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Analytical Methods : Advancing Methods... Dec 2023Methyltestosterone is one of the banned drugs in aquaculture, and it should be monitored in food-producing animals. 17α-Methyl-5β-androstane-3α,17β-diol, as the main...
Methyltestosterone is one of the banned drugs in aquaculture, and it should be monitored in food-producing animals. 17α-Methyl-5β-androstane-3α,17β-diol, as the main metabolite of methyltestosterone in vertebrates, could be used as another marker for controlling the administration of methyltestosterone, due to its high residual concentration and slow elimination rate. In this study, an analytical method based on gas chromatography-mass spectrometry (GC-MS) was developed and validated for the simultaneous determination of methyltestosterone and its main metabolite in fish. After pretreatment by liquid-liquid extraction with -hexane and solid phase extraction with C and NH columns, the target analytes in the muscle tissues were extracted and concentrated, and the influence of the sample matrix was eliminated. Then, the prepared samples were separated and detected with GC-MS in the selected ion monitoring (SIM) mode. Methyltestosterone-D was chosen as the internal standard for quantitation. After optimization, the limits of detection for methyltestosterone and 17α-methyl-5β-androstane-3α,17β-diol were 20 μg kg and 15 μg kg, respectively. The limits of quantitation were both 50 μg kg. The calibration curves showed good linearity in the concentration range from 50.0 ng mL to 500.0 ng mL. The correlation coefficients of methyltestosterone and 17α-methyl-5β-androstane-3α,17β-diol were more than 0.9990. The recoveries of the analytes in real samples were in the range of 99.7-116.6% with the relative standard deviation of 5.2-8.3%. The established method could meet the demand for simultaneous detection of methyltestosterone and its major metabolite, and it could be used to provide more information on the abuse of methyltestosterone in food-producing animals.
Topics: Animals; Gas Chromatography-Mass Spectrometry; Methyltestosterone; Androstanes; Fishes; Calibration
PubMed: 38086623
DOI: 10.1039/d3ay01646c -
Journal of Ethnopharmacology Feb 2024Herba epimedium brevicornu maxim is traditionally known as a sexual enhancement, and has the effect of tonifying kidney yang. Icariin is a flavonoid extracted from...
ETHNOPHARMACOLOGICAL RELEVANCE
Herba epimedium brevicornu maxim is traditionally known as a sexual enhancement, and has the effect of tonifying kidney yang. Icariin is a flavonoid extracted from epimedium brevicornu maxim, and has been shown to improve nephropathy disease.
AIM OF THE STUDY
This study investigated the possible role of icariin in regulating renal EndMT in type 2 diabetic nephropathy (T2DN).
MATERIALS AND METHODS
Male type 2 diabetic Sprague Dawley rats, Male D2.BKS(D)-Leprdb/J (db/db) mice, and mouse glomerular endothelial cells were utilized to evaluate the effect of icariin. Western blotting, Q-PCR, immunohistochemistry, H&E, Masson staining, immunofluorescence, and siRNA transfection, were performed in this study.
RESULTS
The inhibitory function of icariin in renal fibrosis and renal EndMT was verified in type 2 diabetic animals. Methyltestosterone suppressed renal fibrosis and EndMT in db/db mice. Androgen receptor (AR), the major receptor of testosterone, was upregulated by icariin. The AR antagonist MDV3100, blocked the inhibition by icariin in renal EndMT, revealing that icariin repressed renal EndMT by activating AR. In addition, icariin and methyltestosterone upregulated the Raf kinase inhibitor protein (RKIP) in db/db mice. Furthermore, siRNA-RKIP inhibited the effect of icariin on EndMT. The MEK/ERK pathway, as the downstream pathway of RKIP, was suppressed by icariin and methyltestosterone. Of note, the effect of icariin on the MEK/ERK pathway was abolished by MDV3100 or siRNA-RKIP.
CONCLUSIONS
These results supported that icariin targeted AR/RKIP/MEK/ERK pathway to suppress renal fibrosis and EndMT in T2DN.
Topics: Rats; Male; Mice; Animals; Diabetic Nephropathies; Phosphatidylethanolamine Binding Protein; Endothelial Cells; Receptors, Androgen; Methyltestosterone; Rats, Sprague-Dawley; Flavonoids; Fibrosis; RNA, Small Interfering; Diabetes Mellitus, Type 2; Mitogen-Activated Protein Kinase Kinases; Epithelial-Mesenchymal Transition
PubMed: 37977422
DOI: 10.1016/j.jep.2023.117414 -
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 -
Journal of Molecular Endocrinology Aug 2023Built on our recent work that heart rates (HRs) and function in Gambusia holbrooki are sexually dimorphic, this study assessed whether the species is an appropriate...
