentiation appears to be dictated by an oocyte-derived signal via Cyp19a1a aromatase-mediated estrogen production. Androgens and aromatase inhibitors can induce female-to-male sex reversal, however, the mechanisms underlying gonadal masculinisation are poorly understood. We used histological analyses together with RNA sequencing to characterise zebrafish gonadal transcriptomes and investigate the effects of 17α-methyltestosterone on gonadal differentiation.
RESULTS
At a morphological level, 17α-methyltestosterone (MT) masculinised gonads and accelerated spermatogenesis, and these changes were paralleled in masculinisation and de-feminisation of gonadal transcriptomes. MT treatment upregulated expression of genes involved in male sex determination and differentiation (amh, dmrt1, gsdf and wt1a) and those involved in 11-oxygenated androgen production (cyp11c1 and hsd11b2). It also repressed expression of ovarian development and folliculogenesis genes (bmp15, gdf9, figla, zp2.1 and zp3b). Furthermore, MT treatment altered epigenetic modification of histones in zebrafish gonads. Contrary to expectations, higher levels of cyp19a1a or foxl2 expression in control ovaries compared to MT-treated testes and control testes were not statistically significant during early gonad development (40 dpf).
CONCLUSION
Our study suggests that both androgen production and aromatase inhibition are important for androgen-induced gonadal masculinisation and natural testicular differentiation in zebrafish.
Topics: Animals; Female; Male; Methyltestosterone; Ovary; Sex Characteristics; Sex Ratio; Spermatogenesis; Testis; Transcriptome; Zebrafish
PubMed: 28738802
DOI: 10.1186/s12864-017-3915-z -
Heliyon Dec 2022The 17α-methyltestosterone is the most common synthetic hormone used in male mono-sex production of Nile tilapia, . The current research aimed at finding out the most...
The 17α-methyltestosterone is the most common synthetic hormone used in male mono-sex production of Nile tilapia, . The current research aimed at finding out the most effective dose of 17α-methyltestosterone to produce quality Nile tilapia fry. Soon after absorbing the yolk sac, Nile tilapia fry was fed with a mixture of commercial fish feed and 17α-methyltestosterone for 28 days. Five doses of 17α-methyltestosterone, i.e., 0 mg, 50 mg, 60 mg, 70 mg, and 80 mg per kg feed, were used to treat tilapia that has been reared for additional 90 days to compare sex reversal, development, and survival rates. Both gonad histology and Squash test were performed to expose the sex percentage of accurately. The highest male 94.44% was obtained at 60 mg 17α-MT/kg feed dose followed by 91.67%, 88.89%, 86.11%, and 47.22% at 70, 80, 50, and 0 mg 17α-MT/kg feed dose. The groups treated with 17α-methyltestosterone hormone showed superior growth performance in comparison to the control group. The highest weight (14.62 ± 0.59 g) and length (92.18 ± 3.01 mm) were found at 60 mg dose whereas the lowest weight (8.64 ± 0.38 g) and length (70.17 ± 3.75 mm) were in the control group. The group given 60 mg 17α-MT feed represented the highest survival rate (84.10%) among other hormone-treated groups. The study disclosed that 60 mg 17α-MT/kg feed might be treated as the optimal dose for producing quality mono-sex male tilapia in the commercial hatchery.
PubMed: 36536906
DOI: 10.1016/j.heliyon.2022.e12252 -
Journal of Immunological Methods Nov 2023In this study, we have developed bridge heterologous ELISA for the detection of 17α- Methyltestosterone by incorporating aromatic spacers between...
