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Reverse genetics approaches are critical for uncovering complex biological processes and genetic engineering. Clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) system (CRISPR/Cas9) has been widely used for generating mutants in a large number of species. The Nile tilapia (Oreochromis niloticus), a gonochoristic teleost with XX/XY sex determination system, is a good model for studying fish sex determination and reproduction. Here, we describe a detailed procedure for gene mutation in tilapia by CRISPR/Cas9. This protocol covers selection of target sites, in vitro RNA transcription, artificial insemination and microinjection of one cell stage embryos. We also provide details for detection of somatic and germline transmitted mutations, fast establishment of mutant lines and discuss some practical advices. This protocol will facilitate broader applications of CRISPR/Cas9 in studies of tilapia as well as other aquaculture fishes. [ABSTRACT FROM AUTHOR]

• Establishment of gene editing technologies in tilapia enhances our knowledges on fish reproductive endocrinoogy. • Transcription factors Dmrt1 and Foxl3 are key factors involved in germ cell fate decision and sexual plasticity. • Blockage of estrogen synthesis rescues the male to female sex reversal of amhy and gsdf but not dmrt1 mutants. • The deficiency of steroid hormones, such as androgen, DHP and cortisol, impairs reproduction as revealed by gene editing. The Nile tilapia (Oreochromis niloticus), with a system of XX/XY sex determination, is a worldwide farmed fish with a shorter sexual maturation time than that of most cultured fish. Tilapia show a spawning cycle of approximately 14 days and can be artificially propagated in the laboratory all year round to obtain genetically all female (XX) and all male (XY) fry. Its genome sequence has been opened, and a perfect gene editing platform has been established. With a moderate body size, it is convenient for taking enough blood to measure hormone level. In recent years, using tilapia as animal model, we have confirmed that estrogen is crucial for female development because 1) mutation of star2 , cyp17a1 or cyp19a1a (encoding aromatase, the key enzyme for estrogen synthesis) results in sex reversal (SR) due to estrogen deficiency in XX tilapia, while mutation of star1 , cyp11a1 , cyp17a2 , cyp19a1b or cyp11c1 affects fertility due to abnormal androgen, cortisol and DHP levels in XY tilapia; 2) when the estrogen receptors (esr2a / esr2b) are mutated, the sex is reversed from female to male, while when the androgen receptors are mutated, the sex cannot be reversed; 3) the differentiated ovary can be transdifferentiated into functional testis by inhibition of estrogen synthesis, and the differentiated testis can be transdifferentiated into ovary by simultaneous addition of exogenous estrogen and androgen synthase inhibitor; 4) loss of male pathway genes amhy , dmrt1 , gsdf causes SR with upregulation of cyp19a1a in XY tilapia. Disruption of estrogen synthesis rescues the male to female SR of amhy and gsdf but not dmrt1 mutants; 5) mutation of female pathway genes foxl2 and sf-1 causes SR with downregulation of cyp19a1a in XX tilapia; 6) the germ cell SR of foxl3 mutants fails to be rescued by estrogen treatment, indicating that estrogen determines female germ cell fate through foxl3. This review also summarized the effects of deficiency of other steroid hormones, such as androgen, DHP and cortisol, on fish reproduction. Overall, these studies demonstrate that tilapia is an excellent animal model for studying reproductive endocrinology of fish. [ABSTRACT FROM AUTHOR]

