Although the essential role of estrogen in ovarian differentiation has been documented in several species of fish, this remains unknown in allotetraploid common carp (Cyprinus carpio). Here, the gonads from common carp with the cytochrome P450 family 17 subfamily A member 1 (cyp17a1)- /- genotype (knockout, KO), which exhibited complete testicular differentiation regardless of their genetic sex determination (GSD) system (XX or XY), were analyzed for transcriptional profiles at various stages during gonadal differentiation. At 25, 32 and 60 days posthatching (dph), the doublesex and mab-3 related transcription factor 1 (dmrt1)-ovarian aromatase (cyp19a1a) loop pattern could be clustered into high dmrt1-low cyp19a1a and low dmrt1-high cyp19a1a clusters, depending on the presence or absence of the male-determining gene on the Y chromosome (mdy) in genetic male and female cyp17a1+/ + carp (Control, Con), respectively. The transcriptional profile of KO;XY carp continuously exhibited similar patterns to those of Con;XY carp in the dmrt1-cyp19a1a loop (high dmrt1-low cyp19a1a). However, the gene expression profiles of KO;XX carp deviated from those of Con;XX carp and skewed towards those of Con;XY and KO;XY carp from 25 to 60 dph, and clustered with those of Con;XY carp at 90 dph. At the same stage from 25 to 60 dph, Con;XX exhibited upregulated meiosis-related genes, including synaptonemal complex central element protein 2 (syce2), REC8 meiotic recombination protein b (rec8b), and DNA meiotic recombinase 1 (dmc1), among which only dmc1 was observed to be upregulated in KO;XX carp. Administering 17(3-estradiol restored ovarian differentiation in KO;XX carp. Antagonism between Dmrt1 and cyp19a1a/17(3-estradiol was observed in fish gonads due to a direct interaction between Dmrt1 and Steroidogenic factor 1 (Sf-1), whereby Dmrt1 repressed Sf1's ability to activate the cyp19a1aA and cyp19a1aB promoters. The Con;XX fish at 90 dph exhibited comprehensively advanced meiotic processes. We uncovered the genetic basis of a two-phase checkpoint regulatory mechanism of the putative mdy and estrogen sufficiency and elucidated the association between dmrt1 and estrogen signaling in sustaining gonadal differentiation and the meiotic process in juvenile common carp during the period of 32-90 dph.

Sexual dimorphism in growth has been reported in mandarin fish leading to a desire for monosex populations in aquaculture farming. The neomale individuals which were physiological males with chromosome genotype XX were prepared through sex reversal using 17 alpha-methyltestosterone (17 alpha-MT). Additionally letrozole as an aromatase inhibitor was also employed for sex reversal in economically important teleost species. This study aimed to compare the masculinization effect of 17 alpha-MT and letrozole and then elucidate the mechanisms of natural sex determination. The sex reversal rate of 17 alpha-MT (100 mg/kg feed)and letrozole (200 mg/kg feed) had no significant difference. However, the gonadosomatic index (GSI) of the letrozole administration group was elevated by 93% compared with the 17 alpha-MT group. Through comparative transcriptomics analysis the expression of amh was repressed in the gonad following 17 alpha-MT administration in the adult stage. The plasma estrogen (E2) content and the expression of cyp19a1a gene were elevated in the 17 alpha-MT administration group. These results suggested that 17 alpha-MT administration plays a dual role in both dmrt1 stimulation and estrogen synthesis activation, which was insufficient to fully repress estrogen signaling in 17 alpha-MT neomales. The masculine sex reversal process was more effective with the aromatase inhibitor letrozole. Furthermore, both dmrt1 and amh expression and cooperation could be induced by letrozole treatment. Although the female marker Foxl2 was transcriptionally activated at the juvenile stage during letrozole treatment, its expression was restricted at the adult stage. Collectively, our data and practice suggest that the aromatase inhibitor letrozole is superior to the 17 alpha-MT in the masculine sex reversal of mandarin fish. Suppression of estrogen synthesis was sufficient to induce male determination which in turn induces the cooperation of dmrt1 and amh.

