dmrt1 Is Responsible for Androgen-Induced Masculinization in Nile Tilapia.

17α-Methyltestosterone (MT) is a widely used androgen for all-male fish production in aquaculture. However, the molecular mechanism underlying MT-induced masculinization remains unclear. In this study, we aim to identify the key gene responsible for MT-induced masculinization using the Nile tilapia (Oreochromis niloticus) amhy, dmrt1, and gsdf mutants, which exhibit male-to-female sex reversal. Nile tilapia fry from these three mutant lines were treated with 50 µg/g MT from 5 to 30 days after hatching (dah). The results showed that amhy and gsdf mutants, but not dmrt1 mutants, were masculinized by the MT treatment. Gonadal transcriptome analysis revealed that genes involved in steroidogenesis and germ cell development in MT-treated dmrt1 mutants exhibited a similar expression pattern to that of the wild type (WT) XX. In addition, the dmrt1 mutants cannot be masculinized by co-treatment with MT and the aromatase inhibitor fadrozole. The MT treatment completely blocked early steroidogenic enzyme (Star2, Cyp17a2, and Cyp19a1a) expression independent of amhy, gsdf, and dmrt1. A luciferase analysis showed that MT directly suppressed basal and Sf-1-activated cyp19a1a promoter activity through ara and arb in cultured HEK293 cells. Furthermore, MT treatment inhibited germ cell proliferation in amhy and gsdf mutants but not in dmrt1 mutants. Consistently, dmrt1 expression was induced in MT-treated WT XX, -amhy, and -gsdf mutants. Taken together, these results suggest that dmrt1 is indispensable for MT-induced masculinization in Nile tilapia and that MT functions by inhibiting early steroid synthesis and activating dmrt1 to promote testis development.

Comparable to 17α- methyl testosterone, dietary supplements of Tribulus terrestris and Mucuna pruriens promote the development of mono-sex, all-male tilapia fry, growth, survival rate and sex-related genes (Amh, Sox9, Foxl2, Dmrt1).

To evaluate Tribulus terrestris and Mucuna pruriens for inducing all-male tilapia, mixed-sex Nile tilapia, Oreochromis niloticus, (mean weight 0.025 ± 0.009 g; mean length 1.25 ± 0.012 cm), were given a meal supplemented with either T. terrestris powder (commercial fish feed, 40% crude protein) (TT group), M. pruriens seed extract (MP group), MP + TT (mixed group), 17α-methyl testosterone (MT, control positive), or without supplements (control negative). The MP extracts significantly increased (P < 0.05) the final weight, weight gain, weight gain rate, and specific growth rate while feed conversion ratio was significantly decreased (P < 0.05). Plant extracts markedly improved (P < 0.05) the survival rate, proportion of males, and total testosterone compared to control and MT. Estrogen levels were lower in groups with plant extract than other groups. Fifteen days post-feeding, the Amh gene was expressed in the brain of O. niloticus fries with higher levels in MP, TT, and MT groups. Additionally, the expression of the Sox9 and Dmrt1 genes as a male related genes in fish fry gonads revealed significantly (P < 0.05) higher levels in groups fed on MP, TT, and MT compared to control after 30-day post-feeding, whereas; Foxl2 gene expression as a female related gene was significantly (P < 0.05) lower in fish fed on MP, TT, and MT compared to other groups after 30 days post feeding. Histologically, MT, MP, TT, and the mixture all exhibited solely male reproductive traits without noticeable abnormalities. This study concluded that each of the TT or MP extracts can induce sex reversal in tilapia while having no negative health impact compared to MT as the growth and survival rate in the treated groups with TT and MP were higher than control and group treated with MT.

Molecular and Physiological Effects of 17α-methyltestosterone on Sex Differentiation of Black Rockfish, Sebastes schlegelii.

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-ß 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.

Testing androgen-induced immunosuppression: Environmental androgens as a model system for steroid-immune interaction.

