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The Jumonji family (Jmj family) is a large family of proteins that contain the JmjC domain. Various members of this family are lysine (K)-specific demethylases, which remove methylation from the lysines of histones, and thus, are known as Jmj-KDMs. In this study, all Jmj-KDMs were identified in the orange-spotted grouper Epinephelus coioides and compared with those from Homo sapiens, Danio rerio, Takifugu rubripes, Oreochromis niloticus, and Xenopus tropicalis. The comparison revealed the teleost-specific loss of jmjd1a and the teleost-specific expansion of kdm5b, jmjd3, jmjd2a, and kdm6a in E. coioides. Analysis of the temporal expression pattern of Jmj-KDM genes in E. coioides enabled their division into different types according to their sex-related expression patterns. The kdm6 subfamily genes kdm6aa and jmjd3 (kdm6bb), which are also teleost specific, were selected for further analysis. Quantitative PCR analysis revealed the significantly higher expression of kdm6bb in bisexual-phase gonads and testes compared with ovary. Histological expression pattern analysis based on in situ hybridization (ISH) showed the high expression of kdm6aa in primary-growth stage oocytes, cortical-alveolus stage oocytes and granulosa cells, whereas kdm6aa and jmjd3 were highly expressed in Sertoli cells and spermatogonium. These results suggest that Jmj-KDMs, especially the kdm6 subfamily genes kdm6aa and jmjd3, are involved in sex-related processes in E. coioides.

Mandarin fish (Siniperca chuatsi) is an economically important freshwater fish cultured in China. In this species, females grow faster than males. However, due to the lack of available genomic and transcriptome information, the mechanisms of sex differentiation remain poorly understood. In this study, Illumina high-throughput sequencing technology was used to sequence four cDNA libraries; the tissues examined included hypothalamus, pituitary gland, ovary, and testis. A total of 134 124 high-quality unigenes were obtained on average length of 1 361 bp and N50 of 3 312 bp. A search of all-unigene sequences against NR, NT, SwissProt, KOG, KEGG, GO, and InterPro databases resulted in 59 688 (44.50%), 76 329 (56.91%), 50 432 (37.60%), 45 741 (34.10%), 48 760 (36.35%), 5 241 (3.91%), and 46 099 (34.37%) annotations, respectively. In a comparison of ovarian and testicular libraries, 15 289 ovary-biased genes and 10 035 testis-biased genes were identified, including a series of genes related to sex determination and differentiation, such as cyp19a1a, foxl2, sox9, dmrtl, amh, and others. In addition, 49 495 SSRs and 85 899 SNPs were detected in transcriptome data. Quantitative real-time PCR results of 15 sex-related functional genes indicated that RNA-seq data was reliable. This study will contribute to a better understanding of the molecular mechanisms of sex differentiation and development in Mandarin fish.

Sex change in teleost fishes is commonly regulated by social factors. In species that exhibit protogynous sex change, such as the orange-spotted grouper Epinephelus coioides, when the dominant males are removed from the social group, the most dominant female initiates sex change. The aim of this study was to determine the regulatory mechanisms of socially controlled sex change in E. coioides. We investigated the seasonal variation in social behaviours and sex change throughout the reproductive cycle of E. coioides, and defined the behaviour pattern of this fish during the establishment of a dominance hierarchy. The social behaviours and sex change in this fish were affected by season, and only occurred during the prebreeding season and breeding season. Therefore, a series of sensory isolation experiments was conducted during the breeding season to determine the role of physical, visual and olfactory cues in mediating socially controlled sex change. The results demonstrated that physical interactions between individuals in the social groups were crucial for the initiation and completion of sex change, whereas visual and olfactory cues alone were insufficient in stimulating sex change in dominant females. In addition, we propose that the steroid hormones 11-ketotestosterone and cortisol are involved in regulating the initiation of socially controlled sex change.

Gonadotropin-releasing hormone (GnRH) is a key neuropeptide of the reproductive system. However, little is known about the role of GnRH in the spotted scat (Scatophagus argus). Here, three GnRH subtypes (cGnRH-II, sGnRH, and sbGnRH) were identified in the spotted scat. cGnRH-II and sGnRH were only expressed in the brains and gonads of both male and female fish, exhibiting a tissue-specific expression pattern, while sbGnRH was expressed at different transcription levels in all examined tissues. During ovarian maturation, hypothalamus-associated sbGnRH was upregulated, while the expression of sGnRH was variable and cGnRH-II first increased and then decreased. In vivo experiments showed that sbGnRH significantly promoted the expression of fsh and lh genes in a dose-dependent manner and exhibited a desensitization effect on lh expression at high concentrations. For sGnRH and cGnRH-II, only high concentrations could induce fsh and lh expression. Furthermore, treatment with highly concentrated sbGnRH peptide also induced fsh and lh expression, whereas the sGnRH and cGnRH-II peptides only induced fsh expression in vitro. 17β-Estradiol (E2) significantly inhibited the expression of sbGnRH mRNA in a dose-dependent manner and did not impact sGnRH and cGnRH-II mRNA levels in vivo or in vitro. The inhibitory effect of E2 on sbGnRH expression was attenuated by the estrogen receptor (ER) broad-spectrum antagonist (fulvestrant) and the ERα-specific antagonist (methyl-piperidinopyrazole), respectively, implying that the feedback regulation on sbGnRH is mediated via ERα. This study provides a theoretical basis for the reproductive endocrinology of the spotted scat by studying GnRH.

Inhibitor of nuclear factor kappa-B kinase subunit alpha (IKKα) plays crucial roles in regulating activation of nuclear factor kappa-B (NF-κB) in response to pathogens infections. Here, we cloned and identified IKKα gene of orange-spotted grouper (Epinephelus coioides), named as EcIKKα. The gene transcript contained a 2262 bp open reading frame, which encoded 753 amino acids. The typically conserved IKKα structure, including serine kinase domain (KD), leucine chain (LZ) structure, helix-loop-helix (HLH) motif and IKKβ-NEMO-binding domain, was identified in EcIKKα. Phylogenetic analysis suggested that EcIKKα had the closest relationship with large yellow croaker (Larimichthy crocea) IKKα. Ecikkα was ubiquitously expressed in all tissues tested and the highest expression level was in ovary. After lipopolysaccharide (LPS), flagellin, polyinosinic-polycytidylic acid (poly I:C), polyadenylic-polyuridylic acid (poly A:U), and Vibrio parahaemolyticus stimulation, the expression of Ecikkα increased in grouper spleen (GS) cells. In the luciferase assay, NF-κB-luc activity was significantly up-regulated when human embryonic kidney 293T (HEK 293T) cells were transfected with EcIKKα plasmid. Moreover, overexpression of EcIKKα significantly increased LPS- and flagellin-induced proinflammatory cytokines (interleukin-6 (il-6) and tumor necrosis factor-α (tnf-α)) expression, but did not significantly affect poly I:C- and poly A:U-induced cytokines (il-6 and tnf-α) expression. Overall, these results suggested that EcIKKα functions like that of mammals to activate NF-κB, and it could be involved in host defense against invading pathogens.

