It is widely known that all-female fish production holds economic value for aquaculture. Sebastes schlegelii, 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 S. schlegelii for subsequent all-female offspring breeding. Long-term MT administration severely inhibits the growth of S. schlegelii, 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 (cyp19a1a and foxl2) while up-regulating male-related genes (amh) in pseudomales. Furthermore, MT modulated the TGF-β signaling and steroid hormone biosynthesis pathways, indicating its crucial role in S. schlegelii sex differentiation. Therefore, the current study provides a method for achieving sexual reversal using MT in S. schlegelii and offers an initial insight into the underlying mechanism of sexual reversal in this species.

As a synthetic androgen, 17α-methyltestosterone (MT) is widely used in aquaculture to induce sex reversal and may pose a potential risk to aquatic organisms. This ecological risk has attracted the attention of many scholars, but it is not comprehensive enough. Thus, the adverse effects of MT on zebrafish (Danio rerio) were comprehensively evaluated from gonadal histology, as well as the mRNA expression levels of 47 genes related to hypothalamic-pituitary-gonadal (HPG) axis, germ cell differentiation, sex determination, and hypothalamus-pituitary-thyroid (HPT) axis. Adult zebrafish with a female/male ratio of 5:7 were exposed to a solvent control (0.001% dimethyl sulfoxide) and three measured concentrations of MT (5, 51 and 583 ng/L) for 50 days. The results showed that MT had no significant histological effects on the ovaries of females, but the frequency of late-mature oocytes (LMO) showed a downward trend, indicating that MT could induce ovarian suppression to a certain extent. The transcriptional expression of activating transcription factor 4b1 (atf4b1), activating transcription factor 4b2 (atf4b2), calcium/calmodulin-dependent protein kinase II delta 1 (camk2d1), calcium/calmodulin-dependent protein kinase II delta 2 (camk2d2) and calcium/calmodulin-dependent protein kinase II inhibitor 2 (camk2n2) genes in the brain of females increased significantly at all treatment groups of MT, and the mRNA expression of forkhead box L2a (foxl2) and ovarian cytochrome P450 aromatase (cyp19a1a) genes in the ovaries were down-regulated by 5 and 583 ng/L group, which would translate into inhibition of oocyte development. As compared to females, MT had relatively little effects on the reproductive system of males, and only the transcriptional alterations of synaptonemal complex protein 3 (sycp3) and 17-alpha-hydroxylase/17,20-lyase (cyp17) genes were observed in the testes, not enough to affect testicular histology. In addition, MT at all treatments strongly increased corticotropin-releasing hormone (crh) transcript in the brain of females, as well as deiodinase 2 (dio2) transcript in the brain of males. The paired box protein 8 (pax8) gene was significantly decreased at 51 or 583 ng/L of MT in both female and male brains. The above results suggest that MT can pose potential adverse effects on the reproductive and thyroid endocrine system of fish.

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 ED50 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 ED50 were improved and found to be 0.02 ng/mL, 0.086 × 10-8 L/mol and 2.95 ng/mL than homologous assay where Sensitivity, affinity and ED50 were 0.11 ng/mL, 0.02 × 10-8 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, ED50 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 (R2 = 0.96) by comparing with the commercial kit and validated by measuring levels of 17α- methyltestosterone in rat serum after administering them.

The steroid 17α-methyltestosterone (MT) inhibits ovarian function and is often used to induce sex reversal artificially in vertebrates. In the present study, different concentrations of MT were added as dietary supplementation, and the effects on sex ratio, growth, and gonadal development were examined. After 40 days, the sex ratio (male:female) in each group increased at different degrees with 50 (1.36:1), 100 (1.57:1), and 200 (2.61:1) mg/kg MT, and neo-males with testis-ovary coexistence were observed in the 200 mg/kg MT group. Furthermore, 50 and 100 mg/kg MT could induce female reversion in neo-males. Histologically, the development of the testes in experimental groups was slower, but the ovaries of the experimental and control groups had similar developmental rates. The expression levels of DMRT11E, Foxl2, and SoxE1 in males at 200 mg/kg MT were 8.65-, 3.75-, and 3.45-fold greater than those of the control group. In crustaceans, sex reversal through vertebrate sex hormones can be observed. Neo-males (sex-reversed female prawns) were maintained by exogenous androgen, and over-reliance led to slow testis growth, small body size, and low growth rate, but sperm was still produced. In female prawns, MT inhibited ovary development and promoted growth.

