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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.

The leopard coral grouper, Plectropomus leopardus, belonging to the family Epinephelinae, is a carnivorous coral reef fish widely distributed in tropical and subtropical waters of the Indo-Pacific. Due to its appealing body appearance and delicious taste, P. leopardus has become a popular commercial fish for aquaculture in many countries. However, the lack of genomic and molecular resources for P. leopardus has hindered study of its biology and genomic breeding programmes. Here we report the de novo sequencing and assembly of the P. leopardus genome using a combination of 10 × Genomics, high-throughput chromosome conformation capture (Hi-C) and PacBio long-read sequencing technologies. The genome assembly has a total length of 881.55 Mb with a scaffold N50 of 34.15 Mb, consisting of 24 pseudochromosome scaffolds. busco analysis showed that 97.2% of the conserved single-copy genes were retrieved, indicating the assembly was almost entire. We predicted 25,248 protein-coding genes, among which 96.5% were functionally annotated. Comparative genomic analyses revealed that gene family expansions in P. leopardus were associated with immune-related pathways. In addition, we identified 5,178,453 single nucleotide polymorphisms based on genome resequencing of 54 individuals. The P. leopardus genome and genomic variation data provide valuable genomic resources for studies of its genetics, evolution and biology. In particular, it is expected to benefit the development of genomic breeding programmes in the farming industry.

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.

Coronavirus disease 2019 (COVID-19) raises the issue of how hypoxia destroys normal physiological function and host immunity against pathogens. However, there are few or no comprehensive omics studies on this effect. From an evolutionary perspective, animals living in complex and changeable marine environments might develop signaling pathways to address bacterial threats under hypoxia. In this study, the ancient genomic model animal Takifugu obscurus and widespread Vibrio parahaemolyticus were utilized to study the effect. T. obscurus was challenged by V. parahaemolyticus or (and) exposed to hypoxia. The effects of hypoxia and infection were identified, and a theoretical model of the host critical signaling pathway in response to hypoxia and infection was defined by methods of comparative metabolomics and proteomics on the entire liver. The changing trends of some differential metabolites and proteins under hypoxia, infection or double stressors were consistent. The model includes transforming growth factor-β1 (TGF-β1), hypoxia-inducible factor-1α (HIF-1α), and epidermal growth factor (EGF) signaling pathways, and the consistent changing trends indicated that the host liver tended toward cell proliferation. Hypoxia and infection caused tissue damage and fibrosis in the portal area of the liver, which may be related to TGF-β1 signal transduction. We propose that LRG (leucine-rich alpha-2-glycoprotein) is widely involved in the transition of the TGF-β1/Smad signaling pathway in response to hypoxia and pathogenic infection in vertebrates as a conserved molecule.

Promoting angiogenesis to restore circulation to the ischemic tissue is still an important therapeutic target in stroke. Here, we ask whether the Ca2+-regulated, phospholipid-and membrane-binding protein-Annexin A2 (ANXA2) may regulate angiogenesis after stroke.Compared with wild type (WT) mice, the density of microvessels in brain and the number of new vessels sprouting from aortic ring were significantly increased in Anxa2 knock-in (ANXA2+/+) mice. After focal cerebral ischemia, proliferation of brain endothelial cells in ANXA2+/+ mice was significantly elevated at 7 days post-stroke, which further improved behavioral recovery. To assess the pro-angiogenic mechanisms of ANXA2, we used brain endothelial cells cultures to investigate its effects on cell tube-formation and migration. Recombinant ANXA2 increased tube-formation and migration of brain endothelial cells either under normal condition or after OGD injury. These protective effects of recombinant ANXA2 were regulated by interaction with ANXA2 receptor (A2R), and the ability of ANXA2-A2R to activate AKT/ERK pathways. Taken together, our study indicates that ANXA2 might be involved in angiogenesis after ischemic stroke. Further investigation of ANXA2-A2R will provide a new therapeutic target for stroke.