The deficiency or insufficiency of androgen can trigger a range of reproductive diseases as well as other symptoms. Stem Leydig cells (SLCs) are critical for the formation and maintenance of a functional androgen-producing cell (Leydig cell, LC) population throughout adult male life. However, to date, our knowledge about SLCs is poor. Here we report the derivation and characterisation of a clonal stem LC line (designated as TSL) capable of 11- ketotestosterone (11-KT) production from a 3-month-old Nile tilapia (Oreochromis niloticus) testis. The cells retained stable proliferation after 77 generations with normal karyotype and growth factor dependency. They expressed platelet-derived growth factor receptor-α (pdgfr α), nestin and chicken ovalbumin upstream promoter transcription factor II (coup - tfIIa), which are characteristic of SLCs. Upon induction in defined medium, TSLs could undergo differentiation into steroidogenically active LCs and produce 11-KT. When implanted into recipient Nile tilapia testes from which endogenous LCs had been eliminated by ethane dimethanesulphonate (EDS) treatment, the PKH26-labelled TSLs could colonise the interstitium, subsequently express steroidogenic genes and restore 11-KT production. Taken together, our data suggest that TSLs possess the ability of continuous proliferation and potential of differentiation into functional LCs in vitro and in vivo. To the best of our knowledge TSL might represent the first stem LC line capable of 11-KT production to date. Our study may offer new opportunities for investigating the self-renewal of SLCs and steroidogenesis in vitro , and provide an invaluable in vitro model for investigating endocrine disruptors. Stem Leydig cells are critical for the formation and maintenance of a functional androgen-producing cell (Leydig cell) population throughout adult male life. A clonal stem Leydig cell line capable of androgen production from 3-month-old Nile tilapia testis has been successfully established. This cell line may offer new opportunities for investigating the self-renewal of stem Leydig cells and steroidogenesis in vitro , and also provide an invaluable in vitro model for screening and investigating endocrine disruptors. [ABSTRACT FROM AUTHOR]

A novel insulin-like growth factor (igf3), which is exclusively expressed in the gonads, has been widely identified in fish species. Recent studies have indicated that Igf3 regulates spermatogonia proliferation and differentiation in zebrafish; however, detailed information on the role of this Igf needs further in vivo investigation. Here, using Nile tilapia (Oreochromis niloticus) as an animal model, we report that igf3 is required for spermatogenesis and reproduction. Knockout of igf3 by CRISPR/Cas9 severely inhibited spermatogonial proliferation and differentiation at 90 days after hatching, the time critical for meiosis initiation, and resulted in less spermatocytes in the mutants. Although spermatogenesis continued to occur later, more spermatocytes and less spermatids were observed in the igf3−/− testes when compared with wild type of testes at adults, indicating that Igf3 regulates spermatocyte to spermatid transition. Importantly, a significantly increased occurrence of apoptosis in spermatids was observed after loss of Igf3. Therefore, igf3−/− males were subfertile with drastically reduced semen volume and sperm count. Conversely, the overexpression of Igf3 in XY tilapia enhanced spermatogenesis leading to more spermatids and sperm count. Transcriptomic analysis revealed that the absence of Igf3 resulted in dysregulation of many genes involved in cell cycle, meiosis and pluripotency regulators that are critical for spermatogenesis. In addition, in vitro gonadal culture with 17α-methyltetosterone (MT) and 11-ketotestosterone (11-KT) administration and in vivo knockout of cyp11c1 demonstrated that igf3 expression is regulated by androgens, suggesting that Igf3 acts downstream of androgens in fish spermatogenesis. Notably, the igf3 knockout did not affect body growth, indicating that this Igf specifically functions in reproduction. Taken together, our data provide genetic evidence for fish igf3 in the regulation of reproductive capacity by controlling spermatogenesis. [ABSTRACT FROM AUTHOR]

Teleost fish exhibit remarkable plasticity of sexual differentiation as sex reversal can be induced by exogenous steroid hormones. To date, little is known about whether the sex chromosome karyotype and copy number of sex determiners affects sex reversal in fish. The Nile tilapia (XX/XY system with amhy as sex determiner) and blue tilapia (ZZ/ZW system) are two closely cichlids widely used for aquaculture. In this study, we measured the sex reversal rates of XY, YY- amhy +/+, YY- amhy +/−, ZX and ZZ tilapia by 17β-estradiol (E2) treatment, ZW and WW tilapia by aromatase inhibitor (letrozole) treatment and aimed to promote sex reversal of YY fish through blockage of androgen synthesis and administration of E2. Statistically, E2 treatment resulted in lower sex reversal rate in YY and ZZ fish compared with XY and ZX fish, respectively. In addition, letrozole treatment led to lower sex reversal rate in WW fish compared with ZW fish. Importantly, E2 treatment of YY fish with mutation of one copy of amhy resulted in similar sex reversal rate compared with XY fish. These results implied that the two homogametic sex chromosomes in YY and ZZ fish antagonized the E2 induced sex reversal due to the up-regulation of the male pathway genes, and in WW fish antagonized the effects of the letrozole induced sex reversal. Simultaneous treatment of E2 and trilostane, an inhibitor of 3β-hydroxysteroid dehydrogenase, promoted sex reversal of YY fish. Taken together, our results demonstrate that sexual plasticity is associated with the sex chromosome karyotype and copy number of the sex determiner in tilapia. The production of YY neofemale tilapia allowed establishment of a YY maintenance line and large scale breeding of XY genetically male tilapia in aquaculture. • The two homogametic sex chromosomes in YY and ZZ fish enhanced the antagonistic effects to the E2 induced sex reversal. • The two homogametic sex chromosomes in WW fish enhanced the antagonistic effects to the letrozole induced sex reversal. • E2 treatment of YY fish carrying a mutation of one copy of amhy resulted in comparable sex reversal rate to that of XY fish. • An effective method for sex reversal of YY tilapia was developed. [ABSTRACT FROM AUTHOR]