It is well known that hormones influence and direct most facets of physiology; however, there is still contention regarding the directions of certain relationships, for example, between gonadal hormones and immunity. Among the many proposed relationships relating to gonadal-immune interactions, support for immunosuppressive effects of androgens remains prominent within physiological literature. Although ample study has been directed toward the immunosuppressive effects of androgens, considerable disagreement remains regarding their influence on immune function. In this study, we test the hypothesis that androgens inhibit immunocompetence in the American alligator (Alligator mississippiensis). Developing alligators were incubated at female-producing temperatures with a subset of individuals being exposed to 17-α-methyltestosterone (MT) before sexual determination. 17-α-methyltestosterone is a potent androgen, not aromatizable by crocodilians, that has been found to exert masculinizing effects in exposed crocodilian populations in vivo and in vitro. Additionally, a subset of animals was exposed to a novel antigen to quantify innate and acquired immune function. We recovered no significant differences in leukocyte ratios or proportions between groups and found no significant differences in innate immune function as measured by hemolysis-hemagglutination. However, we did find significant differences in acquired immune function, where masculinized individuals expressed greater antibody titers. Our findings reject the hypothesis that androgens suppress immune function; rather, androgens may be immunoenhancing to acquired humoral responses and neutral to innate humoral immunity in crocodilians.

Effects of combined exposure to polystyrene microplastics and 17α-Methyltestosterone on the reproductive system of zebrafish.

Polystyrene microplastics (PS-MPs) are important carriers of pollutants in water. 17α-Methyltestosterone (MT) is a synthetic environmental endocrine disrupting chemical (EDC) with androgenic effects. To study the effects of PS-MPs and MT on zebrafish reproductive systems, zebrafish were exposed to 0 or 50 ng L-1 MT, 0.5 mg∙L-1 PS-MPs, or 50 ng∙L-1 MT + 0.5 mg∙L-1 PS-MPs for 21 d. The results showed that the different exposure reagents caused varying degrees of damage to the reproductive systems in zebrafish, with the extent of damage increasing as the exposure duration increased. Histological analysis of the gonads revealed that the ratio of mature oocytes and mature spermatozoa in the gonad decreased gradually with increased exposure time, with the ratio being Control > PS-MPs > MT > MT + PS-MPs in decreasing order. The results of quantitative real-time PCR (qRT‒PCR) showed that in female fish treated for 7 d, the expression of cyp11a mRNA was significantly reduced in all three treatment groups(MT, PS-MPs, and MT + PS-MPs), while in the group treated for 14 d with MT + PS-MPs, the expression of cyp19a1a and StAR mRNA was significantly increased. In male fish exposed for 21 d, the expression of cyp11a, cyp17a1, cyp19a1a, StAR, 3ß-HSD, and 17ß-HSD3 mRNA was significantly decreased in MT + PS-MPs. ELISA results showed that after 14 d of exposure, the levels of E2, LH, and FSH in the ovaries of female fish were significantly reduced in all three treatment groups. Similarly, the levels of T, E2, LH, and FSH in the testis of male fish were significantly reduced after 14 d of exposure to PS-MPs and MT + PS-MPs. Offspring of zebrafish exposed to MT and MT + PS-MPs exhibited delayed incubation time and slow development. The cross-generational toxicity of PS-MPs themselves may be negligible, but it can exacerbate the toxicity of MT, making the cross-generational effects more pronounced in the offspring, causing offspring mortality and malformations. Offspring of zebrafish exposed to MT and MT + PS-MPs exhibited delayed incubation time and slow development. In addition, MT caused malformations such as pericardial edema, yolk cysts, and spinal deformities in zebrafish during the incubation period.

The effects of 17α-methyltestosterone on gonadal histology and gene expression along hypothalamic-pituitary-gonadal axis, germ cells, sex determination, and hypothalamus-pituitary-thyroid axis in zebrafish (Danio rerio).

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.

Effects of exogenous steroid hormones on growth, body color, and gonadal development in the Opsariichthys bidens.

To investigate the effects of exogenous steroid hormones on growth, body color, and gonadal development in the Opsariichthys bidens (O. bidens), synthetic methyltestosterone (MT) and 17ß-estradiol (E2) were used for 28 days' treatment of 4-month-old O. bidens before the breeding season. Our results suggested that MT had a significant growth-promoting effect (P < 0.05), whereas E2 played an inhibitory role. On the body surface, the females in the MT group showed gray stripes, and the fish in other groups showed no obvious stripes. The males with MT treatment displayed brighter blue-green stripes compared to the CK and E2 groups. The histological analysis showed that the MT significantly promoted testes development in males, blocked oocyte development, and caused massive apoptosis in females, whereas the E2 group promoted ovarian development and inhibited testes development. Based on qRT-PCR analysis, in females, the expression of igf-1, dmrt1, and cyp19a1a genes revealed that E2 treatment resulted in down-regulation of igf-1 expression and up-regulation of cyp19a1a expression. In males, igf-1 and dmrt1 were significantly up-regulated after MT treatment, and E2 treatment led to down-regulation of igf-1. Therefore, this study demonstrates that MT and E2 play an important role in reversing the morphological sex characteristics of females and males.