Vibrio parahaemolyticus is an important marine pathogen that cause inflammation even death in teleost. It has brought huge economic losses to aquaculture and serious threats to the sustainable development of marine fisheries. Here, we isolated the DNA, RNA, and total flagellin from V. parahaemolyticus, and obtained the primary spleen and head kidney cells (including leukocytes) from Tetraodon nigroviridis. V. parahaemolyticus DNA, RNA, and total flagellin were used to treat the T. nigroviridis primary cells described above. The results show that the nitric oxide (NO) production and respiratory burst response were significantly induced after stimulation with V. parahaemolyticus total flagellin in T. nigroviridis head kidney and spleen cells. And total flagellin could promote the gene expression and protein production of IL-1β in T. nigroviridis leukocytes. T. nigroviridis TLR5M (TnTLR5M) and TLR5S (TnTLR5S) ORF sequences were obtained as the main recognition receptor for flagellin. Real-time fluorescent quantitative PCR (qRT-PCR) was performed to detect the expression of pattern recognition receptor TnTLR5M and TnTLR5S, the important signal molecule of inflammatory system TnMyD88 and TnTRAF6, and inflammatory cytokines TnIL-1β and TnIFN-γ2. The results show that there were a significant upregulation after challenge with V. parahaemolyticus total flagellin. We further demonstrated that the total flagellin of V. parahaemolyticus could activate the luciferase activity of the NF-κB reporter gene mediated by TnTLR5M. Overall, our results suggest that V. parahaemolyticus total flagellin activated the NO production, respiratory burst response, and inflammatory cytokines expressions, such as TnIL-1β and TnIFN-γ2, through the TnTLR5M–NF–κB signaling pathway in T. nigroviridis.

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.

The giant grouper (Epinephelus lanceolatus) is the largest coral reef teleost, with a native range that spans temperate and tropical waters in the Pacific and the Indian Oceans. It is cultured artificially and used as a breeding species in aquaculture due to its rapid growth rate. Here we report a giant grouper genome assembled at the chromosome scale from sequences generated using Illumina and high-throughput chromatin conformation capture (Hi-C) technology. The assembly comprised 1.086 Gb, with 98.4% of the scaffold sequences anchored into 24 chromosomes. The contig and scaffold N50 values were 119.9 kb and 46.2 Mb, respectively. The assembly is of high integrity, including 96.4% universal single-copy orthologues based on BUSCO analysis. Through chromosome-scale evolution analysis, we identified alignments of six giant grouper chromosomes to three stickleback chromosomes and some of the genes located within the breakpoints of reshuffling events may related to development and growth. From the 24,718 protein-coding genes, we found that several gene families related to innate immunity and glycan biosynthesis were significantly expanded in the giant grouper genome compared to other teleost genomes. In addition, we identified several genes related to the hormone signalling pathway and innate immunity that have experienced positive selection or accelerated evolution, implicating their roles in immune defence and fast growth of the species. The high-quality genome assembly will provide a valuable genomic resource for further biological and evolutionary studies, and useful genomic tools for breeding of the giant grouper.

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.

It is well-documented that nitric oxide (NO) is an important regulator of oocyte maturation in mammals. Conversely, the function of NO during oocyte maturation has received little attention in nonmammalian vertebrates. NO is produced from L-arginine through the action of the enzyme NO synthase (NOS). Herein, we examined the expression, hormonal regulation, and involvement of NOS in meiotic signaling in zebrafish oocyte maturation. Three types of nos genes, nos1, nos2a, and nos2b, have been identified in zebrafish. We found that the expression of nos1 was highest in the ovary among the three nos genes, with maximal expression in full-grown (FG)-stage follicles during folliculogenesis. In addition, the concentration of NO was reduced during oocyte maturation and this corresponded with the decreased expression of nos1 in the follicular cell layers, suggesting that NOS1-derived NO may be one of the inhibitors of oocyte maturation in zebrafish. This is the first description of nos1 involvement in oocyte maturation in vertebrates. Moreover, the NO donor SNAP (S-nitroso-l-acetyl penicillamine) partially attenuates human chorionic gonadotropin (hCG)- and 17,20β-P-induced GVBD (germinal vesicle breakdown), perhaps by increasing cGMP levels during oocyte maturation. Finally, our results showed that SNAP and the cGMP analog 8-Br-cGMP inhibited hCG-induced mitogen-activated protein kinase (MAPK) activation, further indicating that NO and cGMP block oocyte maturation in zebrafish.