Variation in the TGF-β signaling pathway is emerging as an important mechanism by which gonadal sex determination is controlled in teleosts. Here we show that amhy, a Y-specific duplicate of the anti-Müllerian hormone (amh) gene, induces male sex determination in Nile tilapia. amhy is a tandem duplicate located immediately downstream of amhΔ-y on the Y chromosome. The coding sequence of amhy was identical to the X-linked amh (amh) except a missense SNP (C/T) which changes an amino acid (Ser/Leu92) in the N-terminal region. amhy lacks 5608 bp of promoter sequence that is found in the X-linked amh homolog. The amhΔ-y contains several insertions and deletions in the promoter region, and even a 5 bp insertion in exonVI that results in a premature stop codon and thus a truncated protein product lacking the TGF-β binding domain. Both amhy and amhΔ-y expression is restricted to XY gonads from 5 days after hatching (dah) onwards. CRISPR/Cas9 knockout of amhy in XY fish resulted in male to female sex reversal, while mutation of amhΔ-y alone could not. In contrast, overexpression of Amhy in XX fish, using a fosmid transgene that carries the amhy/amhΔ-y haplotype or a vector containing amhy ORF under the control of CMV promoter, resulted in female to male sex reversal, while overexpression of AmhΔ-y alone in XX fish could not. Knockout of the anti-Müllerian hormone receptor type II (amhrII) in XY fish also resulted in 100% complete male to female sex reversal. Taken together, these results strongly suggest that the duplicated amhy with a missense SNP is the candidate sex determining gene and amhy/amhrII signal is essential for male sex determination in Nile tilapia. These findings highlight the conserved roles of TGF-β signaling pathway in fish sex determination. [ABSTRACT FROM AUTHOR]

Four pairs of XX and XY gonads from Nile tilapia were sequenced at four developmental stages, 5, 30, 90, and 180 days after hatching (dah) using Illumina HiseqTM technology. This produced 28 Gb sequences, which were mapped to 21,334 genes. Of these, 259 genes were found to be specifically expressed in XY gonads, and 69 were found to be specific to XX gonads. Totally, 187 XX- and 1,358 XY-enhanced genes were identified, and 2,978 genes were found to be co-expressed in XX and XY gonads. Almost all steroidogenic enzymes, including cyp19a1a, were up-regulated in XX gonads at 5 dah; but in XY gonads these enzymes, including cyp11b2, were significantly up-regulated at 90 dah, indicating that, at a time critical to sex determination, the XX fish produced estrogen and the XY fish did not produce androgens. The most pronounced expression of steroidogenic enzyme genes was observed at 30 and 90 dah for XX and XY gonads, corresponding to the initiation of germ cell meiosis in the female and male gonads, respectively. Both estrogen and androgen receptors were found to be expressed in XX gonads, but only estrogen receptors were expressed in XY gonads at 5 dah. This could explain why exogenous steroid treatment induced XX and XY sex reversal. The XX-enhanced expression of cyp19a1a and cyp19a1b at all stages suggests an important role for estrogen in female sex determination and maintenance of phenotypic sex. This work is the largest collection of gonadal transcriptome data in tilapia and lays the foundation for future studies into the molecular mechanisms of sex determination and maintenance of phenotypic sex in non-model teleosts. [ABSTRACT FROM AUTHOR]