Effects of dietary aromatase inhibitors on masculinization of rosy barb (Pethia conchonius): Evidence from growth, coloration and gonado-physiological changes.

The objective of this study was to reveal the growth, colouration and gonado-physiological changes due to the exogenous aromatase inhibitor (AIs) in an ornamental fish. 17α-methyltestosterone (MT) and letrozole (LET) were used as potential AIs. The AI were supplemented with a gel-based feed (LET: 50, 100, 150 and MT: 12.5, 25, 37.5 mg/kg feed) in Rosy barb, Pethia conchonius fry. The fishes were reared in a 45-L glass tank using AI treated gel-based feed for 3 months. Growth in AI-based diets was reduced but the reduction was minimal compared to the control. At 25 mg/kg feed of 17 MT, the highest male proportion (84.72% 6.05%) was recorded, which was significantly higher (P≤0.05) than other groups. L*, a*, and b* values showed that 17α-MT-fed groups had brighter coloration (P≤0.05). Histological sections showed that LET-17α-MT suppressed ovarian development, causing atretic oocytes. Testicular development was unaffected. 25 mg/kg-treated feed increased SOD, CAT, GST, and GPX. The AI (MT) at 25 mg/kg gel-based feed could therefore be utilised for musculinization without impacting growth, colour, and antioxidant activity of rosy barb, which serves the entire male population in the ornamental fish sector.

Characterization of Early Gonadal Differentiation and Induction of Sex Reversal in the Rock Bream Oplegnathus fasciatus.

Rock bream (Oplegnathus fasciatus) is a typical fish that has a unique multiple sex chromosome system (♀X1X1X2X2/♂X1X2Y). We examined the early gonadal development in rock bream via continuous histological observations of the gonads at 40-120 days post hatching (dph). The fish was identified as a typical gonochorist, and female gonads were found to differentiate earlier than male gonads. The ovarian cavity of the female was initially observed at 80 dph, whereas the efferent duct of the male was not observed until 100 dph. Immunofluorescence with the vasa-antibody revealed that germ cells were predominantly distributed around the ovarian cavity in females and on the edge of the gonad in males during the early stages of sex differentiation. Sex reversal was induced via the oral administration of letrozole (LTZ), 17α-methyltestosterone (MT), and 17ß-estradiol (E2), respectively, during the labile period of gonadal development. LTZ and MT induced 100% masculinization of genotype-females, whereas E2 induced only 50-60% feminization of genotype-males. Such findings suggest that the fish retained high sexual plasticity despite the existence of the neo-Y chromosome. MT and E2 had negative effect on fish growth, whereas LTZ did not exert such side effect. LTZ and MT could accelerate gonadal development in sex-reversed genotype-males, whereas E2 inhibited gonadal development in genotype-females of rock bream. These findings provide a basis for further research on the mechanisms of sex determination and differentiation in fishes with X1X2Y sex chromosome system and provide a sex reversal protocol for rock bream.

Effects of 17α-Methyltestosterone on the Transcriptome and Sex Hormones in the Brain of Gobiocypris rarus.

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, fshß, 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 effect of androgen exposure on cerebral lateralization in the American alligator (Alligator mississippiensis).

The division of the brain manifests in lateralized physical behaviors, where specific tasks originate from one side of the body. Previous studies have shown that birds and reptiles mediate aggression in their right hemisphere and focus on opponents with their left eye. Degree of lateralization varies between sexes, likely due to androgen inhibition of lateralization in mammals, birds, and fish, but remains untested in herpetofauna. In this experiment, we investigated the effect of androgen exposure on cerebral lateralization in the American Alligator, Alligator mississippiensis. Alligator eggs were collected and incubated at female producing temperature with a subset dosed with methyltestosterone in ovo. Dosed hatchlings were randomly paired with control individuals and their interactions were recorded. The number of bites initiated by focus from each eye and the number of times an animal was bitten on each side of the body was recorded for each individual to elucidate cerebral lateralization in aggression. Control alligators had a significant bias towards left-eye bite initiation whereas androgen exposed alligators used both eyes indiscriminately. No significance was found in injury patterns. This study suggests that androgen exposure inhibits cerebral lateralization in alligator brains and corroborates right-hemisphere mediation of aggression, something previously unstudied in crocodilians.