The kelp grouper, Epinephelus moara, is a carnivorous coral reef fish widely distributing in the Indo-Pacific Oceans. Compared to other grouper species, E. moara is featured for its wide tolerance to environmental stresses, such as temperature and salinity. In addition, it is popularly used as a breeding species for hybrid grouper production. Here, we report the de novo sequencing and assembly of E. moara genome using a combination of Illumina pair-ended, PacBio and Hi-C technologies. We generated a 1.08 Gb genome assembly with a scaffold N50 of 44.93 Mb and contig N50 of 2.22 Mb. The scaffolds and contigs were clustered and oriented into 24 chromosomes, and 24,135 protein-coding genes were predicted, among which 96.8% were annotated. More than 95.6% of the conserved complete genes were successfully retrieved by BUSCO analysis. Comparative genomic analyses showed that some expanded gene families in the E. moara genome were significantly enriched in innate immune pathways. The E. moara genome provides a valuable resource for genetic improvement and genomic breeding of groupers, as well as evolutionary and comparative study with other grouper species.

As a lower vertebrate, the immune defense mechanism of fish mainly depends on the innate immune system. Nucleotide-binding oligomerization domain-like receptors (NLRs) are an important class of pattern recognition receptors in the innate immune system. In this study, NOD1 gene was cloned and characterized in Nile tilapia (Oreochromis niloticus). The ORF of Nile tilapia NOD1 gene was 2826 bp long and encoded 941 amino acid residues with a structure of CARD-NACHT-LRRs that was similar to the other counterparts in mammals and fishes. Phylogenetic and synteny analysis showed that NOD1 was conserved among different fishes and existed at least in the early stage of fish evolution. Expression pattern revealed that NOD1 mRNA was constitutively expressed in the tested tissues, while had high expression level in main immune organs and mucosal immune tissues (liver, head kidney, spleen, blood, gill, and intestine). Following Streptococcus agalactiae challenge, Nile tilapia NOD1 mRNA expression levels were altered in immune organs (liver, head kidney, spleen, blood), and the expression pattern was similar in liver, spleen and blood. Furthermore, the ligand recognition and signaling pathway of Nile tilapia NOD1 were also analyzed, it showed that NOD1 could recognize Tri-DAP intracellularly and activated NF-κB signaling pathway. In summary, our results indicated that the Nile tilapia NOD1 may play an important role in innate immune system and provided a basis for the functional study of NOD1 in teleost.

Toll-like receptors (TLRs) are major pattern recognition receptors (PRRs) that recognize multiple pathogen-associated molecular patterns (PAMPs) through the leucine-rich repeat (LRR) domain and mount effective immune responses. Vibrio parahaemolyticus is the main pathogen that causes vibriosis in aquatic animals, yet the mechanisms of its recognition by innate immune system in teleost fish remain unknown. Here, the results reveal that TLR13 in orange-spotted grouper (Epinephelus coioides) (EcTLR13) recognizes a conserved 23S ribosomal RNA (23S rRNA) sequence in V. parahaemolyticus, and the 13-nucleotide motif near the 23S rRNA ribozyme activation site (VP13) acts as a PAMP. After challenge with RNA and 23S rRNA from V. parahaemolyticus and with the synthetic oligoribonucleotide VP13, the expression of EcTLR13 in grouper spleen cells (GS cells) was significantly increased. EcTLR13-knockdowned GS cells were stimulated with the same stimulants as listed above, the expression of IL-6, IL-12, IL-1β and TNFα was significantly reduced. RNA-protein immunoprecipitation revealed that VP13 could directly bind to EcTLR13. The dual-luciferase reporter assay also showed that EcTLR13 enhanced the fluorescence activity of IFNβ rather than that of NF-κB when the cells were challenged with RNA from V. parahaemolyticus or with synthetic VP13. Our study established the mechanism of fish TLR13-mediated recognition of microbial products during V. parahaemolyticus infection.

