Mutation of spexin2 promotes feeding, somatic growth, adiposity, and insulin resistance in zebrafish.

Spexin2 (spx2) is a newly identified gene in vertebrates, but its biological functions remain unclear. In this study, we cloned the full-length cDNA of spx2 in zebrafish. The 288-bp open reading frame encodes a protein of 95 amino acids that contains a 14 amino acids mature peptide. Spx2 is highly expressed in brain and testis. Its expression was significantly downregulated in the hypothalamus after feeding treatment and 7 days of food deprivation. Using a zebrafish spx2-/- mutant line, we observed a greater amount of food intake and changes in mRNA levels of feeding factors. We found that, SPX2 acts as a satiety factor that inhibits food intake by downregulating the expression of agouti-related neuropeptide (agrp). Moreover, spx2 mutant fish exhibited a larger body size, excessive lipid accumulation, and insulin resistance. Taken together, our results revealed that SPX2 functions as a satiety factor involved in energy metabolic regulation in zebrafish.

Distinct Roles of Estrogen Receptors in the Regulation of Vitellogenin Expression in Orange-Spotted Grouper (Epinephelus coioides).

During their breeding season, estrogen induces vitellogenin (VTG) production in the liver of teleost fish through estrogen receptors (ERs) that support oocyte vitellogenesis. There are at least three ER subtypes in teleost fish, but their roles in mediating E2-induced VTG expression have yet to be ascertained. In this study, we investigated the expression of vtgs and ers in the liver of orange-spotted grouper (Epinephelus coioides). Their expression levels were significantly increased in the breeding season and were upregulated by an estradiol (E2) injection in female fish, except for the expression of erß1. The upregulation of vtgs, erα and erß2 by E2 was also observed in primary hepatocytes, but these stimulatory effects could be abolished by ER antagonist ICI182780 treatment. Subsequent studies showed that ERß antagonist Cyclofenil downregulated the E2-induced expression of vtg, erα, and erß2, while the ERß agonist DPN simulated their expression. Knockdown of erß2 by siRNA further confirmed that ERß2 mediated the E2-induced expression of vtgs and erα. To reveal the mechanism of ERß2 in the regulation of erα expression, the erα promoter was cloned, and its activity was examined in cells. E2 treatment simulated the activity of the erα promoter in the presence of ERß2. Deletions and site-directed mutations showed that the E2 up-regulated transcriptional activity of erα occurs through a classical half-estrogen response element- (ERE) dependent pathway. This study reveals the roles of ER subtypes in VTG expression in orange-spotted grouper and provides a possible explanation for the rapid and efficient VTG production in this species during the breeding season.

A single intronic single nucleotide polymorphism in splicing site of steroidogenic enzyme hsd17b1 is associated with phenotypic sex in oyster pompano, Trachinotus anak.

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.

Knockout of tac3 genes in zebrafish shows no impairment of reproduction.

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.

Physical interactions facilitate sex change in the protogynous orange-spotted grouper, Epinephelus coioides.

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.

Molecular cloning and functional characterization of growth hormone-releasing hormone in Mastacembelus armatus.

Growth hormone-releasing hormone (GHRH) is a neuropeptide that controls growth hormone (GH) synthesis and release. In this study, the full-length cDNA of Mastacembelus armatus ghrh was obtained by rapid amplification of cDNA ends method. Sequence analysis showed that the cloned sequence is 1090 bp in length, containing an open reading frame (ORF) of 429 bp that encodes a precursor protein of 142 amino acids. Sequence alignment revealed that the 27-amino acid mature peptide of Ghrh in M. armatus is conserved. Real-time PCR showed that ghrh is highly expressed in the brain, with very low or no expression in other tissues. During embryonic and larval development, ghrh expression was low in embryos but increased gradually in the stages of larval development. The biological function of Ghrh peptide was further investigated in vivo. Ghrh injection could significantly upregulate the mRNA expression of growth hormone (gh) and insulin-like growth factor-1/2 (igf-1/2) in M. armatus. Our data indicate that Ghrh is able to activate the GH-IGFs axis in M. armatus.