A Simple Method for Inducing Masculinization of Zebrafish Stocks Using 17α-Methyltestosterone.

Severely skewed sex ratios in zebrafish stocks can pose significant hurdles for line propagation and sperm cryopreservation. To overcome female-biased sex ratios in stocks derived from imported sperm samples, the Zebrafish International Resource Center has implemented routine supplementation of larval food with 17α-methyltestosterone to skew gonadal sex differentiation toward masculinization. Resulting stocks averaged 80% males.

Cyp17a effected by endocrine disruptors and its function in gonadal development of Hyriopsis cumingii.

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 17ß-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.

Involvement of IGF-1R-PI3K-AKT-mTOR pathway in increased number of GnRH3 neurons during androgen-induced sex reversal of the brain in female tilapia.

The neuroplastic mechanism of sex reversal in the fish brain remains unclear due to the difficulty in identifying the key neurons involved. Mozambique tilapia show different reproductive behaviours between sexes; males build circular breeding nests while females hold and brood fertilized eggs in their mouth. In tilapia, gonadotropin-releasing hormone 3 (GnRH3) neurons, located in the terminal nerve, regulate male reproductive behaviour. Mature males have more GnRH3 neurons than mature females, and these neurons have been indicated to play a key role in the androgen-induced female-to-male sex reversal of the brain. We aimed to elucidate the signalling pathway involved in the androgen-induced increase in GnRH3 neurons in mature female tilapia. Applying inhibitors to organotypic cultures of brain slices, we showed that the insulin-like growth factor (IGF)-1 receptor (IGF-1R)/PI3K/AKT/mTOR pathway contributed to the androgen-induced increase in GnRH3 neurons. The involvement of IGF-1 and IGF-1R in 11-ketotestosterone (11-KT)-induced development of GnRH3 neurons was supported by an increase in Igf-1 mRNA shortly after 11-KT treatment, the increase of GnRH3 neurons after IGF-1 treatment and the expression of IGF-1R in GnRH3 neurons. Our findings highlight the involvement of IGF-1 and its downstream signalling pathway in the sex reversal of the tilapia brain.

The effect of methyltestosterone (MT) on sex differentiation and growth in juvenile yellow perch (Perca flavescens).

A study was conducted to evaluate the gonad differentiation of juvenile yellow perch (YP, Perca flavencens) and determine the latest labile period related to hormone treatment. Juvenile fish were subjected to two dietary concentrations of methyltestosterone (MT; 20 and 50 mg/kg feed) for 60 days in three (3) age groups of 38-, 46-, and 67-days post-hatching (dph), where control group were fed with standard commercial feed. Following a 10-month on-growing period, sex phenotypes were determined by gross and histological gonad morphology. Results showed the juvenile YP responded to the exogenous hormone when it was applied at 38 dph for both 20 and 50 mg/kg feed resulting in 100% males. At 46 dph, only 50 mg/kg feed resulted in 100% males. Both MT-treated at 38 and 46 dph significantly differed (P < 0.01) from the expected normal population of male:female (1:1). MT-treated at 67 dph resulted in 37% and 25% intersex fish for both 20 and 50 mg/kg feed dosage groups, respectively. MT-treated at 38 and 46 dph promoted growth and showed significantly heavier mean body weight (P < 0.05) compared to control. The gonadosomatic index (GSI) of MT-treated at 38 and 46 dph was significantly lower than that in control. This study provides the first evidence that juvenile YP can be successfully masculinized when the treatment is initiated at the age of up to 46 dph. The result is important for sex control in aquaculture.

Potential role of DNA methylation of cyp19a1a promoter during sex change in protogynous orange-spotted grouper, Epinephelus coioides.