17α-Methyltestosterone (17MT), a synthetic organic compound commonly found in sewage waters, can affect reproduction in aquatic animals, such as tilapia and yellow catfish. In the present study, male Gobiocypris rarus were exposed to 25, 50, and 100 ng/L of 17α-methyltestosterone (17MT) for 7 days. We first analyzed miRNA- and RNA-seq results to determine miRNA-target gene pairs and then developed miRNA-mRNA interactive networks after 17MT administration. Total weights, total lengths, and body lengths were not significantly different between the test groups and control groups. The paraffin slice method was applied to testes of G. rarus in the MT exposure and control groups. We found that there were more mature sperm (S) and fewer secondary spermatocytes (SSs) and spermatogonia (SGs) in the testes of control groups. As 17MT concentration increased, fewer and fewer mature sperm (S) were observed in the testes of male G. rarus. The results showed that FSH, 11-KT, and E2 were significantly higher in individuals exposed to 25 ng/L 17MT compared with the control groups. VTG, FSH, LH, 11-KT, and E2 were significantly lower in the 50 ng/L 17MT exposure groups compared to the control groups. VTG, FSH, LH, 11-KT, E2, and T were significantly lower in the groups exposed to 100 ng/L 17MT. High-throughput sequencing revealed 73,449 unigenes, 1205 known mature miRNAs, and 939 novel miRNAs in the gonads of G. rarus. With miRNA-seq, 49 (MT25-M vs. Con-M), 66 (MT50-M vs. Con-M), and 49 (MT100-M vs. Con-M) DEMs were identified in the treatment groups. Five mature miRNAs (miR-122-x, miR-574-x, miR-430-y, lin-4-x, and miR-7-y), as well as seven differentially expressed genes (soat2, inhbb, ihhb, gatm, faxdc2, ebp, and cyp1a1), which may be associated with testicular development, metabolism, apoptosis, and disease response, were assayed using qRT-PCR. Furthermore, miR-122-x (related to lipid metabolism), miR-430-y (embryonic development), lin-4-x (apoptosis), and miR-7-y (disease) were differentially expressed in the testes of 17MT-exposed G. rarus. This study highlights the role of miRNA-mRNA pairs in the regulation of testicular development and immune response to disease and will facilitate future studies on the miRNA-RNA-associated regulation of teleost reproduction.

17α-Methyltestosterone (MT), a synthetic environmental endocrine disruptor with androgenic effects, has been shown to disrupt the reproductive system and inhibit germ cell maturation in Gobiocypris rarus. To further investigate the regulation of gonadal development by MT through the hypothalamic-pituitary-gonadal (HPG) axis, G. rarus were exposed to 0, 25, 50, and 100 ng/L of MT for 7, 14, and 21 days. We analyzed its biological indicators, gonadotropin-releasing hormone (GnRH), gonadotropins, reproduction-related gene expression, and brain tissue transcriptome profiles. We found a significant decrease in the gonadosomatic index (GSI) in G. rarus males exposed to MT for 21 days compared to the control group. GnRH, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels, as well as the expressions of the gnrh3, gnrhr1, gnrhr3, fshβ, and cyp19a1b genes, were significantly reduced in the brains of both male and female fish when exposed to 100 ng/L MT for 14 days compared to the controls. Therefore, we further constructed four RNA-seq libraries from 100 ng/L MT-treated groups of male and female fish, obtaining 2412 and 2509 DEGs in male and female brain tissue, respectively. Three common pathways were observed to be affected in both sexes after exposure to MT, namely, nicotinate and nicotinamide metabolism, focal adhesion, and cell adhesion molecules. Furthermore, we found that MT affected the PI3K/Akt/FoxO3a signaling pathway through the upregulation of foxo3 and ccnd2, and the downregulation of pik3c3 and ccnd1. Therefore, we hypothesize that MT interferes with the levels of gonadotropin-releasing hormone (GnRH, FSH, and LH) in G. rarus brains through the PI3K/Akt/FoxO3a signaling pathway, and affects the expression of key genes in the hormone production pathway (gnrh3, gnrhr1 and cyp19a1b) to interfere with the stability of the HPG axis, thus leading to abnormal gonadal development. This study provides a multidimensional perspective on the damaging effects of MT on fish and confirms that G. rarus is a suitable model animal for aquatic toxicology.