TSP1 was reported to be involved in multiple biological processes including the activation of TGF-β signaling pathways and the regulation of angiogenesis during wound repair and tumor growth, while its role in ovarian folliculogenesis remains to be elucidated. In the present study, Tsp1a was found to be expressed in the oogonia and granulosa cells of phase I to phase IV follicles in the ovaries of Nile tilapia by immunofluorescence. tsp1a homozygous mutants were generated by CRISPR/Cas9. Mutation of tsp1a resulted in increased oogonia, reduced secondary growth follicles and delayed ovary development. Expression of the cell proliferation marker PCNA was significantly up-regulated in the oogonia of the mutant ovaries. Furthermore, transcriptomic analysis revealed that expressions of DNA replication related genes were significantly up-regulated, while cAMP and MAPK signaling pathway genes which inhibit cell proliferation and promote cell differentiation were significantly down-regulated. In addition, aromatase (Cyp19a1a) expression and serum 17β-estradiol (E2) concentration were significantly decreased in the mutants. These results indicated that lacking tsp1a resulted in increased proliferation and inhibited differentiation of oogonia, which in turn, resulted in increased oogonia, reduced secondary growth follicles and decreased E2. Taken together, our results indicated that tsp1a was essential for ovarian folliculogenesis in Nile tilapia. [ABSTRACT FROM AUTHOR]

The Sox family member Sox30 is highly expressed in the testis of several vertebrate species and has been shown to play key roles in spermiogenesis. However, its transcription regulation remains unclear. Here, we analyzed the Sox30 promoter from the teleost fish Nile tilapia (Oreochromis niloticus) and predicted a putative cis-regulatory element (CRE) for doublesex and mab-3 related transcription factor 1 (Dmrt1), a male-specific transcription factor involved in male sex differentiation. Transcriptional profiling revealed that Sox30 and Dmrt1 similarly exhibited a high expression in tilapia testes from 90 days after hatching (dah) to 300 dah, and the transcription of the Sox30 gene was reduced about one-fold in the testes of male tilapia with Dmrt1 knockdown. Further dual-luciferase reporter assay confirmed that Dmrt1 overexpression significantly promoted transcriptional activity of the Sox30 promoter and this promotion was decreased following the mutation of putative CRE for Dmrt1 within the Sox30 promoter. Chromatin immunoprecipitation-based PCR (ChIP-PCR) and electrophoretic mobility shift assay (EMSA) demonstrated that Dmrt1 directly binds to putative CRE within the Sox30 promoter. These results together indicate that Dmrt1 positively regulates the transcription of the tilapia Sox30 gene by directly binding to specific CRE within the Sox30 promoter. [ABSTRACT FROM AUTHOR]

Abstract The Nile tilapia Sox9b gene is characterized as a homolog of the mammalian Sox9 gene, which exhibits testis-biased expression and is involved in testis development. However, the transcriptional regulation of the Sox9b gene is poorly understood. In this study, we demonstrated that the male sex differentiation gene doublesex and mab-3 related transcription factor 1 (Dmrt1) was predominantly expressed in the Nile tilapia testis and that Dmrt1 knockdown in the Nile tilapia decreased the expression of the Sox9b gene in the testis. An in silico analysis predicted that the proximal promoter of the Nile tilapia Sox9b gene had two potential cis-regulatory elements (CREs) for the Dmrt1 transcription factor. Together, a luciferase reporter analysis and site-directed mutagenesis revealed that Dmrt1 increased the transcriptional activity of the Nile tilapia Sox9b promoter via a specific CRE near the translation start site. A chromatin immunoprecipitation (ChIP) analysis and an electrophoretic mobility shift assay (EMSA) confirmed that Dmrt1 could directly bind to this specific CRE for Dmrt1 in the Nile tilapia Sox9b promoter. Taken together, our results demonstrate that the male sex-differentiation factor Dmrt1 positively regulates the transcription of the Nile tilapia Sox9b gene by directly binding to a specific CRE within the Sox9b promoter. Highlights • Sox9b and Dmrt1 are predominately expressed in the Nile tilapia testis. • Dmrt1 knockdown in Nile tilapia decreases Sox9b transcription in testis. • Dmrt1 promotes the activity of the Nile tilapia Sox9b promoter via a specific CRE for Dmrt1. • Dmrt1 directly binds to a specific CRE within the Nile tilapia Sox9b promoter. [ABSTRACT FROM AUTHOR]