Estrogen has a pivotal role in early female differentiation and further ovarian development. Aromatase (Cyp19a) is responsible for the conversion of androgens to estrogens in vertebrates. In teleosts, cyp19a1a and it paralog cyp19a1b are mainly expressed in the ovary and hypothalamus, respectively. Decreased plasma estrogen levels and lower cyp19a1a expression are associated with the initiation of female-to-male sex change in protogynous grouper. However, an 17α-methyltestosterone (MT)-induced the sex change from a female to a precocious male is a transient phase, and a reversible sex change (induced male-to-female) occurs after chemical withdrawal. Thus, we used this characteristic to study the epigenetic modification of cyp19a1a promoter in orange-spotted grouper. CpG-rich region with a CpG island is located on the putative regulatory region of distal cyp19a1a promoter. Our results showed that cyp19a1a promoter exhibited tissue-specific methylation status. Low methylation levels of distal cyp19a1a promoter and hypomethylated (0-40%) clones of cyp19a1a promoter region were widely observed in the ovary but not shown in testis and other tissues. In femaleness, higher numbers of hypomethylated clones of cyp19a1a promoter region were observed in the vitellogenic oocyte stage compared to the primary oocyte stage. Furthermore, decreased numbers of hypomethylated clones of cyp19a1a promoter region were associated with the maleness during the female-to-male sex change. DNA methylation inhibitor (5-aza-2'-deoxycytidine) delayed the spermatogenesis process (according to germ cell stage and numbers: by decrease of sperm and increase of spermatocytes) but did not influence the reversed sex change in MT-induced bi-directional sex change. These results suggest that epigenetic modification of cyp19a1a promoter may play an important role during the sex change in orange-spotted grouper.

Androgenic Sensitivities and Ovarian Gene Expression Profiles Prior to Treatment in Japanese Eel (Anguilla japonica).

Androgens stimulate ovarian development in eels. Our previous report indicated a correlation between the initial (debut) ovarian status (determined by kernel density estimation (KDE), presented as a probability density of oocyte size) and the consequence of 17MT treatment (change in ovary). The initial ovarian status appeared to be an important factor influencing ovarian androgenic sensitivity. We postulated that the sensitivities of initial ovaries are correlated with their gene expression profiles. Japanese eels underwent operation to sample the initial ovarian tissues, and the samples were stored in liquid nitrogen. Using high-throughput next-generation sequencing (NGS) technology, ovarian transcriptomic data were mined and analyzed based on functional gene classification with cutoff-based differentially expressed genes (DEGs); the ovarian status was transformed into gene expression profiles globally or was represented by a set of gene list. Our results also implied that the initial ovary might be an important factor influencing the outcomes of 17MT treatments, and the genes related with neuronal activities or neurogenesis seemed to play an essential role in the positive effect.

Gene Expression of Takifugu rubripes Gonads During AI- or MT-induced Masculinization and E2-induced Feminization.

Elucidating the global molecular changes that occur during aromatase inhibitor (AI)- or 17α-methyltestosterone (MT)-induced masculinization and estradiol-17ß (E2)-induced feminization is critical to understanding the roles that endocrine and genetic factors play in regulating the process of sex differentiation in fish. Here, fugu larvae were treated with AI (letrozole), MT, or E2 from 25 to 80 days after hatching (dah), and gonadal transcriptomic analysis at 80 dah was performed. The expression of dmrt1, gsdf, foxl2, and other key genes (star, hsd3b1, cyp11c1, cyp19a1a, etc.) involved in the steroid hormone biosynthesis pathway were found be altered. The expression of dmrt1, gsdf, cyp19a1a, and foxl2 was further verified by quantitative polymerase chain reaction. In the control group, the expression of dmrt1 and gsdf was significantly higher in XY larvae than in XX larvae, while the expression of foxl2 and cyp19a1a was significantly higher in XX larvae than in XY larvae (P < .05). AI treatment suppressed the expression of foxl2 and cyp19a1a, and induced the expression of dmrt1 and gsdf in XX larvae. MT treatment suppressed the expression of foxl2, cyp19a1a, dmrt1, and gsdf in XX larvae. E2 treatment suppressed the expression of dmrt1 and gsdf, but did not restore the expression of foxl2 and cyp19a1a in XY larvae. The shared response following AI, MT, and E2 treatment suggested that these genes are essential for sex differentiation. This finding offers some insight into AI or MT-induced masculinization, and E2-induced femininization in fugu.

The reproductive regulation of LPXRFa and its receptor in the hypothalamo-pituitary-gonadal axis of the spotted scat (Scatophagus argus).