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.

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.

Interferon-γ (IFNγ), a type II interferon, is essential to host resistance against various infections. Unlike other vertebrates, fish have two types of IFNγs, IFNγ1 (also named IFNγ-rel) and IFNγ2. MicroRNAs (miRNAs) regulate multiple biological processes by suppressing mRNA translation or inducing mRNA degradation. Among them, miR-29 can directly target IFNγ and affact innate and adaptive immune responses in mice. There are five members of the miR-29 family in orange-spotted grouper (Epinephelus coioides), which share the same miRNA seed region. However, whether miR-29 directly targets E. coioides IFNγs and regulate IFNγ production is still unknown. In the present study, the negative correlation between miR-29b and both IFNγs in immune tissues of healthy E. coioides and grouper spleen cells (GS cells) stimulated with LPS or poly I:C was demonstrated. Moreover, dual-luciferase reporter assays and western blotting were performed to demonstrate that miR-29b suppressed E. coioides IFNγ production. Studies of NO production in GS cells after miR-29b transfection revealed that miR-29b overexpression affected NO production through the downregulation of IFNγ expression. Taken together, our results suggest that miR-29b may directly target E. coioides IFNγs and modulate IFNγ-mediated innate immune responses by suppressing E. coioides IFNγs production.

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.

The effects of the preovulatory luteinizing hormone (LH) surge on the ovulatory process are mediated by prostaglandins (PGs), the synthesis of which involves prostaglandin synthetase and cytosolic phospholipase A2 (cPLA2). In our previous study, we systematically investigated the function of prostaglandin endoperoxide synthase (ptgs) genes on ovulation in zebrafish. However, the role of cPLA2 in ovulation was not determined in zebrafish. In this study, we investigated the function of cpla2α in PGs production and ovulation in periovulatory follicles. Our data showed that the expression of pla2g4aa increased during zebrafish folliculogenesis and the follicular layer was the primary region with expression of pla2g4aa. In addition, the expression of pla2g4aa was regulated by LH in vitro and in vivo. Furthermore, injection of AACOCF3, a specific inhibitor of cPLA2, significantly reduced ovarian PGs level and blocked hCG-induced ovulation. Collectively, these findings suggest that pla2g4aa is related to the ovulation process in zebrafish.

Sexual differentiation and ovotestis development are closely associated with cortisol levels, the principal indicator of stress, via the glucocorticoid receptor (GR) in teleosts. Thus, GR is regarded as a mediator to expound the relationship between social stress and gonad development. In the present study, two gr genes (gr1 and gr2) were cloned and analyzed from a protandrous hermaphroditic teleost, the yellowtail clownfish (Amphiprion clarkii). GR1 was found to display a conserved nine-amino-acid insert, WRARQNTDG, between two zinc finger domains. The phylogenetic tree of GR showed that yellowtail clownfish GR1 and GR2 are clustered to teleost GR1 and teleost GR2 separately, and differ from tetrapod GR. The result of real-time PCR revealed that high-level gr1 was mainly distributed in the cerebellum, hypothalamus and heart. The gr2 gene was abundant in the pituitary and liver of females and nonbreeders, while gr2 was mainly detected in the medulla oblongata and middle kidney of males. Moreover, GRs can be expressed in cultured eukaryotic cells and functionally interact with dexamethasone (exogenous glucocorticoid), thereby triggering downstream signaling pathways of different potentials. GR1 and GR2 can be activated by 10 nM dexamethasone treatment in HEK-293T cells. Notably, real-time PCR analysis among three social status groups demonstrated that gr2 expression was the highest in the hypothalamus of nonbreeders, but gr1 was no difference. We speculate that social stress would increase the expression of gr2 gene expression in the hypothalamus to inhibit sexual development. These data provide evidence of social stress involving reproductive regulation, which may help to elucidate the underlying mechanism of sex differentiation and change.