Insulin-like growth factor 1 (IGF-1) signaling plays a complementary role to insulin signaling in glucose metabolism homeostasis. This study characterized the physiological roles of the IGF1 receptor A (Igf1ra) and B (Igf1rb) in zebrafish. The transcripts of igf1ra and igf1rb were detected in multiple zebrafish tissues, including the liver, muscle, and brain. Zebrafish lacking igf1ra or igf1rb were generated using CRISPR/Cas9 technology. Both igf1ra-/- and igf1rb-/- zebrafish exhibited stunted growth. Reduced BMI was found in igf1ra-/- zebrafish, while BMI increased in igf1rb-/- zebrafish. Hyperglycemia and increased hepatic glycogen were observed in igf1ra-/- zebrafish, while blood glucose levels in igf1rb-/- zebrafish were normal. No significant difference in whole-body or hepatic triglyceride content was observed in igf1ra-/- zebrafish, while the whole-body and hepatic triglyceride content of igf1rb-/- zebrafish increased compared to their wild-type control siblings. Further analyses of the expression patterns of key genes involved in glucose and lipid metabolism were conducted on igf1r mutants. Decreased levels of genes involved in glucose absorption and glycolysis and increased levels of genes involved in gluconeogenesis and glycogen synthesis were observed in igf1ra-/- zebrafish, but not in igf1rb-/- zebrafish. Conversely, significantly decreased levels of transcripts involved in lipolysis and increased levels of transcripts involved in the lipogenesis process were observed in igf1rb-/- zebrafish, but not in igf1ra-/- zebrafish. Restricted cell growth and protein synthesis signaling, including AKT and mTOR activation, was also detected in igf1ra-/- zebrafish, while a moderate elevation in AKT and mTOR activity was seen in igf1rb-/- zebrafish. Taken together, our results suggest that functional divergence occurred after the duplication of the zebrafish igf1r gene, with igf1ra primarily modulating glucose absorption and utilization, and igf1rb primarily affecting lipid metabolism in the somatotropic axis.

In teleosts, gonadal differentiation is a complex and precisely controlled process that requires a proper balance of androgen and estrogen signaling activities. However, the interaction between these steroid signals remains unclear, particularly during the critical stage that determines the sexual fate of the bipotential gonad in zebrafish. In the present study, we investigated the expression of cyp19a1a and foxl2, the amount of primordial germ cells (PGCs), and the status of ovarian differentiation, in response to 17β-estradiol (E2) and 11-ketotestosterone (11-KT) using the Tg(piwil1:EGFP-nanos3’UTR) zebrafish line. The expression of cyp19a1a, foxl2a, foxl2b and foxl2l was significantly higher in the PGC-rich group reared in control medium (CM) at 23 days post-fertilization (dpf) than in the PGC-less group. Treatment with E2 promoted ovarian differentiation in 83% of individuals and increased foxl2 expression. Conversely, 11-KT treatment caused testis-biased differentiation in 86% of individuals and decreased foxl2 and cyp19a1a expression at the same stage. Significant differences were observed when the number of PGCs labeled with green fluorescent protein (GFP) in E2- and 11-KT-treated fish were compared. Furthermore, the inhibitory effect of 11-KT on the expression of foxl2 and ovarian differentiation, which was Androgen receptor (Ar)-dependent, could be completely counteracted in wild-type (WT) fish when E2 was co-administered. In summary, our results showed that estrogen signaling is sufficient to counteract the effect of androgen/Ar signaling on upregulating foxl2 expression and promoting ovarian development and differentiation.