Gonadotropin-inhibitory hormone (GnIH) plays a critical role in regulating gonadotropin-releasing hormone (GnRH), gonadotropin hormone (GtH), and steroidogenesis. The Lpxrfa (the piscine ortholog of GnIH) system has been found to regulate fish reproduction. To gain insight into the role of Lpxrfa in the regulation of spotted scat (Scatophagus argus) reproduction, spotted scat Lpxrfa (ssLpxrfa), and its receptor (ssLpxrfa-r) were cloned and analyzed. Tissue distribution and expression patterns at the hypothalamo-pituitary-gonadal axis (HPG axis) of sslpxrfa and sslpxrfa-r mRNA were also investigated during gonadal development of spotted scat. The open reading frame (ORF) of the sslpxrfa was 606 bp encoding 201 amino acids and includes a putative signal peptide and two mature ssLpxrfa peptides with LPXRFamide motif at their C-terminus. The sslpxrfa-r ORF was 1449 bp encoding 482 amino acids and contracted a seven-hydrophobic transmembrane (TM) domain structure. The tissue distribution showe d that the sslpxrfa was highly expressed in hypothalami, gill, and the gonads. In addition, sslpxrfa-r was highly expressed in hypothalami, pituitaries, and the gonads. Quantitative real-time polymerase chain reaction (qPCR) revealed that sslpxrfa had the highest expression in the hypothalami and pituitaries, and the lowest expression in the gonads in stage V. During gonadal development, the expression of sslpxrfa-r was gradually increased in the hypothalami but reduced in the gonads. However, no obvious trend was observed in the pituitaries. The expression of sslpxrfa and sslpxrfa-r decreased significantly after injection with 17ß-estradiol (E2). However, the expression of both sslpxrfa and sslpxrfa-r was not changed after injection with 17α-methyltestosterone(17α-MT) in the hypothalami. In addition, no changes were observed in the expression of fshß and lhß in the pituitaries after injecting ssLpxrfa-1. However, ssLpxrfa-2 could downregulate the expression of sbgnrh and fshß in the hypothalami and pituitaries, respectively. Taken together, these findings suggested that ssLpxrfa may participate in E2 feedback in reproduction and regulate the reproductive axis of spotted scat.

Controlled release of sex steroids through osmotic pump alters brain GnRH1 and catecholaminergic system dimorphically in the catfish, Clarias gariepinus.

The present study aimed to evaluate osmotic pump-mediated controlled release of estrogen in males and androgen in females to analyze the impact on gonadotropin-releasing hormone (GnRH1), catecholamines (CAs) and other associated genes in the catfish, Clarias gariepinus. During pre-spawning phase, catfish were separately implanted osmotic pumps loaded with 17ß-estradiol (E2) in males and 17α-methyltestosterone (MT) in females at a dose of 10 µg/100 µl or saline (100 µl) controls into both sexes to release for 21 days and all fishes were maintained as per the duration. Further, GnRH1 expression levels were analysed in the discrete regions of brain after E2 and MT treatments in male and female catfish, respectively using qPCR which revealed that GnRH1 expression was significantly higher in E2 treated male as compared to the control. On the other hand, GnRH1 expression was lower in MT treated female when compared to the control in the discrete regions of brain. In addition, certain brain and monoaminergic system related genes showed a differential response. Catfish GnRH1 could be localized in preoptic area-hypothalamus (POA-HYP) that correlated with the expression profile in the discrete regions of catfish brain. Serum levels of sex steroids in the treated male fish indicated that the treatment of E2 could maintain and impart feminization effect even in the presence of endogenous androgen during gonadal recrudescence while such an effect was not seen in females with androgen treatment. Measurement of CAs, L-3,4-dihydroxyphenylalanine, dopamine and norepinephrine levels in the male and female brain after the controlled release of E2 and MT, respectively confirmed the modulation of neurotransmitters in the E2treated male than MT treated female fish. These results collectively suggest the severity of estrogenic over androgenic compounds to alter reproductive status even at a minimal dose by targeting CAs and GnRH1 at the level of brain of catfish. This study provides insights into the reproductive toxicity of sex steroid analogues at the level of brain GnRH1 and CA-ergic system in addition to serum T, 11-KT and E2 levels during gonadal recrudescence, which is a crucial period of gametogenesis preceding spawning.