Nile tilapia display conspicuous differences in growth rates between genders, as males tend to outpace and outgrow females. The production of all male Nile tilapia populations using genetically male tilapia (GMT) technology represents the most efficient and scientifically grounded approach. Most of the Nile tilapia stocks in aquaculture use LG23 as sex chromosome. The crux of GMT lies in the acquisition of YY supermales and YY pseudofemales (pF). Nonetheless, the utilization of GMT is impeded by the lower fertility and even infertility of feminized YY-pF. In this study, we found that YY-pF exhibited male-like genital papillae, and exhibited significantly higher androgen levels and lower E2 levels compared with that of fertile XY-pF. YY-pF carries two copies of Amhy but lacks Amh , and XY-pF possesses one Amhy and one Amh. In XY-pF, the expression of Amh/Amhy was only observed in granulosa cells of phase I and II follicles, while in YY-pF, Amhy expression persisted from phase I to IV. On the contrary, Cyp19a1a was found to be highly expressed in granulosa cells of phase III and IV follicles in XY-pF, but significantly reduced in granulosa cells of phase III and IV follicles in YY-pF, exhibiting a reciprocal expression pattern with Amhy. Consistently, YY-pF with one copy of Amhy mutation (YY- Amhy +/−-pF) were fertile. These results indicated that prolonged expression of Amhy led to a decrease in the expression of Cyp19a1a, followed by a decrease in serum E2 levels and an increase in androgen levels. Based on these data, we treated YY-pF with both E2 and trilostane (androgen synthetase inhibitors) simultaneously at the vitellogenic stage to make it fertile. The acquisition of fertile YY-pF laid the foundation for the widespread application of GMT. • YY-pF displayed male-like genital papillae due to high 11-KT and low E2 levels. • Prolonged expression of Amhy led to the infertility of YY- pF. • Fertile YY-pF were produced by treatment of both trilostane and E2. [ABSTRACT FROM AUTHOR]

Wilms tumor 1 (Wt1) is an essential factor for urogenital system development. Teleosts have two wt1s , named as wt1a and wt1b . In this study, the expression pattern of wt1a and wt1b and their functions on the urogenital system were analyzed by in situ hybridization and CRISPR/Cas9. wt1a was found to be expressed in the glomerulus at 3 dah (days after hatching), earlier than wt1b. wt1a and wt1b were simultaneously expressed in the somatic cells of gonads at 3 dah, while their cell locations were similar, but not identical in adult fish gonads. The wt1a -/- fish displayed pericardial edema and yolk sac edema at 3 dah and subsequently expanded as general body edema at 6 dah, failed to develop glomerulus and died during 6–10 dah, whereas the wt1b -/- fish were phenotypically normal. Immunohistochemical analyses revealed that the germ cell marker Vasa was expressed, while somatic cell genes Cyp19a1a, Amh, Gsdf and Dmrt1 were not expressed in the wt1a -/- gonads at 6 dah. The sex phenotypes of XX and XY in the wt1b -/- fish were not affected. Real-time PCR revealed that the ovarian cyp19a1a expression was up-regulated in XX wt1b -/- fish, compared with XX control at 90 dah. Serum estradiol-17β level was also up-regulated in XX wt1b -/- fish at 90 and 180 dah. The XY wt1b -/- fish had normal serum estradiol-17β and 11-ketotestosterone levels and remained fertile. These results suggest that Wt1a and Wt1b have different functions in the kidneys and gonads of tilapia. [ABSTRACT FROM AUTHOR]

Abstract: Foxp3 is a crucial transcription factor for the development and function of CD4+CD25+ regulatory T cells (Tr) which play a key role in preventing autoimmune diseases and maintaining maternal-fetal tolerance in mammals. However, the knowledge of Foxp3 and its regulation in teleosts is limited. In the present study, the Foxp3 cDNA was cloned from Nile tilapia and characterized. The full length cDNA of Nile tilapia (nt)Foxp3 contained a 5′UTR of 104bp, a 3′UTR of 239bp and an open reading frame of 1134bp. The putative ntFoxp3 contained a C2H2 zinc finger domain, a winged-helix/fork head domain and a leucine zipper-like domain with a consensus of V-x(6)-L-x(6)-L-x(6)-L, which are typical motifs of a Foxp3. The highest mRNA expression of ntFoxp3 was observed in the spleen, with moderate expression in the head kidney, intestine, kidney, liver and brain. Stimulation of peripheral blood mononuclear cells with PHA and LPS led to a significant increase of ntFoxp3 mRNA expression at 6 and 24h, respectively. In contrast, stimulation with PMA had no effect during the sampling period. After injecting 1×107 cells of live Aeromonas hydrophila into adult female Nile tilapias, the mRNA expression of ntFoxp3 significantly increased in the gill and intestine at 6h, while unchanged in the spleen. In all the tissues examined, a clearly up-regulation of ntFoxp3 expression was detected at 24h after the second challenge. These results suggest that ntFoxp3 might be involved into lymphocyte activation and immune responses as reported in mammals. Meanwhile, the correlation between the expression profile of ntFoxp3 and serum estrogen (17-beta-estradiol, E2) concentration was investigated by real-time PCR and enzyme immunoassay. The results showed that E2 could regulate the expression of ntFoxp3 in the intestine and kidney but not in the spleen. This work will help future investigations into Tr cells and extend our understanding of interactions between sex hormones and immune related molecules in teleosts. [Copyright &y& Elsevier]

