Dmrt1 is the only male pathway gene tested indispensable for sex determination and functional testis development in tilapia.

Sex is determined by multiple factors derived from somatic and germ cells in vertebrates. We have identified amhy, dmrt1, gsdf as male and foxl2, foxl3, cyp19a1a as female sex determination pathway genes in Nile tilapia. However, the relationship among these genes is largely unclear. Here, we found that the gonads of dmrt1;cyp19a1a double mutants developed as ovaries or underdeveloped testes with no germ cells irrespective of their genetic sex. In addition, the gonads of dmrt1;cyp19a1a;cyp19a1b triple mutants still developed as ovaries. The gonads of foxl3;cyp19a1a double mutants developed as testes, while the gonads of dmrt1;cyp19a1a;foxl3 triple mutants eventually developed as ovaries. In contrast, the gonads of amhy;cyp19a1a, gsdf;cyp19a1a, amhy;foxl2, gsdf;foxl2 double and amhy;cyp19a1a;cyp19a1b, gsdf;cyp19a1a;cyp19a1b triple mutants developed as testes with spermatogenesis via up-regulation of dmrt1 in both somatic and germ cells. The gonads of amhy;foxl3 and gsdf;foxl3 double mutants developed as ovaries but with germ cells in spermatogenesis due to up-regulation of dmrt1. Taking the respective ovary and underdeveloped testis of dmrt1;foxl3 and dmrt1;foxl2 double mutants reported previously into consideration, we demonstrated that once dmrt1 mutated, the gonad could not be rescued to functional testis by mutating any female pathway gene. The sex reversal caused by mutation of male pathway genes other than dmrt1, including its upstream amhy and downstream gsdf, could be rescued by mutating female pathway gene. Overall, our data suggested that dmrt1 is the only male pathway gene tested indispensable for sex determination and functional testis development in tilapia.

Germline sexual fate is determined by the antagonistic action of dmrt1 and foxl3/foxl2 in tilapia.

Germline sexual fate has long been believed to be determined by the somatic environment, but this idea is challenged by recent studies of foxl3 mutants in medaka. Here, we demonstrate that the sexual fate of tilapia germline is determined by the antagonistic interaction of dmrt1 and foxl3, which are transcriptionally repressed in male and female germ cells, respectively. Loss of dmrt1 rescued the germ cell sex reversal in foxl3Δ7/Δ7 XX fish, and loss of foxl3 partially rescued germ cell sex reversal but not somatic cell fate in dmrt1Δ5/Δ5 XY fish. Interestingly, germ cells lost sexual plasticity in dmrt1Δ5/Δ5 XY and foxl3Δ7/Δ7 XX single mutants, as aromatase inhibitor (AI) and estrogen treatment failed to rescue the respective phenotypes. However, recovery of germ cell sexual plasticity was observed in dmrt1/foxl3 double mutants. Importantly, mutation of somatic cell-specific foxl2 resulted in testicular development in foxl3Δ7/Δ7 or dmrt1Δ5/Δ5 mutants. Our findings demonstrate that sexual plasticity of germ cells relies on the presence of both dmrt1 and foxl3. The existence of dmrt1 and foxl3 allows environmental factors to influence the sex fate decision in vertebrates.

Mutation of mpv17 results in loss of iridophores due to mitochondrial dysfunction in tilapia.

Mpv17 (mitochondrial inner membrane protein MPV17) deficiency causes severe mitochondrial DNA depletion syndrome in mammals and loss of pigmentation of iridophores and a significant decrease of melanophores in zebrafish. The reasons for this are still unclear. In this study, we established an mpv17 homozygous mutant line in Nile tilapia. The developing mutants are transparent due to loss of iridophores and aggregation of pigment granules in the melanophores and disappearance of the vertical pigment bars on the side of the fish. Transcriptome analysis using skin of fish at 30 dpf (days post fertilization) revealed that the genes related to purine (especially pnp4a) and melanin synthesis were significantly downregulated. However, administration of guanine diets failed to rescue the phenotype of the mutants. In addition, no obvious apoptosis signals were observed in the iris of the mutants by TUNEL staining. Significant downregulation of genes related to iridophore differentiation was detected by qPCR. Insufficient ATP, as revealed by ATP assay, α-MSH treatment and adcy5 mutational analysis, might account for the defects of melanophores in mpv17 mutants. Several tissues displayed less mtDNA and decreased ATP levels. Taken together, these results indicated that mutation of mpv17 led to mitochondrial dTMP deficiency, followed by impaired mtDNA content and mitochondrial function, which in turn, led to loss of iridophores and a transparent body color in tilapia.

