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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 -
General and Comparative Endocrinology Jul 2022Estrogens and androgens that coexist in the aquatic environment could potentially affect shellfish, however, endocrine disrupting effects of them in shellfish are...
Estrogens and androgens that coexist in the aquatic environment could potentially affect shellfish, however, endocrine disrupting effects of them in shellfish are significant. As an important aquaculture shellfish in China, Hyriopsis cumingii has remarkable economic benefits. In this study, the effects of endocrine disrupting chemicals on the steroid synthase Hc-Cyp17a in the male and female gonads of the H. cumingii were assessed by exposing juvenile mussels to cultured waters containing 17β-Estradiol (E2) and 17α-Methyltestosterone (MT) for 28 days. At the same time, the E2 content in the four stages of gonadal development, the expression changes of Hc-Cyp17a in gonadal development and its localization in the mature gonad were measured to explore the relationship between genes and hormones. The results showed that both E2 and MT at 50 ng/L and 200 ng/L could affect the transcription level of Hc-Cyp17a, which was inhibited initially and promoted in post-development. E2 content was positively correlated with gonadal development stage, which was in mussel. By tracing the expression of Hc-Cyp17a, difference was found during different developmental periods. The expression level in ovary was higher than that in testis during gonadal development of 1/ 2/ 3-year-old mussels and showed an increasing trend with age. Furthermore, the expression levels in 6 tissues of mature individuals were measured and it showed that there was a significant difference between male and female in the gonads (p < 0.01). In situ hybridization, it suggested that Hc-Cyp17a was significantly signaled in the follicular wall and oocyte of female and in the follicular membrane of testis, respectively. These results could play a vital role in assessing and understanding the effects of aquatic environment on the endocrine system of H. cumingii.
Topics: Animals; Endocrine Disruptors; Estradiol; Estrogens; Female; Gonadal Steroid Hormones; Gonads; Male; Methyltestosterone; Steroid 17-alpha-Hydroxylase
PubMed: 35314150
DOI: 10.1016/j.ygcen.2022.114028 -
Comparative Biochemistry and... 2023Steroidogenesis is an important biological process for gonadal differentiation and development. In mammals, 3β-hydroxysteroid dehydrogenase 7 (HSD3B7) could convert...
Steroidogenesis is an important biological process for gonadal differentiation and development. In mammals, 3β-hydroxysteroid dehydrogenase 7 (HSD3B7) could convert 3β-hydroxy of 7α-hydroxycholesterol into a ketone and form 7α-hydroxy-4-cholesten-3-one, which may affect steroidogenesis. However, in fish, the study of Hsd3b7 is still lacking. In this study, Hsd3b7 was identified in the olive flounder Paralichthys olivaceus, an important mariculture fish. According to bioinformatics analysis, Hsd3b7 belongs to a Rossmann-fold NAD(P)(+)-binding protein and can interact in a predictable manner with Hsd17b2, -3, and - 4, which play a role in steroidogenesis. In the adult flounder, Hsd3b7 was expressed in various tissues, at particularly high level in male muscle. The expression levels of Hsd3b7 at gonadal development stages I-V initially increased and then decreased, with an inflection point in the ovary at stage III and in the testis at stage IV. At stage III, the expression level of Hsd3b7 was significantly higher in the ovary than in the testis (P < 0.01). The results of in situ hybridization (ISH) revealed that it was mainly expressed in oocytes of phases I-IV or around oocytes of phases IV-V in the ovaries and around spermatid lobules at stages IV-V in the testes. Three regulatory sites of SRY-box transcription factor 9 (Sox9), a transcription factor involved in steroidogenesis and gonadal differentiation, were predicted in the promoter of Hsd3b7. After intraperitoneal injection with the recombination flounder Sox9a, the expression of Hsd3b7 was significantly up-regulated (P < 0.01). During the flounder gonadal differentiation, 17β-estradiol (E2, 5 μg/g feed) and 17α-methyltestosterone (T, 5 μg/g feed) were used to obtain the phenotypic female or male flounder, and the results showed that in the E2 group, Hsd3b7 expression was highest at 2 cm TL, the primordial gonad stage, which was significantly higher than that at 12 cm TL (P < 0.05). In the T group, Hsd3b7 expression level was also highest at 2 cm TL and significantly higher than at 10 and 12 cm TL (P < 0.05). Moreover, Hsd3b7 was detected to be localized mainly around oogonia and spermatogonia during the differentiated gonads with ISH. These findings first introduce the expression characteristics of Hsd3b7 and the effect of Sox9a on its expression, which contribute to our understanding of the function of Hsd3b7 in fish gonads.
Topics: Animals; Female; Male; Flounder; Gene Expression Regulation, Developmental; Gonads; Mammals; Testis; Transcription Factors; Spermatogonia
PubMed: 36933762
DOI: 10.1016/j.cbpb.2023.110848 -
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 -
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 -
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 -
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