The intrinsic regulatory network governing the somatic environment and germ cell sexual fate remains elusive. Here, we performed Switching Mechanism at the 5’ end of RNA transcript sequencing (SMART-Seq) on samples of cytochrome p450 family 17 subfamily a member 1 (cyp17a1)-deficient zebrafish at 13 days post-fertilization (dpf), which exhibits an all-testicular differentiation. The integrative analysis of SMART-Seq and RNA-Seq suggested that zinc finger gata like protein 1 (zglp1), rec8 meiotic recombination protein a (rec8a) and meiosis initiator (meiosin) are three putative factors responsible for all-testicular differentiation due to estradiol-insufficiency in cyp17a1-deficient fish, and that zglp1-, rec8a- or meiosin-deficiency also caused all-testicular differentiation. In contrast to estradiol administration, tumor protein 53 (tp53)-depletion restored ovarian differentiation and oocyte development in rec8a-deficient fish, which also exhibited the ability to spawn, despite their impaired fertility as evidenced by the abnormal development of fertilized eggs. The all-testicular differentiation phenotype of the zglp1-deficient fish was accompanied by meiosis delay and germ cell apoptosis, which could be rescued by tp53-depletion or estradiol administration. The promoters of rec8a and meiosin were activated by Zglp1. In addition, zglp1, rec8a, and meiosin were upregulated by estradiol signaling and Forkhead Box L2 (Foxl2) in vitro. Mechanistically, we showed that zglp1 was more highly expressed in ovaries than testes at 3 months post fertilization (mpf), upregulated in primordial germ cell (PGC)-rich compared to PGC-less fish at 23 dpf, induced by exogenous estradiol, and inversely correlated with the male marker doublesex and mab-3 related transcription factor 1 (dmrt1). Collectively, these findings demonstrate that zglp1 is positively regulated by estradiol signaling, negatively associated with male differentiation, and critical for female germ cell development. Tamoxifen (TAM), the antagonist of estrogen receptor (Esr), significantly inhibited the expression of zglp1, rec8a and meiosin in zebrafish. In summary, we demonstrated that estradiol signaling promotes female germ cell fate and ovarian differentiation through a Zglp1-dependent regulatory pathway, and that this pathway is associated with activation of the meiotic program involving rec8a and meiosin.

Teleosts are excellent models for elucidating the regulatory mechanisms of gonadal differentiation. Although the essential role of ovarian aromatase (encoded by cyp19a1a) in ovarian differentiation has been documented in several species of fish, this remains poorly understood in common carp (Cyprinus carpio) due to its long period of sexual maturity and the complexity of the multiple loci in its allotetraploid genome. Here, using the CRISPR/ Cas9 strategy, some neomale carp with chimeric mutation (CM) of cyp19a1aA, cyp19a1aB or cyp19a1aA; cyp19a1aB were found in the F0 population at 1 year post-hatching (yph). The mutation ratios of cyp19a1aA, cyp19a1aB, or cyp19a1aA;cyp19a1aB were positively correlated with the ratio of sex reversal from female to male. Concentrations of estradiol were significantly decreased in female and neomale of chimaeras carp compared to wild-type (WT) control female carp. The progeny from artificial fertilization between WT female carp and these neomale carp were confirmed as all-female (AF) carp, as evidenced by genetic identification of male-specific DNA marker and histological analysis of progeny gonads. Finally, in comparison to the control group, which comprised both male and female carp reared in the same pond, AF carp at 4 months post-hatching (mph) demonstrated enhanced 12.06 % growth performance. In conclusion, we have shown that both Cyp19a1aA and Cyp19a1aB are required for estrogen synthesis, which is essential for ovarian differentiation in common carp. Therefore, a rapid method to generate AF populations using the F0 CM populations has been established by genetic manipulation targeting the loci of the cyp19a1a genes. The present study has revealed the non-redundant roles of cyp19a1aA and cyp19a1aB in ovarian differentiation and also provides an efficient and rapid method of controlling gonadal differentiation in fish without knowledge of the specific sex-determining gene (SDG) of the particular fish.