Teleosts show varied master sex determining (MSD) genes and sex determination (SD) mechanisms, with frequent turnovers of sex chromosomes. Tracing the origins of MSD genes and turnovers of sex chromosomes in a taxonomic group is of particular interest in evolutionary biology. Oyster pompano ( Trachinotus anak ), a marine fish, belongs to the family Carangidae, in which 17b-hydroxysteroid dehydrogenase 1 ( hsd17b1 ) has repeatedly evolved to an MSD gene. Whole-genome resequencing identified a single nucleotide polymorphism (SNP) at chromosome 24 to be strictly associated with phenotypic sex, with females being the heterozygous sex. This SNP is located in a splicing site at the first exon/intron boundary of hsd17b1 . The Z-linked SNP results in malfunction of all spliced isoforms, whereas the W-linked isoforms were predicted to have open reading frames that are conserved among vertebrates, suggesting that hsd17b1 is a female-determining gene. The differential alternative splicing patterns of ZZ and ZW genotypes were consistently observed both in undifferentiated stages and differentiated gonads. We observed elevated recombination around the SD locus and no differentiation between Z and W chromosomes. The extreme diversity of mutational mechanisms that hsd17b1 evolves to an MSD gene highlights frequent in situ turnovers between sex chromosomes in the Carangidae.

Toll-like receptors (TLRs) as essential pattern recognition receptors in innate immunity, can recognize pathogens and trigger immune response to eliminate invading pathogens. MicroRNAs regulates multiple biological processes by suppressing mRNA translation or resulting in mRNA degradation. MiR-182 has previously been implicated in DNA repair, disease and cancer aspects. The potential role of miR-182-3p in TLR signaling pathway against pathogens is unclear. In this study, we found that the expression of miR-182-3p was up-regulated after Vibrio parahaemolyticus flagellin stimulation in grouper spleen (GS) cells, and negatively correlated with the expression of orange-spotted grouper (Epinephelus coioides) TLR5M (EcTLR5M). Then we found that miR-182-3p could directly target EcTLR5M by using bioinformatic analysis and dual-luciferase reporter assay. Dual-luciferase reporter assay also showed that miR-182-3p down-regulated the wild-type EcTLR5M 3'UTR in luciferase activity rather than the mutant group in HEK 293T cells. We further verified the effect of miR-182-3p on the activation of Nuclear factor-κB (NF-κB) signaling pathway, and found that miR-182-3p inhibitors significantly augmented flagellin-induced NF-κB phosphorylation. Additionally, we also demonstrated that the increased expression of miR-182-3p significantly suppressed the flagellin-induced EcTLR5M, pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) mRNA expression. And the endogenous miR-182-3p knockdown experiments reversely verified the regulatory effect of miR-182-3p. These results suggested that miR-182-3p post-transcriptionally controls EcTLR5M expression and thereby suppresses the expression of pro-inflammatory cytokines. This study is the first to demonstrate that miR-182-3p suppresses pro-inflammatory cytokines expression by regulating the TLR signaling pathway.

To ascertain the significance of the Neurokinin B/Tachykinin 3 receptor (NKB/NK3) system in goldfish reproduction, two cDNAs encoding tachykinin 3 receptors, namely tacr3a and tacr3b, were cloned. Subsequent studies revealed that the downstream signalling of both Tac3rs can be activated by different NKB peptides, suggesting that the cloned receptors are biologically functional in goldfish. RT-PCR analysis showed that tacr3s are widely expressed in brain regions. During the gonadal development, tacr3a and tacr3b exhibited different expression patterns in the hypothalamus and pituitary. The actions of NKB peptides on reproductive axis was further investigated in vivo. Intraperitoneal injections of NKB peptides significantly reduced the expression of kiss2 and gonadotropin releasing hormone 3 (gnrh3) in the hypothalamus, and the expression of luteinizing hormone beta subunit (lhb) and follicle stimulating hormone beta subunit (fshb) in the pituitary in sexually immature goldfish. Taken together, our findings revealed that NKB/NK3 system plays a negative role in the reproductive axis of immature goldfish.

Oocyte maturation and hydration are regulated by a complex interplay of various hormones and local factors. We have investigated the morphological changes of follicles and serum steroid levels during the HCG (human choionic gonadotophin)-induced oocyte maturation in the orange-spotted grouper. For the first time, a large-scale transcriptomic analysis of follicles during the maturation has been conducted in a fish species which produce pelagic oocytes. Eight cDNA libraries of follicle samples, from full-grown immature follicles to mature follicles, were constructed. A total of 402,530,284 high-quality clean reads were obtained after filtering, 79.66% of which perfectly mapped to the orange-spotted grouper genome. Real-time PCR results of 12 representative genes related to oocyte maturation and hydration verified the reliability of the RNA-seq data. A large number of genes related to oocyte maturation and hydration were identified in the transcriptome dataset. And the transcriptomic analysis revealed the dynamic changes of the steroid synthesis pathway and the pathway of hydration during oocyte maturation. The present study will facilitate future study on the oocyte maturation and hydration in the orange-spotted grouper and other marine pelagic egg spawner.

The spotted scat (Scatophagus argus) is an economically important cultured marine fish that exhibits a typical sexual size dimorphism (SSD). SSD has captivated considerable curiosity for farmed fish production, however, up till now the exact underlying mechanism remains largely unclear. As an important digestive and metabolic organ, the liver plays key roles in the regulation of fish growth. It is necessary to elucidate its significance as a downstream component of the hypothalamic-pituitary-liver axis in the formation of SSD. In this study, the liver physiological differences between the sexes were evaluated in S. argus, and the activity of several digestive and metabolic enzymes were affected by sex. Females had higher amylase, protease, and glucose-6-phosphate dehydrogenase activities, while males exhibited markedly higher hepatic lipase and antioxidant enzymes activities. A comparative transcriptomics was then performed to characterize the responsive genes. Illumina sequencing generated 272.6 million clean reads, which were assembled into 79,115 unigenes. A total of 259 differentially expressed genes were identified and a few growth-controlling genes such as igf1 and igfbp1 exhibited female-biased expression. Further analyses showed that several GO terms and pathways associated with metabolic process, particularly lipid and energy metabolisms, were significantly enriched. The male liver showed a more active mitochondrial energy metabolism, implicating an increased energy expenditure associated with reproduction. Collectively, the female-biased growth dimorphism of S. argus may be partially attributed to sexually dimorphic metabolism in the liver. These findings would facilitate further understanding of the nature of SSD in teleost fish.

Sex-specific markers are extremely important for unisexual breeding and understanding of sex determination mechanisms. Spotted mandarin fish (Siniperca scherzeri) and big eye mandarin fish (Siniperca kneri) are economically important freshwater fish in China. Here, for the first time, we successfully identified three male-specific markers in the two species. Through genome sequencing and bioinformatic analysis of three males, three females, one male mixed pool and one female mixed pool of spotted mandarin fish, we screened 27 potential male-specific sequences. After PCR validation, three male-specific markers were further identified. Moreover, these markers could also be used to identify genetic sex of spotted mandarin fish from different populations with 100 % accuracy. In addition, these markers were also successfully used in the identification of genetic sex of big eye mandarin fish. Notably, the sequences of male-specific markers were highly similar in the two species and the corresponding sequence of one marker was highly similar to partial genome sequence of amhy gene in mandarin fish (Siniperca chuatsi). In all, the verified male-specific markers suggested XY sex determination system existed in spotted mandarin fish and big eye mandarin fish and would play an important role in future work of sex control breeding.

Chromatin structure plays a pivotal role in maintaining the precise regulation of gene expression. Accessible chromatin regions act as the binding sites of transcription factors (TFs) and cis-elements. Therefore, information from these open regions will enhance our understanding of the relationship between TF binding, chromatin status and the regulation of gene expression. We employed an assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and RNA-seq analyses in the gonads of protogynous hermaphroditic orange-spotted groupers during sex reversal to profile open chromatin regions and TF binding sites. We focused on several crucial TFs, including ZNF263, SPIB, and KLF9, and analyzed the networks of TF-target genes. We identified numerous transcripts exhibiting sex-preferred expression among their target genes, along with their associated open chromatin regions. We then investigated the expression patterns of sex-related genes as well as the mRNA localization of certain genes during sex reversal. We found a set of sex-related genes that&mdash, upon further study&mdash, might be identified as the sex-specific or cell-specific marker genes that trigger sex reversal. Moreover, we discovered the core genes (gnas, ccnb2, and cyp21a) of several pathways related to sex reversal that provide the guideposts for future study.