In this study, we have developed bridge heterologous ELISA for the detection of 17α- Methyltestosterone by incorporating aromatic spacers between 17α-Methyltestosterone-3-Carboxymethyloxime and Horseradish peroxidase label through N-hydroxysuccinimide mediated carbodiimide reaction method. The immunogen 17α-Methyltestosterone-3-Carboxymethyloxime-Bovine serum albumin used to generate the antibody was also prepared by the N-hydroxysuccinimide mediated carbodiimide reaction without using any spacer. We have studied the impact of bridge/aromatic spacers on functional parameters i.e. sensitivity, affinity and ED of the bridge heterologous assay and compared it with homologous assay. The five combinations of bridge heterologous assay using 17α-Methyl testosterone-3-CMO-BSA antiserum and 17α-MT-3-CMO-4,4'-Diaminodiphenyl sulphide-HRP, 17α MT-3-CMO-4,4'-Oxydianiline-HRP, 17α-MT-3-CMO-Benzidine-HRP, 17α- MT-3-CMO-p-Phenylenediamine-HRP and 17α-MT-3-CMO-Dapson-HRP enzyme conjugates were evaluated. Out of these five combinations, the combination 17α-MT-3-CMO-BSA with 17α-MT-3-CMO-Benzidine-HRP showed the best results. Sensitivity, affinity and ED were improved and found to be 0.02 ng/mL, 0.086 × 10 L/mol and 2.95 ng/mL than homologous assay where Sensitivity, affinity and ED were 0.11 ng/mL, 0.02 × 10 L/mol and 5.78 ng/mL respectively. The cross-reactivity for this bridge heterologous assay combination was seen with only 4 steroids (6-hydrotestosterone- 6%, Testosterone-5.14%, Danazol-0.9% and Nandrolone-0.85%) instead of eight steroids (6-hydrotestosterone-43.75%, Testosterone-38.3%, Danazol-25.14%, Androstenediol-19.16%, Nandrolone-19%, Metandienone-5%, Androstenedione-3.52%, and 17α dimethyltestosterone-2%) as in homologous assay out of 59 structurally related steroids. Thus, the results of this study conclude that the incorporation of aromatic spacer (bridge) in enzyme conjugate has a crucial role in improving sensitivity, specificity, ED and affinity of the developed assay. The assay was then studied for parameters such as recovery (97.4%-108.6%), precision (Inter and Intra-assay coefficient of variation <10%), correlation coefficient (R = 0.96) by comparing with the commercial kit and validated by measuring levels of 17α- methyltestosterone in rat serum after administering them.
Topics: Animals; Rats; Methyltestosterone; Danazol; Enzyme-Linked Immunosorbent Assay; Antigens; Steroids; Testosterone; Benzidines; Carbodiimides; Nandrolone
PubMed: 37774776
DOI: 10.1016/j.jim.2023.113572 -
Molecules (Basel, Switzerland) Jan 2023Steroid hormone molecules may exhibit very different functionalities based on the associated functional groups and their 3D arrangements in space, i.e., absolute...
Steroid hormone molecules may exhibit very different functionalities based on the associated functional groups and their 3D arrangements in space, i.e., absolute configurations and conformations. Infrared (IR) and vibrational circular dichroism (VCD) spectra of four different steroid hormones, namely dehydroepiandrosterone (DHEA), 17-methyltestosterone (MTTT), (16α,17)-epoxyprogesterone (Epoxy-P4), and dehydroepiandrosterone acetate (AcO-DHEA), were measured in deuterated dimethyl sulfoxide and some also in carbon tetrachloride. Extensive conformational searches were carried out using the recent developed conformer-rotamer ensemble sampling tool (CREST) which also accounts for solvent effects using an implicit solvation model. All the CREST conformational candidates were then reoptimized at the B3LYP-D3BJ/def2-TZVPD with the PCM of solvent. The good agreements between the experimental IR and VCD spectra and the theoretical simulations provide a conclusive information about their conformational distribution and absolute configurations. The experimental and theoretical IR and VCD spectra of AcO-DHEA in the carbonyl and alkene stretching region showed some discrepancies, and the possible causes related to solvent effects, large amplitude motions and levels of theory used in the modelling were explored in detail. As part of the investigation, additional calculations at the B3LYP-D3BJ/6-31++G (2d,p) and B3LYP-D3BJ/cc-pVTZ levels, as well as some 'mixed' calculations with the double-hybrid functional B2PLYP-D3 were also carried out. The results indicate that the double-hybrid functional is important for predicting the correct IR band pattern in the carbonyl and alkene stretching region.