Chorionic gonadotropin α（Cgα） functions as the shared subunit for thyroid-stimulating hormone subunit β（Tshβ）,luteinizing hormone subunit β（Lhβ）, and follicle-stimulating hormone subunit β（Fshβ）. While these β-subunits have been extensively studied using effective gene knockout models in zebrafish, the biological role of Cgα remains elusive. In this study, cgα-deficient zebrafish generated via transcription activator-like effector nucleases（TALENs）exhibited viability but displayed pronounced developmental abnormalities, including growth retardation,hyperpigmentation, reduced thyroxine（T4） levels, and defective anterior swim bladder inflation during juvenile stages. In adults, cgα deficiency led to disrupted gonadal development, impaired secondary sex characteristics（SSCs）, and severely impacted reproductive behavior in both female and male fish. Notably, both testicular and ovarian differentiation were observed in cgα-deficient fish and lhβ -/- ;fshβ -/- mutants. Gonadal sex differentiation in cgα-deficient zebrafish exhibited a pronounced shift toward testicular fate upon additional disruption of fshβ(cgα -/- ;fshβ -/- ), marked by elevated anti-Müllerian hormone（amh） expression, or following loss of follicle-stimulating hormone receptor（fshr）(cgα -/- ;fshr -/- ). In vitro assays in Chinese hamster ovary（CHO） cells revealed increased cAMP response element（CRE） promoter activity following transfection with constructs encoding Fshr, Fshβ/Fshr, or Cgα/Fshβ/Fshr. Collectively, the phenotypes observed in cgα-deficient fish recapitulate those of thyrotropin-and gonadotropin-disrupted models, highlighting the essential role of Cgα in thyroid and gonadal function. Importantly,these findings uncover the role of Fsh signaling in maintaining proper ovarian differentiation in zebrafish,including Cgα-independent Fshβ activity and the constitutive functionality of Fshr.

The mechanism of fish gonadal sex differentiation is complex and regulated by multiple factors. It has been widely known that proper steroidogenesis in Leydig cells and sex-related genes in Sertoli cells play important roles in gonadal sex differentiation. In teleosts, the precise interaction of these signals during the sexual fate determination remains elusive, especially their effect on the bi-potential gonad during the critical stage of sexual fate determination. Recently, all-testis phenotypes have been observed in the cyp17a1-deficient zebrafish and common carp, as well as in cyp19a1a-deficient zebrafish. By mating cyp17a1-deficient fish with transgenic zebrafish Tg(piwil1:EGFP-nanos3UTR), germ cells in the gonads were labelled with enhanced green fluorescent protein (EGFP). We classified the cyp17a1-deficient zebrafish and their control siblings into primordial germ cell (PGC)-rich and -less groups according to the fluorescence area of the EGFP labelling. Intriguingly, the EGFP-labelled bi-potential gonads in cyp17a1+/+ fish from the PGC-rich group were significantly larger than those of the cyp17a1−/− fish at 23 days post-fertilization (dpf). Based on the transcriptome analysis, we observed that the cyp17a1-deficient fish of the PGC-rich group displayed a significantly upregulated expression of amh and gsdf compared to that of control fish. Likewise, the upregulated expressions of amh and gsdf were observed in cyp19a1a-deficient fish as examined at 23 dpf. This upregulation of amh and gsdf could be repressed by treatment with an exogenous supplement of estradiol. Moreover, tamoxifen, an effective antagonist of both estrogen receptor α and β (ERα and Erβ), upregulates the expression of amh and gsdf in wild-type (WT) fish. Using the cyp17a1- and cyp19a1a-deficient zebrafish, we provide evidence to show that the upregulated expression of amh and gsdf due to the compromised estrogen signaling probably determines their sexual fate towards testis differentiation. Collectively, our data suggest that estrogen signaling inhibits the expression of amh and gsdf during the critical time of sexual fate determination, which may broaden the scope of sex steroid hormones in regulating gonadal sex differentiation in fish.