Sexual patterns of teleosts are extremely diverse and include both gonochorism and hermaphroditism. As a protogynous hermaphroditic fish, all orange-spotted groupers (Epinephelus coioides) develop directly into females, and some individuals change sex to become functional males later in life. This study investigated gonadal restructuring, shifts in sex hormone levels and gene profiles of cultured mature female groupers during the first (main) breeding season of 2019 in Huizhou, China (22° 42' 02.6″ N, 114° 32' 10.1″ E). Analysis of gonadal restructuring revealed that females with pre-vitellogenic ovaries underwent vitellogenesis, spawning and regression and then returned to the pre-vitellogenic stage in the late breeding season, at which point some changed sex to become males via the intersex gonad stage. A significant decrease in the level of serum 17β-estradiol (E2) was observed during ovary regression but not during sex change, whereas serum 11-ketotestosterone (11-KT) concentrations increased significantly during sex change with the highest concentration in newly developed males. Consistent with serum hormone changes, a significant decrease in cyp19a1a expression was observed during ovary regression but not during sex change, whereas the expression of cyp11c1 and hsd11b2 increased significantly during sex change. Interestingly, hsd11b2 but not cyp11c1 was significantly upregulated from the pre-vitellogenic ovary stage to the early intersex gonad stage. These results suggest that a decrease in serum E2 concentration and downregulation of cyp19a1a expression are not necessary to trigger the female-to-male transformation, whereas increased 11-KT concentration and upregulation of hsd11b2 expression may be key events for the initiation of sex change in the orange-spotted grouper.

Beta-hydroxysteroid dehydrogenases (β-HSDs) are a group of steroidogenic enzymes that are involved in steroid biosynthesis and metabolism, and play a crucial role in mammalian physiology and development, including sex determination and differentiation. In the present study, a genome-wide analysis identified the numbers of β-hsd genes in orange-spotted grouper (Epinephelus coioides) (19), human (Homo sapiens) (22), mouse (Mus musculus) (24), chicken (Gallus gallus) (16), xenopus (Xenopus tropicalis) (24), coelacanth (Latimeria chalumnae) (17), spotted gar (Lepisosteus oculatus) (14), zebrafish (Danio rerio) (19), fugu (Takifugu rubripes) (19), tilapia (Oreochromis niloticus) (19), medaka (Oryzias latipes) (19), stickleback (Gasterosteus aculeatus) (17) and common carp (Cyprinus carpio) (27) samples. A comparative analysis revealed that the number of β-hsd genes in teleost fish was no greater than in tetrapods due to gene loss followed by a teleost-specific whole-genome duplication event. Based on transcriptome data from grouper brain and gonad samples during sex reversal, six β-hsd genes had relatively high expression levels in the brain, indicating that these genes may be required for neurogenesis or the maintenance of specific biological processes in the brain. In the gonad, two and eight β-hsd genes were up- and downregulated, respectively, indicating their important roles in sex reversal. Our results demonstrated that β-hsd genes may be involved in the sex reversal of grouper by regulating the synthesis and metabolism of sex steroid hormones.

In this study, we injected cortisol into the protogynous orange-spotted grouper (Epinephelus coioides) to investigate the role of this hormone in sex change. Following injection, we evaluated gonadal changes, serum levels of steroid hormones, and sex-related gene expression during the processes of cortisol-induced sex change and cortisol withdrawal in the orange-spotted grouper. Cortisol treatment caused the degeneration of oocytes and induced sex change in a dose-dependent manner. Over the long-term, we observed a significant increase in serum 11-ketotestosterone (11-KT) levels in all cortisol-treated groups, although levels of 17β-estradiol did not change significantly. Consistent with the elevation of serum 11-KT levels, the expression of genes related to testicular development was also significantly up-regulated in the cortisol-treated groups. Based on our results, we propose that cortisol may trigger masculinization by inducing the synthesis of 11-KT and by directly activating the expression of sex-related genes. Furthermore, we found that cortisol-induced sex change was not permanent and could be reversed after the withdrawal of cortisol treatment.

Background Spermatogenesis is an intricate process regulated by a finely organized network. The orange-spotted grouper (Epinephelus coioides) is a protogynous hermaphroditic fish, but the regulatory mechanism of its spermatogenesis is not well-understood. In the present study, transcriptome sequencing of the male germ cells isolated from orange-spotted grouper was performed to explore the molecular mechanism underlying spermatogenesis. Results In this study, the orange-spotted grouper was induced to change sex from female to male by 17alpha-methyltestosterone (MT) implantation. During the spermatogenesis, male germ cells (spermatogonia, spermatocytes, spermatids, and spermatozoa) were isolated by laser capture microdissection. Transcriptomic analysis for the isolated cells was performed. A total of 244,984,338 clean reads were generated from four cDNA libraries. Real-time PCR results of 13 genes related to sex differentiation and hormone metabolism indicated that transcriptome data are reliable. RNA-seq data showed that the female-related genes and genes involved in hormone metabolism were highly expressed in spermatogonia and spermatozoa, suggesting that these genes participate in the spermatogenesis. Interestingly, the expression of zbtb family genes showed significantly changes in the RNA-seq data, and their expression patterns were further examined during spermatogenesis. The analysis of cellular localization of Eczbtb40 and the co-localization of Eczbtb40 and Eccyp17a1 in different gonadal stages suggested that Eczbtb40 might interact with Eccyp17a1 during spermatogenesis. Conclusions Our study, for the first time, investigated the transcriptome of the male germ cells from orange-spotted grouper, and identified functional genes, GO terms, and KEGG pathways involved in spermatogenesis. Furthermore, Eczbtb40 was first characterized and its role during spermatogenesis was predicted. These data will contribute to future studies on the molecular mechanism of spermatogenesis in teleosts.

Luteinizing hormone receptor (LHR) plays a critical role in reproduction by mediating LH signaling in the gonad. In this study, we cloned a novel lhr gene from the orange-spotted grouper, named glhr2 . The cloned complete open reading frame sequence of glhr2 was 2082 bp in length, encoding a protein of 693 amino acids, sharing approximately 50% amino acid identity with glhr1 . glhr1 and glhr2 were primarily expressed in gonad, brain and hypothalamus with low expression in other tissues such as gill, spleen, etc. The expressions of both glhr1 and glhr2 increased during vitellogenesis, while decreased during natural female to male sex change. The two gLHRs both could be activated by equine LH or human chorionic gonadotropin, but not by human follicle stimulating hormone. Both gLHR1 and gLHR2 activation stimulated the expression of cAMP response element driven reporter gene in a dose-dependent manner, while gLHR2 but not gLHR1 also activated serum response element driven reporter gene expression. This was the first study to demonstrate that two active LHRs exist in fish with possible different functional roles.

A variety of mechanisms are involved in sex determination in vertebrates. The orange-spotted grouper (Epinephelus coioides), a teleost fish, functions first as females and later as a male and is an ideal model to investigate the regulation of sexual fate. Here, we report female-to-male sex reversal in juvenile orange-spotted groupers caused by overexpressing anti-Müllerian hormone (Amh). Tissue distribution analyses showed that amh and amhrII primarily expressed in the gonad, and expression level in the testis was much higher than that in the ovary. In gonads, the expression of amh was located in the Sertoli cells around spermatogonia of the testis and in the zona pellucida of the mature ovary, and the expression of amhrII was located in the Sertoli cells of the testis and in the oocytes of the ovary. Decrease in female-related genes and serum 17β-estradiol level, increase in male-related genes and serum 11-ketotestosterone, ovarian regression, and spermatogonia proliferation were observed during plasmid feeding experiment. These results illustrate that amh overexpression plasmid feeding can induce a female-to-male transition in grouper.