The 17α-methyltestosterone is the most common synthetic hormone used in male mono-sex production of Nile tilapia, Oreochromis niloticus. 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 O. niloticus 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.

17α-Methyltestosterone (MT) is a synthetic steroid that has been widely used to masculinize many fish species when administered early during larval development, however, reports on its efficacy on adults is limited. To this end, this study investigated the efficacy of MT in the masculinization of the eastern mosquitofish (G. holbrooki) at two adult stages (maiden and repeat gravid females). The treated females were fed control or respective MT incorporated feed (0-200 mg/kg diet) for 50 days. Effects of the hormone on secondary sexual characteristics, internal gonad morphology, expression of the Anti-Müllerian Hormone (amh) gene and sexual behavior of the treated females were investigated. The results showed that MT at the dose of 50 mg/kg feed stimulated secondary sexual character development, upregulated expression of amh, formation of testicular tissue and a shift in the behavior similar to those of normal males, prominently so in treated maiden gravid females. Post-treatment, long-term observations indicated that only two masculinized females reverted back to being females and gave birth to young. Induction of masculinizing effects in most individuals suggests that the sexual phenotype of this species appears to be highly plastic with potential to sex reverse at adulthood. This in combination with its small size and short reproductive cycle could provide an ideal system to explore the mechanisms of sequential hermaphroditism in fish and contribute to genetic control of this pest fish.

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.

17α-Methyltestosterone (MT) is a synthetic androgen. The objective of this study was to explore the effects of exogenous MT on the growth and gonadal development of female rare minnow Gobiocypris rarus. Female G. rarus groups were exposed to 25-100 ng/L of MT for 7 days. After exposure for 7 days, the total weight and body length were significantly decreased in the 50-ng/L MT groups. The major oocytes in the ovaries of the control group were vitellogenic oocytes (Voc) and cortical alveolus stage oocytes (Coc). In the MT exposure groups, some fish had mature ovaries with a relatively lower proportion of mature oocytes, and the diameter of the perinucleolar oocytes (Poc) was decreased compared with those of the control group. Ovarian VTG, FSH, LH, 11-KT, E2, and T were significantly increased after exposure to 50 ng/L of MT for 7 days. Unigenes (73,449), 24 known mature microRNAs (miRNAs), and 897 novel miRNAs in the gonads of G. rarus were found using high-throughput sequencing. Six mature miRNAs (miR-19, miR-183, miR-203, miR-204, miR-205, and miR-96) as well as six differentially expressed genes (fabp3, mfap4, abca1, foxo3, tgfb1, and zfp36l1) that may be associated with ovarian development and innate immune response were assayed using qPCR. Furthermore, the miR-183 cluster and miR-203 were differentially expressed in MT-exposed ovaries of the different G. rarus groups. This study provides some information about the role of miRNA-mRNA pairs in the regulation of ovarian development and innate immune system, which will facilitate future studies of the miRNA-RNA-associated regulation of teleost reproduction.