Testosterone is closely associated with lipid metabolism and known to affect body fat composition and muscle mass in males. However, the mechanisms by which testosterone acts on lipid metabolism are not yet fully understood, especially in teleosts. In this study, cyp17a1-/- zebrafish ( Danio rerio) exhibited excessive visceral adipose tissue (VAT), lipid content, and up-regulated expression and activity of hepatic de novo lipogenesis (DNL) enzymes. The assay for transposase accessible chromatin with sequencing (ATAC-seq) results demonstrated that chromatin accessibility of DNL genes was increased in cyp17a1-/- fish compared to cyp17a1+/+ male fish, including stearoyl-CoA desaturase ( scd) and fatty acid synthase ( fasn). Androgen response element (ARE) motifs in the androgen signaling pathway were significantly enriched in cyp17a1+/+ male fish but not in cyp17a1-/- fish. Both androgen receptor ( ar)-/- and wild-type (WT) zebrafish administered with Ar antagonist flutamide displayed excessive visceral adipose tissue, lipid content, and up-regulated expression and activity of hepatic de novo lipogenesis enzymes. The Ar agonist BMS-564929 reduced the content of VAT and lipid content, and down-regulated acetyl-CoA carboxylase a ( acaca), fasn, and scd expression. Mechanistically, the rescue effect of testosterone on cyp17a1-/- fish in terms of phenotypes was abolished when ar was additionally depleted. Collectively, these findings reveal that testosterone inhibits lipid deposition by down-regulating DNL genes via Ar in zebrafish, thus expanding our understanding of the relationship between testosterone and lipid metabolism in teleosts.

Natural and synthetic environmental estrogens (EEs) are widespread and have received extensive attention. Our previous studies demonstrated that depletion of the cytochrome P450 17a1 gene (cyp17a1) leads to all-testis differentiation phenotype in zebrafish and common carp. In the present study, cyp17a1-deficient zebrafish with defective estrogen biosynthesis were used for the evaluation of EEs, as assessed by monitoring vitellogenin (vtg) expression. A rapid and sensitive assessment procedure was established with the 3-day administration of estradiol (E2), followed by examination of the transcriptional expression of vtgs in our cyp17a1-deficient fish. Compared with the control fish, a higher E2-mediated vtg upregulation observed in cyp17a1-deficient zebrafish exposed to 0.1 μg/L E2 is known to be estrogen receptor-dependent and likely due to impaired in vivo estrogen biosynthesis. The more responsive vtg expression in cyp17a1-deficient zebrafish was observed when exposed to 200 and 2000 μg/L bisphenol A (BPA) and perfluoro-1-octanesulfonate (PFOS). The estrogenic potentials of E2, BPA, and PFOS were compared and assessed by the feminization effect on ovarian differentiation in cyp17a1-deficient zebrafish from 18 to 50 days postfertilization, based on which a higher sensitivity of E2 in ovarian differentiation than BPA and PFOS was concluded. Collectively, through the higher sensitivity to EEs and the capacity to distinguish chemicals with different estrogenic potentials exhibited by the all-male cyp17a1-deficient zebrafish with impaired estrogen biosynthesis, we demonstrated that they can be used as an excellent in vivo model for the evaluation of EEs. Environ Toxicol Chem 2024;43:1062-1074. © 2024 SETAC.