Steroid hormones are essential for the normal reproductive activity in females and males, such as early sex differentiation, gonad development and sex reversal, which works by binding with the steroid receptors. In the present study, the full-length of estrogen receptors (ERα, ERβ1 and ERβ2) and androgen receptors (ARα and ARβ) cDNAs were isolated from the mandarin fish (Siniperca chuatsi). Real time quantitative PCR (RT-qPCR) revealed that ERα and ERβ2 expressed mainly in the liver of females and males, and ERβ1 mostly distributed in ovary and testis. Additionally, the high expressions of ARα and ARβ in both females and males were detected in gonads and livers respectively. During the early sex differentiation stage, the expressions of ERs and ARs in female were higher than in male during 5–30 day after hatching (dah), and reached the peak level at 30 dah. In males, the expressions of ERβ1, ERβ2, and ARβ increased largely during 30 dah to 60 dah, and revealed significantly higher levels than in females during 60–180 dah. In female, the expression of ERβ2 in ovary increased sharply at 180 dah. Moreover, the result of gonadal histology revealed that the spermatocytes were observed at 45 dah and the spermatozoon could be found largely at 60 dah in males and oil droplets appeared for the first time in the oocytes at 180 dah. The expression levels of three ERs and ARβ were up-regulated and the ARα was down-regulated during female to male sex reversal induced by 17α-methyltestosterone (MT). Furthermore, after MT treatment for 30 and 35 days, the expressions of ERβ1, ERβ2 and ARβ were significantly up-regulated. These results suggest that ERβ1, ERβ2 and ARβ may play critical roles in spermatogenesis and masculinization, while ERβ2 and ERβ1 may be essential for vitellogenesis and ovary development in female.

Neurokinin B (NKB) plays a pivotal role in the regulation of reproduction in vertebrates. However, whether this neuropeptide is dispensable for reproduction in teleosts remains unknown. In order to reveal its authentic functions in fish, in this study, two tachykinin 3 (tac3) genes encoding Nkbs were functional mutated in zebrafish using the Transcription Activator-like Effector Nucleases (TALEN) technology. We established tac3a-/-, tac3b-/- and tac3a-/-;tac3b-/- mutant lines, and investigated their reproductive performance and ontogeny. According to our study, spermatogenesis and folliculogenesis were not impaired in tac3a-/-, tac3b-/- and tac3a-/-;tac3b-/- mutant lines, but changes in the expression of genes related to reproductive axis were observed after loss of Tac3, suggesting that possible compensatory response was activated to maintained the reproductive function in zebrafish. In summary, our results indicate that mutation of tac3 genes do not disrupt the reproduction in zebrafish unlike in mammals, revealing the plasticity of reproductive neuroendocrine system in the brain of zebrafish.

Dead end (dnd), vertebrate-specific germ cell marker, had been demonstrated to be essential for primordial germ cell (PGC) migration and survival, and the link between PGC number and sex change had been revealed in some teleost species, but little is known about dnd in hermaphroditic vertebrates. In the present study, a protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides) dnd homologue (Ecdnd) was identified and characterized. Quantitative real-time PCR and in situ hybridization analysis revealed a dynamic and sexually dimorphic expression pattern in PGCs and germ cells of gonads. During sex changing, the Ecdnd transcript sharply increased in early transitional gonad, reached the highest level at late transitional gonad stage, and decreased after testis maturation. Visualization of zebrafish PGCs by injecting with RFP-Ecdnd-3'UTR RNA and GFP-zfnanos3-3'UTR RNA confirmed importance of Ecdnd 3'UTR for the PGC distribution. In addition, knockdown of EcDnd by using antisense morpholinos (MO) caused the ablation of PGCs in orange-spotted grouper. Therefore, the current data indicate that Ecdnd is essential for PGCs survival and may serve as a useful germ cell marker during gametogenesis in hermaphroditic grouper.

It is generally believed that kisspeptin and gonadotropin-releasing hormone (GnRH) are required for reproduction in vertebrates. In this study, we generated gnrh3-null zebrafish and found that gnrh3 mutation did not impair gonad development and reproductive capacity. Moreover, zebrafish triple knockout mutant lacking gnrh3 and the 2 kiss1s genes undergo normal puberty and gonad maturation. The expression of follicle-stimulating hormone beta (fshβ) and luteinizing hormone beta (lhβ) was not significantly altered whereas the expression of neuropeptide Y (npy), tachykinin 3 (tac3), and secretogranin-II (sgII) was significantly increased in the triple knockout mutant, suggesting that compensation mechanisms exist to stimulate the reproductive axis in the absence of kiss and gnrh. Our results challenge the prevailing view that GnRH is indispensable for reproduction across species. These data provide genetic evidence that different mechanisms have evolved for the neuroendocrine control of reproduction between mammals and fish: pulsatile release of GnRH to the portal system is the final gateway to stimulate the reproductive axis in mammals, whereas multiple factors act in parallel with GnRH to stimulate the reproductive axis in certain fish species.

Using Ringer solution as extender, the present study examined the protective effect of dimethyl sulphoxide (Me2SO; 8-12%, v/v) on the cryopreservation of giant grouper (Epinephelus lanceolatus) sperm. The cryopreserved sperm was then successfully applied in interspecific hybridization with tiger grouper (E. fuscoguttatus). Higher motility (90.56 ± 6.58%) and fertilization rate (69.61 ± 4.83%) was achieved in 10% Me2SO with Ringer solution as extender (dilution ratio 1:1), which should no significant difference in comparison with fresh sperm (95.88 ± 1.64% and 73.10 ± 1.28%). There were no statistical differences in both fertilization and hatching rates between hybrid and non-hybrid tiger grouper by using cryopreserved sperm for fertilization, but malformation rate of the hybrid was higher than non-hybrid (17%) (P

Vasa and dazl genes have been reported to play pivotal roles in germ cell development and differentiation both in vertebrates and invertebrates; however, little is known about their functions in germ cell differentiation during gametogenesis and sex reversal in hermaphroditic fish. In the present study, vasa (Ecvasa) and dazl (Ecdazl) cDNA were cloned from orange-spotted grouper (Epinephelus coioides). The full-length cDNA sequences of Ecvasa and Ecdazl were 2162 and 2101 bp, and encoded 646 and 214 amino acid residues, respectively. Reversal transcription PCR showed that Ecvasa and Ecdazl mRNA were highly expressed in the gonads. Further, in situ hybridization revealed that Ecvasa and Ecdazl RNA were dynamically expressed in germ cells at different stages during oogenesis, sex reversal, and spermatogenesis in orange-spotted grouper. Intriguingly, the signals for Ecvasa and Ecdazl mRNA became weaker in oocytes of ovo-testes gonads, indicating that the expression of germ cell genes could be suppressed in oocytes during sex reversal in the orange-spotted grouper. Our study is the first time to describe the expression profiles of vasa and dazl mRNA in germ cells during gametogenesis and sex reversal in the orange-spotted grouper. These findings will provide new insights into understanding the mechanisms through which vasa and dazl regulate germ cell differentiation in hermaphrodite fish species.