PubMed: 36677830
DOI: 10.3390/molecules28020771 -
General and Comparative Endocrinology Jan 2022Gonadotropin-releasing hormone (GnRH) is an important neuropeptide in the reproductive system. Although GnRH analogues have been used to artificially spawn pompano...
Gonadotropin-releasing hormone (GnRH) is an important neuropeptide in the reproductive system. Although GnRH analogues have been used to artificially spawn pompano (Trachinotus sp.), the native forms of GnRH have not been described in this species. In this study three GnRH subtypes [sea bream GnRH (sbGnRH), chicken GnRH-Ⅱ (cGnRH-Ⅱ) and salmon GnRH (sGnRH)] were identified in pompano (Trachinotus ovatus). cgnrh-Ⅱ and sgnrh were mainly expressed in the brain of male and female fish, showing a tissue-specific expression pattern, while sbgnrh was expressed at different transcriptional levels in all tested tissues. In vivo injection experiment showed that sbGnRH significantly increased fsh and lh genes expression in a dose-dependent manner, but a high concentration of sbGnRH could desensitize the expression of lh. High concentrations of cGnRH-Ⅱ and sGnRH could induce the expression of fsh and lh. In addition, the results of in vitro incubation experiments showed that the high concentration of sbGnRH peptide could induce the expression of fsh and lh, while cGnRH-Ⅱ and sGnRH peptides could only induce the expression of fsh. 17β-estradiol (E) and 17α-methyltestosterone (MT) significantly inhibited sbgnrh mRNA expression in a dose-dependent manner, but did not affect the expression of cgnrh-Ⅱ and sgnrh mRNA. sbGnRH is the main GnRH subtype in pompano. E and MT can play a negative role in the regulation of sbgnrh. This study provides a theoretical basis for the reproductive endocrinology of pompano.
Topics: Animals; Female; Fishes; Gonadotropin-Releasing Hormone; Gonadotropins; Male; Perciformes; Pituitary Gland
PubMed: 34861278
DOI: 10.1016/j.ygcen.2021.113958 -
Theriogenology Oct 2018Endocrine effects as 11-ketotestosterone (11-KT), an unaromatizable androgen, regulating the follicles growth in the previtellogenic stage of eel reproduction have been...
Endocrine effects as 11-ketotestosterone (11-KT), an unaromatizable androgen, regulating the follicles growth in the previtellogenic stage of eel reproduction have been widely elucidated. However, the influence of aromatizable androgens on the brain-pituitary-gonad axis during oogenesis in A. japonica has not been clearly elaborated. In the study, androstenedione (AD) and 17α-methyltestosterone (MT) were employed together to induce ovary development of seven-year-old female Anguilla japonica through feeding or exposure in the migration season. After female A. japonica had been fed with commercial diet containing 5 mg AD and MT kg d body weight respectively for 45 d in fresh water (Trial I), the development of oocytes still remained at the oil droplet stage, but the GSI and follicle diameter increased significantly. The serum 11-KT level and expression of liver vitellogenin mRNA were significantly elevated. After female fish had been exposed to seawater containing 50 μg L AD and MT respectively for 45 d (Trial II), the ovaries of A. japonica almost reached midvitellogenic stage and the GSI and follicle diameter increased significantly. Yolk granular layer was observed in the peripheral ooplasm. The serum 11-KT level maintained consistently low, and the serum E2 level declined significantly to a relatively low level. The expression levels of ovarian arα and cyp19a1, brain (with pituitary together) mGnRH and lhβ increased significantly. The results showed that A. japonica in Trial II appeared a higher ovarian development than those in Trial I. These findings indicated that AD and MT increased the oil droplet and enlarged follicle diameter in previtellogenic stage, while the vitellogenesis and gonadotropin release did not occur in Trial I. In Trial II, AD and MT promoted vitellogenesis by stimulating the ovary expression of arα and by up-regulating brain mGnRH and pituitary lhβ expression.