The regulatory mechanism of gonadal sex differentiation, which is complex and regulated by multiple factors, remains poorly understood in teleosts. Recently, we have shown that compromised androgen and estrogen synthesis with increased progestin leads to all-male differentiation with proper testis development and spermatogenesis in cytochrome P450 17a1 (cyp17a1)-/- zebrafish. In the present study, the phenotypes of female-biased sex ratio were positively correlated with higher Fanconi anemia complementation group L (fancl) expression in the gonads of doublesex and mab-3 related transcription factor 1 (dmrt1)-/- and cyp17a1-/-;dmrt1-/- fish. The additional depletion of fancl in cyp17a1-/-;dmrt1-/- zebrafish reversed the gonadal sex differentiation from all-ovary to all-testis (in cyp17a1-/-;dmrt1-/-;fancl-/- fish). Luciferase assay revealed a synergistic inhibitory effect of Dmrt1 and androgen signaling on fancl transcription. Furthermore, an interaction between Fancl and the apoptotic factor Tumour protein p53 (Tp53) was found in vitro. The interaction between Fancl and Tp53 was observed via the WD repeat domain (WDR) and C-terminal domain (CTD) of Fancl and the DNA binding domain (DBD) of Tp53, leading to the K48-linked polyubiquitination degradation of Tp53 activated by the ubiquitin ligase, Fancl. Our results show that testis fate in cyp17a1-/- fish is determined by Dmrt1, which is thought to stabilize Tp53 by inhibiting fancl transcription during the critical stage of sexual fate determination in zebrafish.

The implementation of a controllable sterility strategy is crucial for the commercialization of precise trait improvements in farmed fish using genome editing and sustainable development of fisheries. Our previous research has demonstrated that females deficient in pituitary gonadotropin luteinizing hormone β-subunit (lhβ) or gonadal steroidogenesis gene steroidogenic acute regulatory protein (star) exhibit sterility due to impaired oocyte maturation and ovulation. Nevertheless, the effective restoration of fertility in lhβ- or star-deficient females remains unsolved. This study has discovered that the administration of exogenous 17α,20β-dihydroxy-4-pregnen-3-one (DHP) at 100 and 300 μg/L for 6 h (from 02:00 to 08:00 a.m.) effectively restores the fertility of lhβ- or star-deficient females. Fertilized eggs from these mutant females can be raised without noticeable developmental defects for up to 3 weeks post-fertilization (wpf) compared to the wild-type (WT) control zebrafish. The increased expression levels of adamts9 and adam8b in lhβ- or star-deficient zebrafish females treated with DHP demonstrate a positive correlation with oocyte maturation and ovulation restoration. In contrast, exogenous DHP administration did not rescue the sterility phenotype observed in progesterone receptor (pgr)-deficient females. Building on our recent success in generating an all-female carp population through cytochrome P450, family 17, subfamily A, polypeptide 1 (cyp17a1)-depletion, our research presents a promising and effective strategy for an “off-on” switch for managing fertility in genome-edited cyprinids. The strategy would offer practical guidance and theoretical justification for developing “controllable fertility” in all-female fish, which would support the sustainable development of fisheries by promoting the use of novel biotechnologies in aquaculture in an eco-friendly manner.

Introduction1α,25-dihydroxyvitamin D3 (1α,25[OH]2VD3) is a hormone known for its key roles in calcium absorption and nutrient metabolism. In teleost fishes, 1α,25(OH)2VD3 insufficiency causes impaired glucose metabolism and lipid oxidation. However, the cascade and mechanisms of 1α,25(OH)2VD3 and the vitamin d receptor (VDR) signaling are unclear.ResultsIn this study, two genes (vdra and vdrb) encoding paralogs of VDRs were genetically knocked out in zebrafish. Growth retardation and accumulated visceral adipose tissue have been observed in vdra-/-;vdrb-/- deficient line. In the liver elevated accumulation of triglycerides and suppressed lipid oxidation were detected. Morover significantly elevated 1α,25(OH)2VD3 levels were detected in vdra-/-;vdrb-/- zebrafish due to cyp24a1 transcription repression. Furthermore VDRs ablation Enhanced insulin signaling including elevated insulin/insra trancriptional levels, glycolysis, lipogenesis and promoted AKT/mTOR activity.DiscussionIn conclusion, our present studies provides a zebrafish model with an elevated 1α,25(OH)2VD3 levels in vivo. The 1α,25(OH)2VD3/VDRs signaling promote lipid oxidation activity. However 1α,25(OH)2VD3 activity of regulation of glucose homeostasis through Insulin/Insr was independent of nuclear VDRs in teleosts.