The red-spotted grouper (Epinephelus akaara) is one of the most commercially important aquatic species in China. However, its seedstock has low larval survival rates, and its stability is confronted with the danger of overexploitation. In this study, a high-density genetic map was constructed using 3435 single nucleotide polymorphisms (SNPs) from 142 first generation (F1) full-sib offspring and two parents of a red-spotted grouper population. The total genetic length of the map was 2300.12 cM with an average intermarker distance of 0.67 cM. Seventeen genome-wide significant quantitative trait loci (QTLs) for growth-related traits were detected on 24 linkage groups, including 5 QTLs for full length, 7 QTLs for body length, and 5 QTLs for body weight. The contribution values of explained phenotypic variance ranged from 10.7% to 12.9%. Moreover, 13 potential candidate genes for growth-related traits were identified. Collectively, these findings will be useful for conducting marker-assisted selection of the red-spotted grouper in future studies.

Meiosis is essential for germ cells development for all sexually reproducing species. Retinoic acid (RA) is the key factor controlling the sex-specific timing of meiotic initiation in mammals, birds and tetrapods. Here, we investigated the effects of RA on meiotic initiation and sex determination in protogynous hermaphrodite orange-spotted grouper (Epinephelus coioides). Expression profile investigations of meiotic marker genes during gonadal development indicated that germ cells undergone meiosis approximately at 180 days after hatching in the orange-spotted grouper. RA synthase inhibitor treatments on juvenile orange-spotted groupers resulted in impeded germ cells development and delayed meiotic initiation with simultaneous down-regulation of vasa, dazl, sycp3 and rec8, which was rescued by exogenous RA administration. Additionally, exogenous androgen treated fish showed a delayed meiotic initiation consistent with decreased sycp3 and rec8 expression and were directed to a spermiogenesis fate. Our results imply that meiotic initiation in the orange-spotted grouper is strongly influenced by RA and androgen, and the regulation of meiotic initiation may involve in the spermatogenesis induced by exogenous androgen.

Background: Spermatogenesis is an intricate process regulated by a finely organized network. The orange-spotted grouper (Epinephelus coioides) is a protogynous hermaphroditic fish, but the process of its spermatogenesis is not well-understood. In the present study, transcriptome sequencing of the male germ cells from orange-spotted grouper was performed to explore the molecular mechanisms underlying spermatogenesis. Results: In this study, the orange-spotted grouper was induced to change sex from female to male by 17alpha-methyltestosterone implantation. During the artificial spermatogenesis, different cell types from cysts containing spermatogonia, spermatocytes, spermatids, and spermatozoa were isolated by laser capture microdissection. Subsequently, transcriptomic analysis for the isolated cells were performed. A series of genes was used to verify and investigate the expression patterns in spermatogenesis. Furthermore, we also analyzed the expression of the same set of genes involved with steroid metabolism and sex throughout spermatogenesis (early-mid, late, and maturing stages) in the orange-spotted grouper. Several generally female-related genes took significantly changes in sex reversal hinted that the female-related genes in previously recognized may also play vital roles in spermatogenesis and sex reversal. In the transcriptomic data, we focused on zbtb family genes, which may be related to the process of spermatogenesis. Their expression patterns and cellular localization were examined, and the location of Eczbtb40 in different gonadal stages was investigated. We found that Eczbtb40 was expressed throughout spermatogenesis. These preliminary findings suggest that Eczbtb40 is highly conserved during vertebrate evolution and plays roles in spermatogenesis. Besides, the expression of Eczbtb40 and Eccyp17a1a overlapped in male germ cells, especially spermatogonium and spermatocyte, which suggested that Eczbtb40 might interact with Eccyp17a1a participant in spermatogenesis and sex reversal. Conclusions: The present study first depicted RNA sequencing of the male germ cells from orange-spotted grouper, and identified many important functional genes and pathways involved in spermatogenesis. The Eczbtb40 gene was subjected to molecular characterization and expression pattern analysis. These results will contribute to future studies of the molecular mechanism of spermatogenesis and sex reversal.

Follicle-stimulating hormone (FSH) signaling is considered to be essential for early gametogenesis in teleosts, but its functional roles during sex differentiation are largely unknown. In this study, we investigated the effects of long-term and short-term FSH injection on sex differentiation in the protogynous orange-spotted grouper (Epinephelus coioides). Long-term FSH treatment initially promoted the formation of ovaries but subsequently induced a male fate. The expression of female pathway genes was initially increased but then decreased, whereas the expression of male pathway genes was up-regulated only during long-term FSH treatment. The genes related to the synthesis of sex steroid hormones, as well as serum 11-ketotestosterone and estradiol, were also up-regulated during long-term FSH treatment. Short-term FSH treatment activated genes in the female pathway (especially cyp19a1a) at low doses but caused inhibition at high doses. Genes in the male pathway were up-regulated by high concentrations of FSH over the short term. Finally, we found that low, but not high, concentrations of FSH treatment activated cyp19a1a promoter activities in human embryonic kidney (HEK) 293 cells. Overall, our data suggested that FSH may induce ovarian differentiation or a change to a male sex fate in the protogynous orange-spotted grouper, and that these processes occurred in an FSH concentration-dependent manner.

Sex differentiation in teleost fishes occurs in response to sex determination signals, which induce the gonad to develop as either an ovary or testis. However, sex differentiation mechanisms in fishes are diverse, and information on gonad differentiation in sex changing fishes remains limited. The orange-spotted grouper (Epinephelus coioides) is a protogynous hermaphroditic fish that provides an ideal model for investigating gonad differentiation in vertebrates. In this study, Transcriptome data showed that expression levels of amh and amhrII in gonads were increased during sex differentiation. Then we investigated the effect of overexpression anti-Mullerian hormone (Amh) on gonad development in juvenile orange-spotted groupers. Expression levels of female-related genes and serum 17β-estradiol levels were decreased, while expression of male-related genes and serum 11-ketotestosterone levels were increased in fish fed with amh-plasmid. Overexpression of Amh was also promoted the spermatogonia proliferation and induced the development of male gonads in undifferentiated orange-spotted groupers, but that this male tendency was preceded by female differentiation. In summary, these results illustrated that Amh overexpression by amh-plasmid feeding induced male gonad development in undifferentiated groupers.

Sex dimorphism is a common phenomenon in animals. Blotched snakehead (Channa maculata) is an economically important freshwater fish with a faster growth rate in males. However, existing sex-specific markers were too few and difficult to use in production practice, which hindered the development of all-male breeding. Here, we generated 15.64, 20.77 and 29.03 Gb paired-end genomic sequence reads for three male libraries, 12.82, 19.36 and 28.33 Gb for three female libraries and 30.93 Gb for a female mixed pool libraries to screen male-specific sequences. After assembly, we obtained a male reference genome with 541,932 assembled sequences and a female reference genome containing 853,706 assembled sequences. After mapping, 23 male-specific sequences were identified. Six candidate male-specific markers were successfully validated using PCR reaction. Moreover, based on homology analysis and coverage information, we found a candidate X chromosome sequence that was partially similar to the corresponding Y chromosome sequence, and two resulting primers were successfully used in the identification of female (XX), male (XY) and supermale (YY) individuals. In general, a deep genome sequencing method was first provided to effectively enrich large sex-specific segments of Blotched snakehead, which would greatly contribute to elucidation of its sex determination mechanisms and development of all-male breeding.