Built on our recent work that heart rates (HRs) and function in Gambusia holbrooki are sexually dimorphic, this study assessed whether the species is an appropriate model to study sex-hormone effects on heart physiology. With a hypothesis that 17β-estradiol (E2) and 17α-methyltestosterone (MT) regulate the HR of juvenile G. holbrooki in a sex-specific manner, genetic males and females were treated with E2 and MT, respectively, and the HR; (bpm) was measured an hour following treatment using light-cardiogram. Results showed the HRs (bpm) of both sexes were significantly (P < 0.05) altered compared to controls. Specifically, the E2 accelerated HR in the males and conversely MT decelerated the HR in the females. The normal expression levels of estrogen (erα and erβ) and G protein-coupled estrogen (gper) receptor genes were significantly higher (P < 0.05) in female than male hearts. Interestingly, the activity of the erβ in the heart of the MT-treated females reversed and was significantly lower (P < 0.05) than those of males while erα and gper were non-responsive. In contrast, significant down- and up-regulation of erα and gper, respectively, occurred in the liver of MT-treated females. Morphological observations suggest that MT caused hepatomegaly, somewhat resembling an inflating balloon, perhaps induced by the accumulation of unexpelled gases. E2-induced ventricular angiogenesis in males was likely due to an influx of blood supply caused by the increased HRs. Collectively, the results demonstrate that the juvenile G. holbrooki heart readily responds to E2/MT in a sex-specific manner.
Topics: Male; Female; Animals; Estrogen Receptor alpha; Estrogen Receptor beta; Heart Rate; Estrogens; Estradiol; Cyprinodontiformes
PubMed: 37159386
DOI: 10.1530/JME-23-0021 -
Drug Testing and Analysis Dec 2023Because of their performance-enhancing effect, anabolic androgenic steroids (AAS) are often misused in sports. Nearly half of the adverse analytical findings (AAF) in...
Because of their performance-enhancing effect, anabolic androgenic steroids (AAS) are often misused in sports. Nearly half of the adverse analytical findings (AAF) in 2022 doping controls are correlated to AAS misuse. Metabolites play a crucial role in the bioanalysis of endogenous and exogenous steroids. Therefore, one important field in antidoping research is the investigation on drug metabolizing and steroidogenic enzymes. The introduction of a hydroxy group is the most common reaction, which is catalyzed by cytochrome P450 (CYP) enzymes in phase-I metabolism. Analysis of AAS metabolites is commonly performed using gas chromatography mass spectrometry (GC-MS) systems. Laborious sample preparation and extended run times compared to liquid chromatography (tandem) mass spectrometry (LC-MS/MS) methods are usually correlated with this type of analysis. On the other hand, liquid chromatography (tandem) mass spectrometry (LC-MS[/MS]) methods have a lower separation efficiency than GC-MS methods. Both techniques lack selectivity for hydroxylated 17α-methyltestosterone metabolites. Therefore, as an orthogonal analytical approach, a supercritical fluid chromatography tandem mass spectrometry method was developed to separate four hydroxy metabolites of 17α-methyltestosterone (2α-/2β-/4-/6β-hydroxy-17α-methyltestosterone). This project aimed to get a more in-depth look at the metabolization and analysis of 17α-methyltestosterone and its hydroxylated metabolites. The developed method revealed lower limits of quantitation between 0.6 and 6 ng/ml at an accuracy of 85-115% using a matrix matched calibration. An in vitro study with human liver microsomes shows 6β-hydroxy-17α-methyltestosterone as main metabolite (15.9%) as well as the metabolite 2β-hydroxy-17α-methyltestosterone (0.5%). The results show that the developed method is sensitive and robust. In addition, the method allows a previously missing discrimination of the hydroxylated metabolites in a short analysis time without prior, complex derivatizations.
PubMed: 38048816
DOI: 10.1002/dta.3620 -
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 -
Genes May 2024It is widely known that all-female fish production holds economic value for aquaculture. , a preeminent economic species, exhibits a sex dimorphism, with females...
It is widely known that all-female fish production holds economic value for aquaculture. , a preeminent economic species, exhibits a sex dimorphism, with females surpassing males in growth. In this regard, achieving all-female black rockfish production could significantly enhance breeding profitability. In this study, we utilized the widely used male sex-regulating hormone, 17α-methyltestosterone (MT) at three different concentrations (20, 40, and 60 ppm), to produce pseudomales of for subsequent all-female offspring breeding. Long-term MT administration severely inhibits the growth of , while short term had no significant impact. Histological analysis confirmed sex reversal at all MT concentrations; however, both medium and higher MT concentrations impaired testis development. MT also influenced sex steroid hormone levels in pseudomales, suppressing E2 while increasing T and 11-KT levels. In addition, a transcriptome analysis revealed that MT down-regulated ovarian-related genes ( and ) while up-regulating male-related genes () in pseudomales. Furthermore, MT modulated the TGF-β signaling and steroid hormone biosynthesis pathways, indicating its crucial role in sex differentiation. Therefore, the current study provides a method for achieving sexual reversal using MT in and offers an initial insight into the underlying mechanism of sexual reversal in this species.
Topics: Animals; Methyltestosterone; Male; Female; Sex Differentiation; Perciformes; Testis; Fishes; Fish Proteins
PubMed: 38790234
DOI: 10.3390/genes15050605 -
Aquatic Toxicology (Amsterdam,... Mar 2024The interactions between estrogen and androgen in aquatic animals remain largely unknown. In this study, two generations (F0 and F1) of western mosquitofish (Gambusia...