Insulin-like growth factor 1 (IGF1) is an essential effector of the growth hormone (GH)/IGF1 axis for somatic growth regulation in mammals. However, its functions have not been thoroughly investigated in zebrafish in vivo. In this study, the igf1-deficient zebrafish model was developed using the CRISPR/Cas9 technique. In this study all the results were performed on both male and female animals. The growth of both male and female igf1-deficient zebrafish were reduced. The igf1 deficiency leads to significant complementary up-regulation of transcriptional expression levels of insulin, igf2 and igf3. This suggested that igf2 and igf3 may act with redundant functions. While the upregulation of gh1 expression can only be detected in igf1-deficient females. At the same time, significant growth retardation, fatty liver, reduced activated levels of ribosomal S6 (S6) are seen only in igf1-deficient males. On the other hand, significant hyperglycemia, elevated transcriptional expression levels of phosphenolpyruvate carboxykinase (pepck) and levels of phosphorylated extracellular signal-regulated kinase (ERK1/2), with additional reduced hepatic lactate/pyruvate (L/P) ratios can only observed in igf1-deficient females. Impaired glucose uptake has been recorded in the primary cultured hepatocytes from igf1-deficient females, but not males. Intriguingly, exposure to 17beta-estroadiol (E2) can partially ameliorated the defects of fatty liver and activation of AKT/mTOR signaling in igf1-deficient males. Our studies demonstrate the significant functions of IGF1 on somatic regulation in zebrafish, with asymmetric gender-related consequences. Our data thus suggest that the zebrafish IGF1 is preferentially required for the activation of AKT/mTOR signaling in male zebrafish, but glucose uptake in females.

Unlike the Cytochrome P450, family 17, subfamily A, member 1 (Cyp17a1), which possesses both 17α-hydroxylase and 17,20-lyase activities involved in the steroidogenic pathway that produces androgens and estrogens, Cytochrome P450, family 17, subfamily A, polypeptide 2 (Cyp17a2) possesses only 17α-hydroxylase activity and is known essential for the synthesis of cortisol. Besides with expressed in testes and ovaries, where the cyp17a1 is mainly expressed, cyp17a2 is also expressed in the interrenal gland in fish. Until now, the roles of cyp17a2 in fish, especially in sexual traits development and hypothalamic-pituitary-interrenal (HPI) axis, are poorly studied. To investigate the roles of Cyp17a2 in teleosts, the cyp17a2 -null zebrafish was generated and analyzed by us. The significantly decreased cortisol concentration was observed both in the cyp17a2 -deficient males and females at adult stage. The interrenal gland enlargement, increased pituitary proopiomelanocortin a ( pomca ) expression, decreased locomotion activity and response to light-stimulated stress were observed in cyp17a2 -deficient fish. Intriguingly, the cyp17a2 -deficient males were fertile and with normal breeding tubercles on the pectoral fin, but females were infertile, deficient in genital papilla and with decreased gonadosomatic index (GSI). The increased progesterone (P4), 17α,20β-dihydroxy-4-pregnen-3-one (DHP) and 11-ketotestosterone (11-KT) in the cyp17a2 -deficient males and females were observed. The increased concentration of testosterone (T) and estradiol (E2) was observed in cyp17a2 -/- females and cyp17a2 -/- males, respectively. By examining the ovaries development of cyp17a2 -deficient fish at 3 months postfertilization (mpf), we observed that the oocytes were over-activated. Taken together, our findings demonstrate that Cyp17a2 is indispensable for production and physiology of cortisol, and cyp17a2 -deficiency resulted in diminished cortisol but accumulated P4 and DHP, which may result in the over-activated oocytes in cyp17a2 -deficient females.