Leptin is an essential hormone for the regulation of energy metabolism and food intake in vertebrate animals. To better understand the physiological roles of leptin in nutrient regulation in paternal ovoviviparous fish (family Syngnathidae), the present study cloned the full-length of leptin-a and leptin receptor (lepr) genes in lined seahorse (Hippocampus erectus). Results showed that there was a 576-bp intron between two exons in leptin-a gene but no leptin-b gene in seahorse. Although the primary amino acid sequence conservation of seahorse leptin-a was very low, the 3-D structure modeling of seahorse leptin-a revealed strong conservation of tertiary structure with other vertebrates. Seahorse leptin-a mRNA was highly expressed in brain, whereas lepr mRNA was mainly expressed in ovary and gill. Interestingly, both leptin-a and lepr mRNA were expressed in the brood pouch of male seahorse, suggesting the leptin system plays a role during the male pregnancy. Physiological experiments showed that the expression of hepatic leptin-a and lepr mRNA in unfed seahorses was significantly higher than that in those fed 100%, as well as 60%, of their food during the fasting stage, showing that seahorse might initiate the leptin system to regulate its energy metabolism while starving. Moreover, the expression of leptin-a in the brood pouch of pregnant seahorse was significantly upregulated compared with non-pregnant seahorse, whereas the expression of lepr was downregulated, suggesting that the leptin system might be involved in the male pregnancy. In conclusion, the leptin system plays a role in the energy metabolism and food intake, and might provide new insights into molecular regulation of male pregnancy in seahorse., Summary: This study provided insights into the function of the leptin system in energy metabolism regulation and male pregnancy in lined seahorse (Hippocampus erectus).

Spexin is a newly discovered neuropeptide in vertebrates. Comprehensive comparative studies are required to unveil its biological functions. In order to ascertain the neuroendocrine function of spexin in orange-spotted grouper, its full-length cDNA and genomic DNA sequences were cloned and analyzed. Sequence analyses showed that the spexin gene structure is composed of six exons and five introns, and the amino acids of mature peptide (spexin-14) in grouper are identical to that of other fish. Tissue expression analysis found that grouper spexin is highly expressed in the brain, liver and ovary. Real time-PCR analysis demonstrated that the hypothalamic expression of spexin declined gradually during the ovarian development, and was up-regulated by food deprivation. Intraperitoneal administration of spexin-14 peptides to grouper significantly elevated the mRNA levels of proopiomelanocortin (pomc) and suppressed the orexin expression in the hypothalamus, but could not change the hypothalamic expression of gonadotropin releasing hormone 1 (gnrh1). Both in vivo and in vitro administration of spexin could not significantly influence the expression of follicle-stimulating hormone β (fshβ) and luteinizing hormone β (lhβ) in the pituitary with the exception of an inhibition of gh expression. Our data suggested that the spexin has a significant role in the regulation of energy metabolism and food intake in orange-spotted grouper.

Growth hormone (GH) is the most important endocrine factor to regulate somatic growth. Spotted scat (Scatophagus argus) is a famous marine aquaculture species in China with a typical sexual growth dimorphism in which females grow faster and larger than males. In this study, gh messenger RNA (gh mRNA) and GH protein expression were examined in the pituitary glands of female and male spotted scat. Based on qPCR analysis, gh mRNA was mainly expressed in the pituitary gland, and weakly in the gonads and hypothalamus. Furthermore, gh mRNA expression in the pituitary gland was significantly higher in females at stages II-IV than in males at stages III-V. In addition, gh mRNA was highly expressed in the ovary and testis during mature development stages. In this study, spotted scat GH polyclonal antibody was produced. Western blot analysis showed that the molecular weight of spotted scat GH was about 21 KDa. Immunohistochemistry (IHC) in pituitary glands showed that GH was mainly expressed in the proximal pars distal (PPD) and a few cells were distributed in the rostral pairs distal (RPD). After injecting 17β-Estradiol (E2) in vivo, gh mRNA expression was significantly up-regulated in the pituitary gland, whereas igf1 and ghr1 mRNA levels were down-regulated in the liver, which might regulate gh mRNA expression in the pituitary gland. These results provide valuable insight into the molecular mechanisms of E2 regulating gh expression in spotted scat.

Sex-specific markers are the prerequisite for interpreting the mechanisms of sex determination and sex control breeding in fish. Here, we provided a simple next generation sequencing strategy to identify potential sex-specific sequences in mandarin fish (Siniperca chuatsi) that is a greatly important freshwater fish in China. Through genome assembly, reads mapping, sequencing depth and coverage analysis of sequenced genomes of three males and three females, we identified 212 potential male-specific sequences. After two rounds of verification, a total of eight primer pairs were identified as potential candidate markers to be able to distinguish the genetic sex of mandarin fish. Subsequently, the best three markers were further tested in another four different farmed populations of mandarin fish, and the authenticity was 100%. Moreover, based on these markers, eight neomales were demonstrated to be genetic females and about 1: 1 sex ratio was observed in 215 juvenile offspring from two full-sib families. Notably, a duplicate of anti-Mullerian hormone, a gene participating in sex determination process in fish, was identified from the verified male-specific sequences. Overall, this study provided a simple and effective next generation sequencing method to identify sex-specific sequences and the male-specific molecular markers suggested an XY sex determination system in mandarin fish.

In aquaculture, oocyte maturation, ovulation and spawning are important for successful breeding and seed production. Most marine teleost fish can be induced to spawn regularly using intramuscular injections of human chorionic gonadotropin (HCG). In this study, an intra-ovarian injection method was developed in orange-spotted grouper to deal with the broodstocks unresponsive to HCG. Successful oocyte maturation and ovulation in the unilateral injected ovary was induced within 35 h of treatment. An in vitro method was then established to induce HCG-primed oocyte maturation. HCG or maturation-inducing hormones (MIHs) induced the maturation of oocytes with a diameter between 490 and 560 μm. Biochemical, physiological and molecular changes during oocyte maturation and hydration were also characterized. The concentration of free amino acids except the cysteine increased during oocyte maturation, whereas Na+ decreased steadily and K+ increased. The expression of genes encoding cathepsin B, D, L, and the subunits of Na+, K+-ATPase were upregulated during hydration. Specific inhibitors for cathepsin and Na+, K+-ATPase were used to further confirm their crucial roles in oocyte hydration both in vivo and in vitro. These results provide valuable data for inducing oocyte maturation and evaluating oocyte quality in marine teleost fish.

Cyp19a1a is a key gene responsible for the production of estradiol-17β (E2), the main functional estrogen and a major downstream regulator of reproduction in teleost fish. It is widely known that CYP19 gene expression, aromatase activity, and E2 production can influence gonadal differentiation and sex reversal in teleost fish, but the feedback mechanisms whereby E2 regulates cyp19a1a remain poorly understood, especially regarding the potential roles of endogenous small RNA molecules (miRNAs). Here, we identified miR-26a-5p as a regulatory factor of its predicted target gene (cyp19a1a). In vitro and in vivo studies showed that miR-26a-5p can decrease cyp19a1a expression. Furthermore, high doses of E2 act as a repressor of miR-26a-5p. This study proposes a regulatory feedback loop whereby E2 regulates cyp19a1a through miR-26a-5p, and suggests that this positive feedback is an important aspect of the control of E2 production.

The kisspeptin/GPR54 system plays a crucial role in the regulation of the reproductive axis in vertebrates. Male pregnancy and ovoviviparity are special reproductive phenomena among vertebrates. To better understand the neuroendocrine mechanisms of male pregnancy, cDNAs encoding kiss2 and GPR54 were cloned and functionally characterized from the lined seahorse, Hippocampus erectus, an ovoviviparous teleost with male pregnancy. The core mature peptide of seahorse Kiss2 is high conserved among seahorses, but unique among vertebrate Kiss orthologs. In the phylogenic analysis, the seahorse Kiss clustered with the teleost Kiss2 clade. The kiss2 transcripts were shown to be widely expressed in various tissues, notably in the brain and gonad of the seahorse, while GPR54-2 mRNA was expressed exclusively in the brain. In addition, kiss2 mRNA found in male seahorse brain tissue increased significantly at the early pubertal stage, and decreased significantly during pregnancy. Intraperitoneal administration of seahorse Kiss2-10 to sexual mature male seahorses demonstrated to stimulate lutropin β (LHβ) and follitropin β (FSHβ) release and increased serum testosterone levels. In summary, we first identified the kisspeptin/GPR54 system in an ovoviviparous fish with male pregnancy, which might be involved in the regulation of the reproductive functions of pubertal onset, gonadal development, and male pregnancy via regulating the synthesis of both gonadotropic hormone (GTH) and testosterone.