Topics: Androstenedione; Anguilla; Animals; Female; Follicle Stimulating Hormone, beta Subunit; Luteinizing Hormone; Methyltestosterone; Ovary; Ovulation Induction; Vitellogenins
PubMed: 30081244
DOI: 10.1016/j.theriogenology.2018.07.009 -
Steroids Jan 2021There is some evidence that marketable supplements contain hormones not declared on the product label. The presence of these androgenic anabolic steroids (AAS) in sports...
There is some evidence that marketable supplements contain hormones not declared on the product label. The presence of these androgenic anabolic steroids (AAS) in sports supplements can be considered an adulteration and affect the health of consumers, who are predominantly athletes. This study aimed to measure anabolic hormones (methyltestosterone and 4-androstenedione) in sport supplements. Ultra Performance Liquid chromatography coupled mass spectrometry (UPLC-MS/MS) with electrospray ionization (ESI) in positive mode was employed under the Multiple Reaction Monitoring (MRM) ion program. To overcome matrix effects and quantify the selected analyte, the calibration curve was made using Matrix Match method. The LOQ and LOD were 1 ng/g and 0.3 ng/g for both analytes. The recovery of 4-androstenedione and methyltestosterone was in the range of 86.87-107.35 and 77.31-113.98, respectively. In terms of reproducibility, CV % for 4-androstenedione and methyltestosterone ranged from 6.56 to 16.87% and 1.45-15.12%, respectively. 4-androstenedione was found in 11 samples including 9 whey as 1.578 ± 0.154 ng/g and 2 whey albumin samples with an amount of 1.134 ng/g and 1.474 ng/g. Consequently, continuous controlling of sport supplements comprising intentionally or unintentionally added androgens could be important for health and discuss in the context of compliance with anti-doping.
Topics: Androstenedione; Doping in Sports; Methyltestosterone; Reproducibility of Results
PubMed: 33161054
DOI: 10.1016/j.steroids.2020.108758 -
Journal of Fish Biology Jul 2021The cyp11 includes cyp11a and cyp11b in most mammals and teleosts, encoded cholesterol side chain lyase and 11β-hydroxylase, respectively. It is essential in steroid...
The cyp11 includes cyp11a and cyp11b in most mammals and teleosts, encoded cholesterol side chain lyase and 11β-hydroxylase, respectively. It is essential in steroid hormone synthesis. However, studies on the regulation of cyp11 are limited, especially in teleosts. In this study, the molecular characterization and function of cyp11a and cyp11b of black rockfish was investigated. Both of them showed high homology with other teleost counterparts by phylogenetic analysis. The expression of cyp11a and cyp11b exhibited a clear sexually dimorphic pattern, with a higher expression level in testis than that of in ovaries. During the different developmental stages (40 dpf, 80 dpf, 190 dpf, 360 dpf, 720 dpf), the expression of cyp11a was earlier than cyp11b. In situ hybridization results showed that cyp11a and cyp11b were mainly expressed in oogonia and oocytes of the ovary. They were located in spermatogonia and interstitial compartment in the 1.5-year-old gonads, and spermatocytesgonia and the peritubular myoid cell of the testis in the 2.5-year-old gonads. To explore the distinct roles of cyp11a and cyp11b in gonads, oestrogen and androgens were used to stimulate the primary testicular and ovarian cells. The expressions of cyp11a and cyp11b were tested under different dose of 17α-methyltestosterone (17α-MT) and 17β-estradiol (E2). The results showed cyp11a was significantly increased at 10 mol ml of 17α-MT and 10 mol ml of E2 in ovary and 10 mol ml of 17α-MT and E2 in testis, while cyp11b was significantly decreased after 17α-MT and E2 treatment. These results indicated that cyp11a and cyp11b were likely to have different functions, and also implied they might play an important roles in the differentiation of gonads and the synthesis of steroids in black rockfish.
Topics: Animals; Female; Male; Methyltestosterone; Ovary; Perciformes; Phylogeny; Testis
PubMed: 33252824
DOI: 10.1111/jfb.14631