Disruption of androgen signaling is known to cause testicular malformation and defective spermatogenesis in zebrafish. However, knockout of cyp17a1, a key enzyme responsible for the androgen synthesis, in ar-/- male zebrafish paradoxically causes testicular hypertrophy and enhanced spermatogenesis. Because Cyp17a1 plays key roles in hydroxylation of pregnenolone and progesterone (P4), and converts 17α-hydroxypregnenolone to dehydroepiandrosterone and 17α-hydroxyprogesterone to androstenedione, we hypothesize that the unexpected phenotype in cyp17a1-/-;androgen receptor (ar)-/- zebrafish may be mediated through an augmentation of progestin/nuclear progestin receptor (nPgr) signaling. In support of this hypothesis, we show that knockout of cyp17a1 leads to accumulation of 17α,20β-dihydroxy-4-pregnen-3-one (DHP) and P4. Further, administration of progestin, a synthetic DHP mimetic, is sufficient to rescue testicular development and spermatogenesis in ar-/- zebrafish, whereas knockout of npgr abolishes the rescue effect of cyp17a1-/- in the cyp17a1-/-;ar-/- double mutant. Analyses of the transcriptomes among the mutants with defective testicular organization and spermatogenesis (ar-/-, ar-/-;npgr-/- and cyp17a-/-;ar-/-;npgr-/-), those with normal phenotype (control and cyp17a1-/-), and rescued phenotype (cyp17a1-/-;ar-/-) reveal a common link between a downregulated expression of insl3 and its related downstream genes in cyp17a-/-;ar-/-;npgr-/- zebrafish. Taken together, our data suggest that genetic or pharmacological augmentation of the progestin/nPgr pathway is sufficient to restore testis organization and spermatogenesis in zebrafish with the depletion of androgen signaling.

为研究雄激素受体(Androgen receptor,Ar)在卵巢发育中的作用机制,文章以雄性激素受体敲除的雌性斑马鱼(Daniorerio;ar-/-)为研究对象,利用ELISA和蛋白质免疫印迹等方法分析Ar对斑马鱼肝脏中卵黄蛋白原(Vitellogenin,Vtg)产生、母体营养通过Vtg运输、卵黄形成和卵巢成熟的影响.研究发现在ar-/-雌性斑马鱼的肝脏中,Vtg的产生和雌激素受体的表达水平显著降低.在ar-/-雌性斑马鱼中的卵巢质量、脂质含量和类胡萝 卜素含量均显著下降,表明Ar的缺失可导致雌性斑马鱼通过Vtg转运到卵巢的脂质和类胡萝 卜素等营养物质供应减少.此外,ar-/-雌性斑马鱼中与卵巢发育相关的几个基因转录表达水平显著下调,这与卵巢发育异常存在相关性.研究结果表明,Ar的缺乏可通过对肝脏中的雌激素受体表达的影响,降低肝脏Vtg蛋白的合成,从而损害由Vtg向卵巢的营养物质运输和卵黄的正常形成,影响卵巢发育.研究阐明了雄激素信号通路与体内肝脏Vtg产生之间的联系.

Human-directed domestication of terrestrial animals traditionally requires thousands of years for breeding. The most prominent behavioral features of domesticated animals include reduced aggression and enhanced tameness relative to their wild forebears, and such behaviors improve the social tolerance of domestic animals toward both humans and crowds of their own species. These behavioral responses are primarily mediated by the hypothalamic-pituitary-adrenal (inter-renal in fish) (HPA/I) endocrine axis, which is involved in the rapid conversion of neuronal-derived perceptual information into hormonal signals. Over recent decades, growing evidence implicating the attenuation of the HPA/I axis during the domestication of animals have been identified through comprehensive genomic analyses of the paleogenomic datasets of wild progenitors and their domestic congeners. Compared with that of terrestrial animals, domestication of most farmed fish species remains at early stages. The present review focuses on the application of HPI signaling attenuation to accelerate the domestication and genetic breeding of farmed fish. We anticipate that deeper understanding of HPI signaling and its implementation in the domestication of farmed fish will benefit genetic breeding to meet the global demands of the aquaculture industry.