In this study, we systematically investigated the process of sex reversal induced by 17-methyltestosterone (MT) feeding and MT-feeding withdrawal at the ovary differentiation stage in orange-spotted groupers, Epinephelus coioides. Gonadal histology showed that MT feeding induced a precocious sex reversal from immature ovaries to testes, bypassing the formation of an ovarian cavity, and MT-feeding withdrawal led to an ovarian fate. In both the MT feeding and MT-feeding withdrawal phases, cytochrome P450 family 11 subfamily B (cyp11b) gene expression and serum 11-KT levels were not significantly changed, suggesting that the MT-treated fish did not generate endogenous steroids, even though active spermatogenesis occurred. Finally, by tracing doublesex-expressing and Mab-3-related transcription factor 1 (dmrt1)-expressing cells and TUNEL (terminal deoxynucleotidyl transferase 2-deoxyuridine, 5-triphosphate nick end labeling) assays, we found that the efferent duct formed first, and then, the germ cells and somatic cells of the testicular tissue were generated around the efferent duct during MT-feeding-induced precocious sex reversal. Collectively, our findings provide insights into the molecular and cellular mechanisms underlying sex reversal induced by exogenous hormones during sex differentiation in the protogynous orange-spotted grouper.

Chemokine receptor Cxcr4 evolved two paralogs in the teleost lineage. However, cxcr4a and cxcr4b have been characterized only in a few species. In this study, we identified two cxcr4 paralogs from the orange-spotted grouper, Epinephelus coioides. The phylogenetic relationship and gene structure and synteny suggest that the duplicated cxcr4a/b should result from the teleost-specific genome duplication (Ts3R). The teleost cxcr4 gene clusters in two paralogous chromosomes exhibit a complementary gene loss/retention pattern. Ec_cxcr4a and Ec_cxcr4b show differential and biased expression patterns in grouper adult tissue, gonads, and embryos at different stages. During embryogenesis, Ec_cxcr4a/b are abundantly transcribed from the neurula stage and mainly expressed in the neural plate and sensory organs, indicating their roles in neurogenesis. Ec_Cxcr4a and Ec_Cxcr4b possess different chemotactic migratory abilities from the human SDF-1α, Ec_Cxcl12a, and Ec_Cxcl12b. Moreover, we uncovered the N-terminus and TM5 domain as the key elements for specific ligand–receptor recognition of Ec_Cxcr4a-Ec_Cxcl12b and Ec_Cxcr4b-Ec_Cxcl12a. Based on the biased and divergent expression patterns of Eccxcr4a/b, and specific ligand–receptor recognition of Ec_Cxcl12a/b–Ec_Cxcr4b/a, the current study provides a paradigm of sub-functionalization of two teleost paralogs after Ts3R.

Socially controlled sex change in teleosts is a dramatic example of adaptive reproductive plasticity. In many cases, the occurrence of sex change is triggered by a change in the social context, such as the disappearance of the dominant individual. The orange-spotted grouper Epinephelus coioides is a typical protogynous hermaphrodite fish that changes sex from female to male and remains male throughout its life span. In this study, male-to-female sex reversal in male Epinephelus coioides was successfully induced by social isolation. The body length and mass, gonadal change, serum sex steroid hormone levels and sex-related gene expression patterns during the process of socially controlled male-to-female sex reversal in E. coioides were systematically examined. This report investigates the physiological mechanisms of the socially controlled male-to-female sex reversal process in a protogynous hermaphrodite grouper species. The results enable us to study the physiological control of sex change, not only from female to male, but also from male to female.

Estrogen plays a pivotal role in the sex differentiation of teleosts, whereas the precise function of androgens is more controversial. In this study, orange-spotted grouper (Epinephelus coioides) fry were treated with letrozole (an aromatase inhibitor, AI), 17α-methyltestosterone (MT), or MT and 17β-estradiol (E2) simultaneously, during the period of gonadal formation and sex differentiation. MT feeding at 50 days after hatching resulted in gonadal dysgenesis, which could be rescued by E2 supplementation. Different doses of AI treatment led to different phenotypes: undifferentiated gonads were maintained in the AI group fed a low dose (5 mg/kg diet), whereas female-to-male sex reversal was observed in the AI group fed a high dose (100 mg/kg diet). MT and MT + E2 treatment could induce female-to-male sex reversal during sex differentiation (90 days after hatching). The expression of female pathway genes was suppressed, while the expression of genes in the male pathway was up-regulated in the MT + E2 group. Consistent with the expression of sex-related genes, the serum 11- ketotestosterone level was also upregulated in MT and MT + E2 group. Finally, we examined the expression of male-specific mark (DMRT1) and proliferating cell nuclear antigen in MT and MT + E2 induced sex reversal, and the result indicated that male germ cells and somatic cells may origin from the gonium and proliferative somatic cells surrounding the efferent duct, respectively. Overall, our data suggested that estrogen acts as a natural inducer of female differentiation, and that the co-administration of estrogen and androgen during sex differentiation leads to a male sex fate in the protogynous orange-spotted grouper.

In our previous studies, diploid and triploid hybrids have been detected from the hybridization of Epinephelus coioides ♀ × E. lanceolatus ♂. The triploid groupers have been found to be delayed in gonadal development , but the mechanism remains poorly understood. In this study, we examined the gonadal development, assayed the serum steroid hormone levels, and compared the BP (brain and pituitary) and G (gonad) transcriptomes of 18-month-old diploid and triploid hybrids. The results showed that levels of serum estradiol-17β and testosterone were significantly higher in triploid groupers. The RNA-seq data revealed that 1518 and 14,963 differentially expressed genes were identified in the BP and G transcriptome, respectively. Further analysis revealed that the expression levels of genes involved in the sexual differentiation pathway and sex steroid synthesis pathway are significantly higher in triploid hybrids. Our findings provided a comprehensive insight into a better understanding of the regulatory mechanisms of sterility in triploid hybrid fish.

Neurokinin B (NKB) is a member of the tackykinin (TAC) family known to play a critical role in the neuroendocrine regulation of reproduction in mammals. However, its biological functions in teleosts are less clear. The aim of this study was to determine the role of NKB in fish reproduction using goldfish as a model. Two transcripts, TAC3a and TAC3b, which encode several NKBs, including NKBa-13, NKBa-10, NKBb-13, and NKBb-11, were cloned. Phylogenetic analysis revealed that NKBa-10 and NKBb-11 are closely related to mammalian NKB, while NKB-13s are more conserved in teleosts. Quantitative real-time PCR analyses in various tissues showed that TAC3a and TAC3b mRNAs were mainly expressed in the brain. In situ hybridization further detected TAC3a and TAC3b mRNAs in several regions of the brain known to be involved in the regulation of reproduction and metabolism, as well as in the neurohypophysis of the pituitary. To investigate the potential role of NKBs in reproduction, goldfish were injected intraperitoneally with synthetic NKBa-13, -10, NKBb-13, or -11 peptides and the mRNA levels of hypothalamic gonadotropin-releasing hormone (GnRH) and pituitary gonadotropin subunits were measured. NKBa-13, -10, or NKBb-13, but not -11, significantly increased hypothalamic salmon GnRH and pituitary FSHβ and LHβ mRNA levels in both female and male goldfish. Finally, ovariectomy increased, while estradiol replacement reduced, TAC3a mRNA levels without affecting TAC3b expression in the hypothalamus. These data suggest that NKBa-13, -10, and NKBb-13 play a role in mediating the estrogen negative feedback regulation of gonadotropins.