The interactions between estrogen and androgen in aquatic animals remain largely unknown. In this study, two generations (F0 and F1) of western mosquitofish (Gambusia affinis) were continuously exposed to 17α-ethinylestradiol (EE2, 10 ng/L), methyltestosterone (MT, 10 ng/L (MTL); 50 ng/L (MTH)), and mixtures (EE2+MTL and EE2+MTH). Various endpoints, including sex ratio (phenotypic and genetic), secondary sex characteristics, gonadal histology, and transcriptional profile of genes, were examined. The results showed that G. affinis exposed to MTH and EE2+MTH had a > 89.7 % of phenotypic males in F1 generation, with 34.5 and 50.0 % of these males originated from genetic females, respectively. Moreover, females from F0 and F1 generations exposed to MTH and EE2+MTH exhibited masculinized anal fins and skeletons. The combined effect of MT and EE2 on most endpoints was dependent on MT. Furthermore, significant transcriptional alterations in certain target genes were observed in both the F0 and F1 generations by EE2 and MT alone and by mixtures, showing some degree of interactions. These findings that the effects of EE2+MTH were primarily on the phenotypic sex of G. affinis in offspring generation suggest that G. affinis under chronic exposure to the binary mixture contaminated water could have sex-biased populations.
Topics: Male; Female; Animals; Ethinyl Estradiol; Methyltestosterone; Water Pollutants, Chemical; Estrogens; Cyprinodontiformes
PubMed: 38309221
DOI: 10.1016/j.aquatox.2024.106854 -
Biology of Reproduction Dec 2023Male contraceptive development has included use of testosterone (T) with or without a progestin or the use of a single molecule such as progestogenic androgens (PA) for...
Male contraceptive development has included use of testosterone (T) with or without a progestin or the use of a single molecule such as progestogenic androgens (PA) for suppression of testicular T production. Expanding upon the vast amount of data accumulated from nortestosterone (NT), NT analogs, and their prodrugs, a new series of PA, the C7 methyl, and ethyl α-substituted T analogs 7α-Methyltestosterone (7α-MT) and 7α-Ethyltestosterone (7α-ET), respectively, were hypothesized and designed to have superior androgenic and progestogenic activities when compared with parent T. Results from androgen receptor and progesterone receptor competitive binding and transcriptional activation assays showed favorable activities for these T analogs. Additionally, 7α-MT and 7α-ET were shown to be active substrates for aromatase in vitro, mitigating a potential negative impact on bone mineral density with long-term use. In conjunction with this observation, the diminished metabolism of these T analogs by 5α-reductase may reduce potential concerns for prostatic growth. In the Hershberger in vivo rat bioassay, 7α-MT and 7α-ET showed superior androgenic and anabolic activities as compared with T. These C7 α-substituted T analogs also showed clear progestogenic activity in the McPhail bioassay which evaluated endometrial glandular arborization in a rabbit model. The discovery of aromatizable molecules with reduced metabolism by 5α-reductase that have androgenic, anabolic, and progestogenic properties indicates that the core and/or prodrugs of 7α-MT and 7α-ET are promising molecules for further development as male contraceptive PAs.
Topics: Male; Rats; Rabbits; Animals; Humans; Androgens; Testosterone; Progestins; Nandrolone; Methyltestosterone; Contraception; Contraceptive Agents, Male; Prodrugs
PubMed: 37669128
DOI: 10.1093/biolre/ioad111 -
The Journal of Steroid Biochemistry and... Mar 2024In comparison to well-known drug-metabolizing organs such as the liver, the metabolic capacity of human skin is still not well elucidated despite the widespread use of...
In comparison to well-known drug-metabolizing organs such as the liver, the metabolic capacity of human skin is still not well elucidated despite the widespread use of topical drug application. To gain a comprehensive insight into anabolic steroid metabolism in the skin, six structurally related anabolic androgenic steroids, testosterone, metandienone, methyltestosterone, clostebol, dehydrochloromethyltestosterone, and methylclostebol, were applied to human keratinocytes and fibroblasts derived from the juvenile foreskin. Phase I metabolites obtained from incubation media were analyzed by gas chromatography-mass spectrometry. The 5α-reductase activity was predominant in the metabolic pathways as supported by the detection of 5α-reduced metabolites after incubation of testosterone, methyltestosterone, clostebol, and methylclostebol. Additionally, the stereochemistry structures of fully reduced metabolites (4α,5α-isomers) of clostebol and methylclostebol were newly confirmed in this study by the help of inhouse synthesized reference materials. The results provide insights into the steroid metabolism in human skin cells with respect to the characteristics of the chemical structures.
Topics: Humans; Methyltestosterone; Anabolic Androgenic Steroids; Anabolic Agents; Testosterone Congeners; Testosterone; Biotransformation; Doping in Sports
PubMed: 38092130
DOI: 10.1016/j.jsbmb.2023.106444