Tilapia, a good model for studying reproductive endocrinology.

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.

Genome-wide analysis of two different regions of brain reveals the molecular changes of fertility related genes in rln3a-/- mutants in male Nile tilapia (Oreochromis niloticus).

Relaxin3 (rln3) has been associated with various emotional and cognitive processes, including stress, anxiety, learning, memory, motivational behavior, and circadian rhythm. Notably, previous report revealed that Rln3a played an indispensable role in testicular development and male fertility in Nile tilapia (Oreochromis niloticus). However, the underlying molecular mechanisms remain largely unknown. We found that Rln3a is expressed exclusively in the diencephalon* (Di*) of the brain. Deficiency of Rln3a resulted in a significant increase in serum dopamine level and an upregulation of gene expression of gnrh1 and kisspeptin2. To further elucidate the role of Rln3a in fish fertility, we collected two different regions of Di* and hypothalamus (Hyp) tissues for subsequent RNA-seq analysis of both wild-type (rln3a+/+) and rln3a-/- male tilapia. Upon the transcriptomic data, 1136 and 755 differentially expressed genes (DEGs) were identified in the Di* and Hyp tissues, respectively. In Di*, the up-regulated genes were enriched in circadian rhythm, chemical carcinogenesis, while the down-regulated genes were enriched in type II diabetes mellitus, dopaminergic synapse, and other pathways. In Hyp, the up-regulated genes were enriched in circadian rhythm, pyrimidine metabolism, while the down-regulated genes were enriched in type I diabetes mellitus, autoimmune thyroid disease, and other pathways. Subsequently, the results of both qRT-PCR and FISH assays highlighted a pronounced up-regulation of core circadian rhythm genes, cry1b and per3, whereas genes such as clocka, clockb, and arntl exhibited down-regulation. Furthermore, the genes associated with dopamine biosynthesis were significantly increased in the Hyp. In summary, the mutation of rln3a in male tilapia resulted in notable changes in circadian rhythm and disease-linked signaling pathways in the Di* and Hyp. These changes might account for the fertility defects observed in rln3a-/- male mutants in tilapia.

Nile Tilapia (Oreochromis niloticus) Patched1 Mutations Disrupt Cardiovascular Development and Vascular Integrity through Smoothened Signaling.

Hedgehog (Hh) signaling is crucial in cardiovascular development and maintenance. However, the biological role of Patched1 (Ptch1), an inhibitory receptor of the Hh signaling pathway, remains elusive. In this study, a Ptch1 ortholog was characterized in Nile tilapia (Oreochromis niloticus), and its function was investigated through CRISPR/Cas9 gene knockout. When one-cell embryos were injected with CRISPR/Cas9 targeting ptch1, the mutation efficiency exceeded 70%. During 0-3 days post fertilization (dpf), no significant differences were observed between the ptch1 mutant group and the control group; at 4 dpf (0 day after hatching), about 10% of the larvae showed an angiogenesis defect and absence of blood flow; from 5 dpf, most larvae exhibited an elongated heart, large pericardial cavity, and blood leakage and coagulation, ultimately dying during the 6-8 dpf period due to the lack of blood circulation. Consistently, multiple differentially expressed genes related to angiogenesis, blood coagulation, and heart development were enriched in the ptch1 mutants. Furthermore, Smoothened (Smo) antagonist (cyclopamine) treatment of the ptch1 mutants greatly rescued the cardiovascular disorders. Collectively, our study suggests that Ptch1 is required for cardiovascular development and vascular integrity via Smo signaling, and excessive Hh signaling is detrimental to cardiovascular development.

Smoothened mediates medaka spermatogonia proliferation via Gli1-Rgcc-Cdk1 axis†.