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.

In this study, we cloned and determined IGFBP-1a cDNA from common carp (Cyprinus carpio) liver. The 1655 bp full-length cDNA consisted of a 96 bp 5-untranslated region (UTR), a 789 bp open reading frame encoding 262 amino acid residues and a 770 bp 3-UTR containing seven mRNA instability motifs. Northern blot revealed a 1.8 kb IGFBP-1a transcript. IGFBP-1a mRNA was widely distributed in all tissues examined and predominantly expressed in the liver. During embryogenesis, IGFBP-1a mRNA was firstly observed in blastula stage, and significant increases were observed in body segment stage, lens formation stage and blood cycling stage. After hatching, its expression increased more than twenty times. Furthermore, hypoxia could significantly up-regulate IGFBP-1a expression in the liver and brain. IGFBP-1a expression increased with ovarian maturation and decreased at regressed stage. In testis, IGFBP-1a mRNA maintained relatively higher levels at recrudescing and matured stages, while it sharply declined at regressed stage. In primary cultured hepatocytes, IGFBP-1a gene was greatly down-regulated by growth hormone via the MAPK and PI3 kinase signaling pathways. These results suggest that IGFBP-1a may be involved in the IGF system regulating growth, development and reproduction in common carp.

Toll-like receptors (TLRs) are important innate immune receptors that recognize multiple pathogen-associated molecular patterns (PAMPs) and activate the immune responses to resist the invasion of pathogens. Many TLRs need assistance from trafficking chaperones to transport to the specific cell compartments and then are processed before they are activated. In this study, we identified an important trafficking chaperone, Unc-93 homolog B1 (unc93b1), from the Epinephelus coioides (orange-spotted grouper). The deduced protein sequence of Eco.unc93b1 was 632 amino acids, containing 12 transmembrane domains, consistent with other UNC93B1 proteins from other species. Phylogenetic analysis showed that Eco.Unc93b1 was clustered with teleost Unc93b1 and had the closest relationship with Larimichthys crocea (large yellow croaker) Unc93b1. Eco.unc93b1 was expressed the highest in the spleen, and its protein was co-localized with the endoplasmic reticulum and early endosomes in both human embryonic kidney 293T cells and grouper spleen cells (GS cells). Moreover, the stimulation of lipopolysaccharide (LPS), high-molecular-weight poly (I:C) (HMW), imidazoquinoline (R848), polyadenylic-polyuridylic acid (poly AU), and 19-mer Staphylococcus aureus 23S rRNA-derived oligoribonucleotide (ORN Sa 19) promoted the mRNA expression of unc93b1 in GS cells with different patterns. Furthermore, the cytokine expression induced by these PAMPs was suppressed, while Eco.unc93b1 was knocked down, by small interfering RNA. In conclusion, these results suggest that Eco.unc93b1 plays an essential role in several PAMP-induced immune responses.