• Mutation of rln3a gene severely affected the spermatogenesis, sperm maturation and fertility in XY male fish. • The deficiency of Rln3a suppressed the expressions of steroidogenic enzyme genes, subsequently declined 11-KT synthesis. • hCG failed to rescue the decrease of 11-KT, while hRLN3 considerably improved the sperm motility in rln3a−/− XY fish. The essential roles of Relaxin3 (RLN3) in energy homeostasis had been well investigated, while the mechanisms of RLN3 regulating reproduction remain to be elusive in mammals. Although two rln3 paralogues have been characterized in several teleosts, their functions still remain largely unknown. In this study, two paralogous rln3 genes, represented as rln3a and rln3b , were identified from the testis of Nile tilapia (Oreochromis niloticus). Rln3a was dominantly expressed in testis, while the most abundant rln3b expression was in brain. In situ hybridization demonstrated that rln3a is abundantly expressed in the Leydig cells of the testis. To understand the role of Rln3 in the testicular development, homologous null- rln3a gene mutant line was constructed by CRISPR/Cas9 technology. Morphological observation demonstrated that null mutation of rln3a gene caused testicular hypertrophy and a significant increase of GSI. However, a significant decrease of spermatogenic cells at different phases, i.e. spermatogonia, spermatocytes, spermatids and sperms was found. Silencing of rln3a gene repressed the expression of key genes in germ cell and Leydig cell. Deficiency of Rln3a led to the significant decrease of 11-KT production, which stimulated the up-regulation of both FSH and LH production in the pituitary via a negative feedback manner possibly. Mutation of rln3a in XY fish led to the hypogonadism with sperm deformation, significant decrease of fertility, and sperm motility, revealing as the high mortality of the offspring obtained by crossing the wild type female and rln3a−/− XY fish. Interestingly, recombinant human RLN3 injection significantly enhanced the sperm motility in rln3a−/− XY fish. Moreover, hCG treatment stimulated the expression of steroidogenic enzyme genes and 11-KT production, which were repressed by rln3a mutation in XY fish. Taken together, this study, for the first time by using a gene knockout model, proved that Rln3a is an indispensable mediator for androgen production in testis via HPG axis, and plays an essential role in spermatogenesis, sperm motility and male fertility in fish. [ABSTRACT FROM AUTHOR]

In the present study, Amh was found to be abundantly expressed in the granulosa cells of the primary growth follicles, and Amhr2 in the granulosa cells, oogonia and phase I oocytes in tilapia by immunohistochemistry. In addition, Amh and Amhr2 were also found to be expressed in the brain and pituitary. Heterozygous mutation of either amh or amhr2 resulted in increased primary growth follicles and decreased fertility, and homozygous mutation resulted in hypertrophic ovaries with significantly increased primary follicles and failed transition from primary to vitellogenic follicles. Expression of gnrh3 in the brain, fsh and lh in the pituitary and serum E2 concentration were significantly decreased in both mutants. Significantly increased apoptosis of follicle cells was observed in both mutants. However, administration of E2 failed to rescue the folliculogenesis defects of the mutants. Our results suggested that Amh acts in a dose-dependent manner by binding Amhr2 in tilapia. • Amh signaling is Amhr2 dependent in teleosts. • Homozygous mutation resulted in excessive proliferation of germ cells. • Homozygous mutants failed to transition from primary to vitellogenic follicles. • Increased apoptosis of follicle cells in both amh and amhr2 mutants. [ABSTRACT FROM AUTHOR]