The proliferation of spermatogonia directly affects spermatogenesis and male fertility, but its underlying molecular mechanisms are poorly understood. In this study, Smoothened (Smo), the central transducer of Hedgehog signaling pathway, was characterized in medaka (Oryzias latipes), and its role and underlying mechanisms in the proliferation of spermatogonia were investigated. Smo was highly expressed in spermatogonia. In ex vivo testicular organ culture and a spermatogonial cell line (SG3) derived from medaka mature testis, Smo activation promoted spermatogonia proliferation, while its inhibition induced apoptosis. The expression of glioma-associated oncogene homolog 1 (gli1) and regulator of cell cycle (rgcc) was significantly upregulated in SG3 after Smo activation. Furthermore, Gli1 transcriptionally upregulated the expression of rgcc, and Rgcc overexpression rescued cell apoptosis caused by Smo or Gli1 inhibition. Co-immunoprecipitation assay indicated that Rgcc could interact with cyclin-dependent kinase 1 (Cdk1) to regulate the cell cycle of spermatogonia. Collectively, our study firstly reveals that Smo mediates the proliferation of spermatogonia through Gli1-Rgcc-Cdk1 axis. In addition, Smo and Gli1 are necessary of the survival of spermatogonia. This study deepens our understanding of spermatogonia proliferation and survival at the molecular level, and provides insights into male fertility control and reproductive disease treatment.

Network architecture and sex chromosome turnovers: Do epistatic interactions shape patterns of sex chromosome replacement?

Recent studies have revealed an astonishing diversity of sex chromosomes in many vertebrate lineages, prompting questions about the mechanisms of sex chromosome turnover. While there is considerable population genetic theory about the evolutionary forces promoting sex chromosome replacement, this theory has not yet been integrated with our understanding of the molecular and developmental genetics of sex determination. Here, we review recent data to examine four questions about how the structure of gene networks influences the evolution of sex determination. We argue that patterns of epistasis, arising from the structure of genetic networks, may play an important role in regulating the rates and patterns of sex chromosome replacement.

Gata2a Mutation Causes Progressive Microphthalmia and Blindness in Nile Tilapia.

The normal development of lens fiber cells plays a critical role in lens morphogenesis and maintaining transparency. Factors involved in the development of lens fiber cells are largely unknown in vertebrates. In this study, we reported that GATA2 is essential for lens morphogenesis in Nile tilapia (Oreochromis niloticus). In this study, Gata2a was detected in the primary and secondary lens fiber cells, with the highest expression in primary fiber cells. gata2a homozygous mutants of tilapia were obtained using CRISPR/Cas9. Different from fetal lethality caused by Gata2/gata2a mutation in mice and zebrafish, some gata2a homozygous mutants of tilapia are viable, which provides a good model for studying the role of gata2 in non-hematopoietic organs. Our data showed that gata2a mutation caused extensive degeneration and apoptosis of primary lens fiber cells. The mutants exhibited progressive microphthalmia and blindness in adulthood. Transcriptome analysis of the eyes showed that the expression levels of almost all genes encoding crystallin were significantly down-regulated, while the expression levels of genes involved in visual perception and metal ion binding were significantly up-regulated after gata2a mutation. Altogether, our findings indicate that gata2a is required for the survival of lens fiber cells and provide insights into transcriptional regulation underlying lens morphogenesis in teleost fish.

Origin of a Giant Sex Chromosome.

Chromosome size and morphology vary within and among species, but little is known about the proximate or ultimate causes of these differences. Cichlid fish species in the tribe Oreochromini share an unusual giant chromosome that is ∼3 times longer than the other chromosomes. This giant chromosome functions as a sex chromosome in some of these species. We test two hypotheses of how this giant sex chromosome may have evolved. The first hypothesis proposes that it evolved by accumulating repetitive elements as recombination was reduced around a dominant sex determination locus, as suggested by canonical models of sex chromosome evolution. An alternative hypothesis is that the giant sex chromosome originated via the fusion of an autosome with a highly repetitive B chromosome, one of which carried a sex determination locus. We test these hypotheses using comparative analysis of chromosome-scale cichlid and teleost genomes. We find that the giant sex chromosome consists of three distinct regions based on patterns of recombination, gene and transposable element content, and synteny to the ancestral autosome. The WZ sex determination locus encompasses the last ∼105 Mb of the 134-Mb giant chromosome. The last 47 Mb of the giant chromosome shares no obvious homology to any ancestral chromosome. Comparisons across 69 teleost genomes reveal that the giant sex chromosome contains unparalleled amounts of endogenous retroviral elements, immunoglobulin genes, and long noncoding RNAs. The results favor the B chromosome fusion hypothesis for the origin of the giant chromosome.

Identification of sex chromosome and sex-determining gene of southern catfish (Silurus meridionalis) based on XX, XY and YY genome sequencing.