Toll-like receptors (TLRs) are one of the most important innate immune receptors, which recognize various pathogen-associated molecular patterns and activate the downstream immune response. Mouse TLR13 has been found to recognize a highly conserved sequence from bacterial or viral RNA and activate the myeloid differentiation primary response gene 88-dependent signaling response. The function of teleost tlr13 is still not fully understood, especially its relationship with bacterial RNA. In our study, we identified and characterized a tlr13 from Epinephelus coioides (orange-spotted grouper). The full-length cDNA of Eco. tlr13 contained a 2844 bp open reading frame, encoding 947 amino acids. The polypeptide was constitutive of a signal peptide, 13 leucine-rich repeats domains, a C-terminal leucine-rich repeats, a transmembrane domain and a conserved Toll/interleukin (IL)-1 receptor domain, indicating that Eco. Tlr13 exhibited a typical TLR structure. Multiple alignments showed that the Toll/IL-1 receptor domain of Eco. Tlr13 was identical with other homologues, and the phylogenetic tree suggested that Eco. Tlr13 was clustered with other TLR13s and had the closest relationship with predicted Lates calcarifer (sea bass) Tlr13. Subcellular localization analysis revealed that Eco. Tlr13 colocalized with the endoplasmic reticulum and early endosome. Moreover, Eco. tlr13 was broadly observed in all tested tissues with the relatively high expressions in the brain and immune-related tissues. After challenged with 19-mer Staphylococcus aureus 23S ribosomal RNA-derived oligoribonucleotide (ORN Sa19), the expression of Eco. tlr13 was significantly up-regulated in grouper spleen cells. Also, the luciferase assay further revealed that with the overexpression of Eco. Tlr13 in human embryonic kidney 293T cells, ORN Sa19 activated the promoter activity of interferon-β in a dose-dependent pattern. These results indicate that Eco. tlr13 may involve in the recognition of bacterial RNA.

TLR22, a typical member of the fish-specific TLRs, is a crucial sensor in virally triggered innate immune signalling retained from natural selection. To elucidate the role of the TLR22-specific signalling cascade mechanism, we provide evidence that the double-stranded (ds) RNA-sensor TLR22 positively regulates the ERK pathway and negatively regulates the JNK, p38 MAP kinase and NF-κB pathway. Here, we show that TLR22 restrains NF-κB activation and IFN (interferon) β and AP-1 (activator protein-1) promoter binding (impairing “primary response” genes (TNF and IL-1)), induces “secondary response” genes (IL-12 and IL-6) and mediates the irregular expression of inflammatory genes. Therefore, TLR22 promotes ERK phosphorylation but impairs the JNK and p38 MAP kinases and IκB phosphorylation. Additionally, TLR22 controls the excessive generation of reactive oxygen species (ROS) to avoid damaging the organism. The specific kinetics of TLR22 depends on its distinct cellular localization. We demonstrate that TLR22 is an intracellular receptor localized in the endosome, and the TLR22-TIR domain is the functional structure inducing the signalling cascade post-viral replication in the body. As mentioned above, our data reveal a novel mechanism whereby TLR22-induced positive adjustment and negative regulation evolved independently to avoid harmful and inappropriate inflammatory responses.

The pivotal role of androgen receptor (AR) in regulating male fertility has attracted much research attention in the past two decades. Previous studies have shown that total AR knockout would lead to incomplete spermatogenesis and lowered serum testosterone levels in mice, resulting in azoospermia and infertility. However, the precise physiological role of ar in controlling fertility of male fish is still poorly understood. In this study, we have established an ar knockout zebrafish line by transcription activator-like effectors nucleases. Homozygous ar mutant male fish with smaller testis size were found to be infertile when tested by natural mating. Intriguingly, a small amount of mature spermatozoa was observed in the ar mutant fish. These mature spermatozoa could fertilize healthy oocytes, albeit with a lower fertilization rate, by in vitro fertilization. Moreover, the expression levels of most steroidogenic genes in the testes were significantly elevated in the ar mutants. In contrast, the levels of estradiol and 11-ketotestosterone (11-KT) were significantly decreased in the ar mutants, indicating that steroidogenesis was defective in the mutants. Furthermore, the protein level of LHβ in the serum decreased markedly in the ar mutants when compared with wild-type fish, probably due to the positive feedback from the diminished steroid hormone levels.

Spermatogenesis is a process of self-renewal and differentiation in spermatogonial stem cells. During this process, germ cells and somatic cells interact intricately to ensure long-term fertility and accurate genome propagation. Spermatogenesis has been intensely investigated in mammals but remains poorly understood with regard to teleosts. Here, we performed single-cell RNA sequencing of ~9500 testicular cells from the male, orange-spotted grouper. In the adult testis, we divided the cells into nine clusters and defined ten cell types, as compared with human testis data, including cell populations with characteristics of male germ cells and somatic cells, each of which expressed specific marker genes. We also identified and profiled the expression patterns of four marker genes (calr, eef1a, s100a1, vasa) in both the ovary and adult testis. Our data provide a blueprint of male germ cells and supporting somatic cells. Moreover, the cell markers are candidates that could be used for further cell identification.