Transcriptome profiling of laser-captured germ cells and functional characterization of zbtb40 during 17alpha-methyltestosterone-induced spermatogenesis in orange-spotted grouper (Epinephelus coioides).

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.

Molecular characterization and functional analysis of IKKα in orange-spotted grouper (Epinephelus coioides).

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 IKKß-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.

Retinoic acid and androgen influence germ cells development and meiotic initiation in juvenile orange-spotted grouper, Epinephelus coioides.

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.

Hybridization of tiger grouper (Epinephelus fuscoguttatus ♀) x giant grouper (Epinephelus lanceolatus ♂) using cryopreserved sperm.

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 < 0.05). Survival rate of the hybrid was lower than that of the controls at 15 days post hatching (23% vs 48%). However, hybrids showed survival rate equal to the controls at the end of the 60-day study period. Hybridization of E. fuscoguttatus x E. lanceolatus was successfully achieved using cryopreserved sperm from giant grouper. The cryopreservation of giant grouper sperm and its application in hybridization provided a technical support for further grouper breeding work.

Comparative transcriptome analysis of diploid and triploid hybrid groupers (Epinephelus coioides♀ × E. lanceolatus♂) reveals the mechanism of abnormal gonadal development in triploid hybrids.

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-17ß 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.

Integration of ATAC-seq and RNA-seq Unravels Chromatin Accessibility during Sex Reversal in Orange-Spotted Grouper (Epinephelus coioides).

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-upon further study-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.

Natural sex change in mature protogynous orange-spotted grouper (Epinephelus coioides): gonadal restructuring, sex hormone shifts and gene profiles.

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 17ß-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.

Identification, functional characterization, and estrogen regulation on gonadotropin-releasing hormone in the spotted scat, Scatophagus argus.

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. 17ß-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.

The co-administration of estradiol/17α-methyltestosterone leads to male fate in the protogynous orange-spotted grouper, Epinephelus coioides.

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 17ß-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.

MicroRNA-182-3p negatively regulates cytokines expression by targeting TLR5M in orange-spotted grouper, Epinephelus coioides.

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.

NKB/NK3 system negatively regulates the reproductive axis in sexually immature goldfish (Carassius auratus).

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.

Whole Genome Sequencing of the Giant Grouper (Epinephelus lanceolatus) and High-Throughput Screening of Putative Antimicrobial Peptide Genes.

Giant groupers, the largest grouper type in the world, are of economic importance in marine aquaculture for their rapid growth. At the same time, bacterial and viral diseases have become the main threats to the grouper industry. Here, we report a high-quality genome of a giant grouper sequenced by an Illumina HiSeq X-Ten and PacBio Bioscience Sequel platform. A total of 254 putative antimicrobial peptide (AMP) genes were identified, which can be divided into 34 classes according to the annotation of the Antimicrobial Peptides Database (APD3). Their locations in pseudochromosomes were also determined. Thrombin-, lectin-, and scolopendin-derived putative AMPs were the three largest parts. In addition, expressions of putative AMPs were measured by our transcriptome data. Two putative AMP genes (gapdh1 and gapdh2) were involved in glycolysis, which had extremely high expression levels in giant grouper muscle. As it has been reported that AMPs inhibit the growth of a broad spectrum of microbes and participate in regulating innate and adaptive immune responses, genome sequencing of this study provides a comprehensive cataloging of putative AMPs of groupers, supporting antimicrobial research and aquaculture therapy. These genomic resources will be beneficial to further molecular breeding of this economically important fish.

Socially controlled male-to-female sex reversal in the protogynous orange-spotted grouper, Epinephelus coioides.

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.

Female-to-male sex reversal in orange-spotted grouper (Epinephelus coioides) caused by overexpressing of Amh in vivo.

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 17ß-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.