Teleosts are important models to study sex chromosomes and sex-determining (SD) genes because they present a variety of sex determination systems. Here, we used Nanopore and Hi-C technologies to generate a high-contiguity chromosome-level genome assembly of a YY southern catfish (Silurus meridionalis). The assembly is 750.0 Mb long, with contig N50 of 15.96 Mb and scaffold N50 of 27.22 Mb. We also sequenced and assembled an XY male genome with a size of 727.2 Mb and contig N50 of 13.69 Mb. We identified a candidate SD gene through comparisons to our previous assembly of an XX individual. By resequencing male and female pools, we characterized a 2.38 Mb sex-determining region (SDR) on Chr24. Analysis of read coverage and comparison of the X and Y chromosome sequences showed a Y specific insertion (approx. 500 kb) in the SDR which contained a male-specific duplicate of amhr2 (named amhr2y). amhr2y and amhr2 shared high-nucleotide identity (81.0%) in the coding region but extremely low identity in the promotor and intron regions. The exclusive expression in the male gonadal primordium and loss-of-function inducing male to female sex reversal confirmed the role of amhr2y in male sex determination. Our study provides a new example of amhr2 as the SD gene in fish and sheds light on the convergent evolution of the duplication of AMH/AMHR2 pathway members underlying the evolution of sex determination in different fish lineages.

Desert hedgehog mediates the proliferation of medaka spermatogonia through Smoothened signaling.

Desert hedgehog (DHH) signaling has been reported to be involved in spermatogenesis and the self-renewal of spermatogonial stem cells (SSCs). However, the role of DHH in proliferation of spermatogonia including SSCs remains to be elucidated. Here, we report that Dhh from medaka (Oryizas latipes) (named as OlDhh) could directly mediate the proliferation of spermatogonia via Smoothened (Smo) signaling. Oldhh is 1362 bp in length and encodes 453 amino acid (aa) residues with more than 50% identity with the homologs in other species. It has expression predominantly restricted to testis. The soluble and tag-free 176-aa mature OlDhh (named as mOlDhh) were successfully obtained by fusing with the N-terminal tag of cleavable 6-histidine and small ubiquitin-related modifier and then removing the tag. Notably, mOlDhh significantly promoted the proliferation of SG3 (a spermatogonial stem cell line from medaka testis) in a dose-dependent manner and spermatogonia in testicular organ culture. Furthermore, the proliferation of SG3 in the presence of mOlDhh could be inhibited by Smo antagonist (cyclopamine) resulting in apoptosis. Additionally, mOlDhh significantly upregulated the expression of smo as well as the pluripotent-related genes bcl6b and sall4. These data suggest that Smo is an indispensable downstream component in the Dhh signaling pathway. In conclusion, our findings unambiguously demonstrate that Dhh could directly mediate the proliferation of spermatogonia through Smo signaling. This study provides new knowledge about the proliferation regulation of spermatogonia.

CRISPR Knockouts of pmela and pmelb Engineered a Golden Tilapia by Regulating Relative Pigment Cell Abundance.

Premelanosome protein (pmel) is a key gene for melanogenesis. Mutations in this gene are responsible for white plumage in chicken, but its role in pigmentation of fish remains to be demonstrated. In this study, we found that most fishes have 2 pmel genes arising from the teleost-specific whole-genome duplication. Both pmela and pmelb were expressed at high levels in the eyes and skin of Nile tilapia. We mutated both genes in tilapia using CRISPR/Cas9. Homozygous mutation of pmela resulted in yellowish body color with weak vertical bars and a hypopigmented retinal pigment epithelium (RPE) due to significantly reduced number and size of melanophores. In contrast, we observed an increased number and size of xanthophores in mutants compared to wild-type fish. Homozygous mutation of pmelb resulted in a similar, but milder phenotype than pmela-/- mutants. Double mutation of pmela and pmelb resulted in loss of additional melanophores compared to the pmela-/- mutants, and also an increase in the number and size of xanthophores, producing a golden body color. The RPE pigmentation of pmela-/-;pmelb-/- was similar to pmela-/- mutants, with much less pigmentation than pmelb-/- mutants and wild-type fish. Taken together, our results indicate that, although both pmel genes are important for the formation of body color in tilapia, pmela plays a more important role than pmelb. To our knowledge, this is the first report on mutation of pmelb or both pmela;pmelb in fish. Studies on these mutants suggest new strategies for breeding golden tilapia, and also provide a new model for studies of pmel function in vertebrates.