Neuroinflammation is a key factor of the pathology of various neurological diseases (brain injury, depression, neurodegenerative diseases). It is a complex and orderly process that relies on various types of glial cells and peripheral immune cells. Inhibition of neuroinflammation can reduce the severity of neurological diseases. The initiation, progression, and termination of inflammation require gene activation, epigenetic modification, transcriptional translation, and post-translational regulation, all of which are tightly regulated by different enzymes. Epigenetics refers to the regulation of epigenetic gene expression by epigenetic changes (DNA methylation, histone modification, and non-coding RNAs such as miRNA) that are not dependent on changes in gene sequence and are heritable. Histone deacetylases (HDACs) are a group of important enzymes that regulate epigenetics. They can remove the acetyl group on the lysine ϵ-amino group of the target protein, thereby affecting gene transcription or altering protein activity. HDACs are involved in the regulation of immunity and inflammation. HDAC inhibitor (HDACi) has also become a new hotspot in the research of anti-inflammatory drugs. Therefore, the aim of the current review is to discuss and summarize the role and mechanism of different HDACs in neuroinflammation and the corresponding role of HDACi in neurological diseases, and to providing new ideas for future research on neuroinflammation-related diseases and drug development.

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.

Aromatase (encoded by the cyp19a1a and cyp19a1b genes) plays a central role in sex differentiation in fish, but its precise roles during sex differentiation are still largely unknown. Here, we systematically generated cyp19a1a and cyp19a1b mutant lines as well as a cyp19a1a;cyp19a1b double mutant line in zebrafish using transcription activatorlike effector nucleases. Our results showed that cyp19a1a mutants and cyp19a1a;cyp19a1b double mutants, but not cyp19a1b mutants, had impaired sex differentiation, and all cyp19a1a mutants and cyp19a1a;cyp19a1b double mutants were males. During sex differentiation, the ovary-like gonads were not observed and the male sex differentiation program was delayed in the cyp19a1a-null fish, and these phenotypes could be partially rescued by 17β-estradiol treatment. Gene expression analysis indicated that male and female sex differentiation-related genes were significantly decreased in the cyp19a1a mutant. Collectively, our results revealed dual functions of the cyp19a1a gene during sex differentiation: cyp19a1a is not only indispensable for female sex differentiation but also required for male sex differentiation.

It is well known that ovulation is induced by luteinizing hormone (LH) surge. However, the down-stream factors that mediating LH surge induced ovulation are less clear. The cyclooxygenases (also known as PTGS) as key enzymes for prostaglandins synthesis appear to be important for ovulation in mammals, but their functional roles and molecular mechanism in regulation of fish ovulation are largely unexplored. In this study, we have systematically investigated the expression, regulation and functional roles of cox genes during zebrafish ovulation. Three types of cox genes including ptgs1, ptgs2a and ptgs2b have been identified in zebrafish. The ptgs2a was dominantly expressed in the ovary with a maximal level at the maturation stage of the follicles. In addition, the ptgs2a expression is up-regulated by LH signaling in vitro and in vivo. Moreover, co-injection of a selective Ptgs2 inhibitor and non-selective Ptgs inhibitor with hCG could significantly block the stimulatory effect of hCG induced ovulation in vivo. Collectively, our findings indicate that LH signaling induced ptgs2a expression is required for ovulation in zebrafish.