Insulin-like growth factor (Igf) is the key regulator for development, growth, and reproduction. In most vertebrate species, the Igf family has two forms: Igf1 and Igf2. A novel form of Igf, termed Igf3, was recently discovered in fish. In the present study, we isolated igf3 from the orange-spotted grouper (Epinephelus coioides). The orange-spotted grouper igf3 consisted of a full-length cDNA of 1014 nucleotides with an open reading frame (ORF) of 597 bp, encoding for proteins of 199 amino acid residues in length. Tissue distribution analysis showed that igf1 widely expressed with the highest expression in the pituitary and liver. igf2 was expressed highly in all the tissues except the olfactory bulb, while igf3 showed the highest expression in the ovary, and moderate expression in brain areas. The expression profiles of three igf genes during the ovarian development and growth hormone (Gh) and human chorionic gonadotropin (hCG) treatment were also investigated. Three igf genes exhibited different expression patterns during the ovarian development, and showed different responses to the Gh and hCG treatments, appearing to play distinct roles in ovarian development. The present study provides further evidence for the existence of an intraovarian Igf system in orange-spotted grouper.

Neuromedin U (NMU) and neuromedin S (NMS) play inhibitory roles in the regulation of food intake and energy homeostasis in mammals. However, their functions are not clearly established in teleost fish. In the present study, nmu and nms homologs were identified in several fish species. Subsequently, their cDNA sequences were cloned from the orange-spotted grouper (Epinephelus coioides). Sequence analysis showed that the orange-spotted grouper Nmu proprotein contains a 21-amino acid mature Nmu peptide (Nmu-21). The Nms proprotein lost the typical mature Nms peptide, but it retains a putative 34-amino acid peptide (Nmsrp). In situ hybridization revealed that nmu- and nms-expressing cells are mainly localized in the hypothalamic regions associated with appetite regulation. Food deprivation decreased the hypothalamic nmu mRNA levels but induced an increase of nms mRNA levels. Periprandial expression analysis showed that hypothalamic expression of nmu increased significantly at 3 h post-feeding, while nms expression was elevated at the normal feeding time. I.p. injection of synthetic Nmu-21 peptide suppressed the hypothalamic neuropeptide y (npy) expression, while Nmsrp administration significantly increased the expression of npy and orexin in orange-spotted grouper. Furthermore, the mRNA levels of LH beta subunit (lhβ) and gh in the pituitary were significantly down-regulated after Nmu-21 peptide administration, while Nmsrp was able to significantly stimulate the expression of FSH beta subunit (fshβ), prolactin (prl), and somatolaction (sl). Our results indicate that nmu and nms possess distinct neuroendocrine functions and pituitary functions in the orange spotted grouper.

The grass carp is an important farmed fish, accounting for similar to 16% of global freshwater aquaculture, and has a vegetarian diet. Here we report a 0.9-Gb draft genome of a gynogenetic female adult and a 1.07-Gb genome of a wild male adult. Genome annotation identified 27,263 protein-coding gene models in the female genome. A total of 114 scaffolds consisting of 573 Mb are anchored on 24 linkage groups. Divergence between grass carp and zebrafish is estimated to have occurred 49-54 million years ago. We identify a chromosome fusion in grass carp relative to zebrafish and report frequent crossovers between the grass carp X and Y chromosomes. We find that transcriptional activation of the mevalonate pathway and steroid biosynthesis in liver is associated with the grass carp's adaptation from a carnivorous to an herbivorous diet. We believe that the grass carp genome could serve as an initial platform for breeding better-quality fish using a genomic approach.

Wnt4 (Wingless-type MMTV integration site family member 4) has been demonstrated to play critical roles in ovarian development in mammals, but its function in fish reproduction is still unclear. In the present study, two full-length wnt4 cDNA sequences (named wnt4a and wnt4b) were cloned from the orange-spotted grouper (Epinephelus coioides). Amino acid alignment analysis showed that both orange-spotted grouper Wnt4s proteins had the typical characteristics of the Wnt family. RT-PCR revealed that both wnt4a and wnt4b were highly expressed in the ovaries of the orange-spotted grouper. Temporal expression profiles of both wnt4 genes during embryonic and ovarian development were examined. The expressions of wnt4a and wnt4b genes were first detected at the embryonic morula stage, but the gens showed different expression patterns. During ovarian development, high expression of wnt4a was observed in the ovarian lumen formation and gonium proliferation stage, while wnt4b exhibited strong expression in the early developmental stage of oocytes. Taken together, the present study indicates that the two wnt4 genes are involved in the regulation of ovarian development in the orange-spotted grouper.

Gonadotropin-inhibitory hormone (GnIH) and its receptor (GnIHR) play an important role in reproduction regulation in birds, mammals and some teleost species. In protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides), the GnIH/GnIHR signaling pathway and its reproductive function have not been addressed yet. In this study, GnIH and GnIHR in orange-spotted grouper were characterized. gGnIH possessed three putative peptides (gGnIH-I, -II, -III), while gGnIHR showed the characteristics of G protein-coupled receptor and was clustered with GPR147. Functional assays demonstrated that three synthetic gGnIH peptides significantly decreased the forskolin-induced CRE promoter activity, but only gGnIH-I could significantly decrease SRE promoter activity in COS-7 cells transfected with gGnIHR. During the process of ovarian differentiation and development, gGnIH mRNA level in hypothalamus was low at the gonadal primordium stage with gonia, then increased significantly at the early differentiated gonad with primary growth oocytes, while decreased significantly at the developing gonads with cortical-alveolus and vitellogenic stage oocytes. During MT-induced sex reversal, gGnIH mRNA level in hypothalamus increased significantly when the fish completely reversed from female to male. However, gGnIHR mRNA level in pituitary decreased significantly in intersex and completely reversed male fish. Intraperitoneal injection (i.p.) of three gGnIH peptides significantly decreased GnRH1 mRNA levels in hypothalamus, and gGnIH-II significantly inhibited synthesis of LHβ in pituitary. In summary, we firstly identified the GnIH/GnIHR signal in protogynous orange-spotted grouper, which might be involved in the regulation of the reproductive function of sex differentiation, gonadal development and sex reversal via regulating the synthesis of both GnRH and GtH.

Leptin actions at the pituitary level have been extensively investigated in mammalian species, but remain insufficiently characterized in lower vertebrates, especially in teleost fish. Prolactin (PRL) is a pituitary hormone of central importance to osmoregulation in fish. Using goldfish as a model, we examined the global and brain-pituitary distribution of a leptin receptor (lepR) and examined the relationship between expression of lepR and major pituitary hormones in different pituitary regions. The effects of recombinant goldfish leptin-AI and leptin-AII on PRL mRNA expression in the pituitary were further analysed, and the mechanisms underlying signal transduction for leptin-induced PRL expression were determined by pharmacological approaches. Our results showed that goldfish lepR is abundantly expressed in the brain-pituitary regions, with highly overlapping PRL transcripts within the pituitary. Recombinant goldfish leptin-AI and leptin-AII proteins could stimulate PRL mRNA expression in dose- and time-dependent manners in the goldfish pituitary, by both intraperitoneal injection and primary cell incubation approaches. Moreover, the PI3K/Akt/mTOR, MKK3/6/p38MAPK, and MEK1/2/ERK1/2-but not JAK2/STAT 1, 3 and 5 cascades-were involved in leptin-induced PRL mRNA expression in the goldfish pituitary.