Amantadine, a promising therapeutic agent against viral encephalopathy and retinopathy.

Outbreaks of viral encephalopathy and retinopathy (VER) in marine and freshwater species severely devastate the aquaculture worldwide. The causative agent of VER is nervous necrosis virus (NNV), which mainly infects the early developmental stages of fish, limiting the effectiveness of vaccines. To counter this case, the anti-NNV potentials of nine drugs with broad-spectrum antiviral activity were explored using ribavirin as a positive drug. Toxicity of the selected drugs to SSN-1 cells and grouper was firstly evaluated to determine the safety concentrations. For screening in vitro, amantadine and oseltamivir phosphate can relieve the cytopathic effects and inhibit NNV replication with the 90% inhibitory concentrations (IC90 ) of 38.74 and 106.75 mg/L, respectively. Amantadine has a stronger anti-NNV activity than ribavirin at the with- and post-NNV infection stages, indicating that it is a potential therapeutic agent against VER by acting directly on NNV. Similarly, amantadine also has a strong anti-NNV activity in vivo with the IC90 of 27.91 mg/L at the 7 days post-infection, while that was 73.25 mg/L for ribavirin. Following exposure to amantadine (40 mg/L) and ribavirin (100 mg/L) for 7 days, the survival rates of NNV-infected grouper were increased to 44% and 39%, respectively. The maximum amantadine content (11.88 mg/Kg) in grouper brain was reached following exposure for 24 hr, and amantadine can be quickly excreted from fish, reducing the risk of drug residue. Results so far indicated that amantadine is a promising therapeutic agent against NNV in aquaculture, providing an effective strategy for VER control at the early developmental stages of fish.

Igf3: a novel player in fish reproduction†.

As in other vertebrates, fish reproduction is tightly controlled by gonadotropin signaling. One of the most perplexing aspects of gonadotropin action on germ cell biology is the restricted expression of gonadotropin receptors in somatic cells of the gonads. Therefore, the identification of factors conveying the action of gonadotropins on germ cells is particularly important for understanding the mechanism of reproduction. Insulin-like growth factors (Igfs) are recognized as key factors in regulating reproduction by triggering a series of physiological processes in vertebrates. Recently, a novel member of Igfs called Igf3 has been identified in teleost. Different from the conventional Igf1 and Igf2 that are ubiquitously expressed in a majority of tissues, Igf3 is solely or highly expressed in the fish gonads. The role of Igf3 in mediating the action of gonadotropin through Igf type 1 receptor on several aspects of oogenesis and spermatogenesis have been demonstrated in several fish species. In this review, we will summarize existing data on Igf3. This new information obtained from Igf3 provides insight into elucidating the molecular mechanism of fish reproduction, and also highlights the importance of Igf system in mediating the action of gonadotropin signaling on animal reproduction.

Rbm46, a novel germ cell-specific factor, modulates meiotic progression and spermatogenesis.

It has been suggested that many novel RNA-binding proteins (RBPs) are required for gametogenesis, but the necessity of few of these proteins has been functionally verified. Here, we identified one RBP, Rbm46, and investigated its expression pattern and role in zebrafish reproduction. We found that rbm46 is maternally provided and specifically expressed in the germ cells of gonadal tissues using in situ hybridization, reverse transcription-PCR, and quantitative real-time polymerase chain reaction (qRT-PCR). Two independent rbm46 mutant zebrafish lines were generated via the transcription activator-like effector nuclease technique. Specific disruption of rbm46 resulted in masculinization and infertility in the mutants. Although the spermatogonia appeared grossly normal in the mutants, spermatogenesis was impaired, and meiosis events were not observed. The introduction of a tp53M214K mutation could not rescue the female-to-male sex-reversal phenotype, indicating that rbm46 acts independently of the p53-dependent apoptotic pathway. RNA sequencing and qRT-PCR subsequently indicated that Rbm46 might be involved in the posttranscriptional regulation of functional genes essential for germ cell development, such as nanos3, dazl, and sycp3, during gametogenesis. Together, our results reveal for the first time the crucial role of rbm46 in regulating germ cell development in vivo through promotion of germ cell progression through meiosis prophase I.

Nile Tilapia: A Model for Studying Teleost Color Patterns.

The diverse color patterns of cichlid fishes play an important role in mate choice and speciation. Here we develop the Nile tilapia (Oreochromis niloticus) as a model system for studying the developmental genetics of cichlid color patterns. We identified 4 types of pigment cells: melanophores, xanthophores, iridophores and erythrophores, and characterized their first appearance in wild-type fish. We mutated 25 genes involved in melanogenesis, pteridine metabolism, and the carotenoid absorption and cleavage pathways. Among the 25 mutated genes, 13 genes had a phenotype in both the F0 and F2 generations. None of F1 heterozygotes had phenotype. By comparing the color pattern of our mutants with that of red tilapia (Oreochromis spp), a natural mutant produced during hybridization of tilapia species, we found that the pigmentation of the body and eye is controlled by different genes. Previously studied genes like mitf, kita/kitlga, pmel, tyrb, hps4, gch2, csf1ra, pax7b, and bco2b were proved to be of great significance for color patterning in tilapia. Our results suggested that tilapia, a fish with 4 types of pigment cells and a vertically barred wild-type color pattern, together with various natural and artificially induced color gene mutants, can serve as an excellent model system for study color patterning in vertebrates.

Role of sex steroids in fish sex determination and differentiation as revealed by gene editing.

The involvement of sex steroids in sex determination and differentiation is relatively conserved among non-mammalian vertebrates, especially in fish. Thanks to the advances in genome sequencing and genome editing, significant progresses have been made in the understanding of steroidogenic pathway and hormonal regulation of sex determination and differentiation in fish. It seems that loss of function study of single gene challenges the traditional views that estrogen is required for ovarian differentiation and androgen is needed for testicular development, but it is not so in essence. Steroidogenic enzymes can be classified into two categories based on expression and enzyme activities in fish. One type, encoded by star2, cyp17a1 and cyp19a1a, is involved in estrogen production and exclusively expressed in the gonads. Mutation of these genes results in the up-regulation of male pathway genes and sex reversal from genetic female to male. The other type, encoded by the duplicated paralogs of the above genes, including star1, cyp11a1, cyp17a2 and cyp19a1b, as well as cyp11c1 gene, is dominantly expressed both in gonads and extra-gonadal tissues. Mutation of these genes alters the steroids (androgen, DHP and cortisol) production and spermatogenesis, fertility, secondary sexual characteristics and sexual behavior, but usually does not affect the sex differentiation. For the estrogen receptors (esr1, esr2a and esr2b), single mutation failed to, but double and triple mutation leads to sex reversal from female to male, indicating that at least Esr2a and Esr2b are required to mediate the role of estrogen in sex determination proved by gene editing experiments. Taken together, results from gene editing enrich our understanding of steroid synthesis pathways and further confirm the critical role of estrogen in female sex determination by antagonizing the male pathway in fish.

Regulation of spermatogenesis and reproductive capacity by Igf3 in tilapia.

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.

Dnmt3aa but Not Dnmt3ab Is Required for Maintenance of Gametogenesis in Nile Tilapia (Oreochromis niloticus).

Dnmt3a, a de novo methyltransferase, is essential for mammalian germ line DNA methylation. Only one Dnmt3a is identified in mammals, and homozygous mutants of Dnmt3a are lethal, while two Dnmt3a paralogs, dnmt3aa and dnmt3ab, are identified in teleosts due to the third round of genome duplication, and homozygous mutants of dnmt3aa and dnmt3ab are viable in zebrafish. The expression patterns and roles of dnmt3aa and dnmt3ab in gonadal development remain poorly understood in teleosts. In this study, we elucidated the precise expression patterns of dnmt3aa and dnmt3ab in tilapia gonads. Dnmt3aa was highly expressed in oogonia, phase I and II oocytes and granulosa cells in ovaries and spermatogonia and spermatocytes in testes, while dnmt3ab was mainly expressed in ovarian granulosa cells and testicular spermatocytes. The mutation of dnmt3aa and dnmt3ab was achieved by CRISPR/Cas9 in tilapia. Lower gonadosomatic index (GSI), increased apoptosis of oocytes and spermatocytes and significantly reduced sperm quality were observed in dnmt3aa-/- mutants, while normal gonadal development was observed in dnmt3ab-/- mutants. Consistently, the expression of apoptotic genes was significantly increased in dnmt3aa-/- mutants. In addition, the 5-methylcytosine (5-mC) level in dnmt3aa-/- gonads was decreased significantly, compared with that of dnmt3ab-/- and wild type (WT) gonads. Taken together, our results suggest that dnmt3aa, not dnmt3ab, plays important roles in maintaining gametogenesis in teleosts.