Kisspeptins are considered critical regulators in the hypothalamic-pituitary-gonadal axis because they can stimulate secretion of Gonadotropin-releasing hormone in mammals, and may also mediate the feedback regulation of sex steroids in the hypothalamus. Two kiss1 paralogues (kiss1 and kiss2) were identified in teleosts, hinting at their increased complexity of signaling for sex-steroid feedback regulation. In the present study, molecular pathways by which 17β-estradiol (E2 ) exerted feedback regulation on two kiss genes, via three types of estrogen receptors, were investigated in the protogynous orange-spotted grouper (Epinephelus coioides). kiss2 expression in the brain significantly increased in ovariectomized orange-spotted groupers, while E2 replacement in ovariectomized fish reversed these changes to levels in the sham-surgery group; conversely, kiss1 expression did not change. Dual-label in situ hybridization showed that kiss1 and kiss2 neurons express erα, erβ1, and erβ2, indicating that E2 may directly regulate kiss1 and kiss2. Indeed, E2 treatment of transiently transfected HEK293T cells decreased the activity of both kiss promoters in the presence of erβ1 and erβ2 rather than erα. Further deletion and site-directed mutagenesis of the kiss promoters indicated that kiss1 is regulated by E2 via an estrogen-responsive element (ERE)-dependent, classical pathway utilized by Erβ1, as well as via an Activator protein 1 (Ap1)-dependent, non-classical pathway utilized by Erβ2. kiss2 was also differently regulated by E2 through the Creb transcription factor, utilized by Erβ1 as well as a half-ERE-dependent, classical pathway utilized by Erβ2. Taken together, multiple signaling pathways in orange-spotted grouper are clearly involved in the feedback regulation of E2 on kiss genes via different estrogen receptors.

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.

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.

Mechanistic target of rapamycin complex 1 (mTORC1) plays crucial roles in male fertility. In mammals, deregulation of mTORC1 led to disordered spermatogonia proliferation and spermatogenesis, which eventually caused infertility in males. However, its roles in male fertility of non-mammalian species remain unclarified. In the present study, it was found that treatment of rapamycin, an mTORC1 inhibitor, resulted in infertility with decreased milt production and sperm motility in zebrafish. However, it is surprising to find that spermatogenesis was normal in these fish. All types of germ cells were found and the proliferation of spermatogonia and spermatocyte were normal. These results suggested that maturation of sperm may be impaired in males treated with rapamycin. Increased apoptosis was found surrounding the lumen containing spermatozoa, implicating a loss of Sertoli cells in testes treated with rapamycin. Moreover, LH/hCG mediated up-regulation of steroidogenic genes was abolished. The expression of npr and ar induced by LH/hCG was also blocked, which further suppressed the signaling of progestin and androgen. Collectively, mTORC1 maintains male fertility via different mechanisms in fish and mammals. mTORC1 is dispensable for spermatogenesis in zebrafish, but possibly supports the maintenance of Sertoli cells and mediates the signaling of hormones, which are crucial for sperm maturation.

Vibriosis is the most common bacterial diseases and brings great economic loss on aquaculture. Vibrio parahaemolyticus ( V. parahaemolyticus) , a gram-negative bacterium, has been identified as one main pathogens of Vibriosis. The pathogenic mechanism of V. parahaemolyticus is not entirely clear now. In our study, a model of V. parahaemolyticus infection of green-spotted puffer fish ( Tetraodon nigroviridis ) was established . T. nigroviridis were injected intraperitoneally (i.p.) with 200 μL of V. parahaemolyticus (8 × 10 10 CFU/mL). V. parahaemolyticus infection caused 64% mortality and infected some organs of T. nigroviridis . Histopathology studies revealed V. parahaemolyticus infection induced tissue structural changes, including adipose hollow space in the liver. Immunohistochemistry showed V. parahaemolyticus were present in infected tissue such as liver, head kidney and spleen. In livers of T. nigroviridis infected by V. parahaemolyticus , the alkaline phosphatases (ALP) activity first gradually increased and then backed to normal level, a trend that was on the contrary to the expression profile of the miR-29b. Quantitative real-time PCR analysis showed that the expression level of TLR1, TLR2, TLR5, TLR9, TLR21, NOD1, NOD2 and IL-6 in response to V. parahaemolyticus infection decreased compared to that of non-infected fish. The establishment of the T. nigroviridis model of V. parahaemolyticus infection further confirmed V. parahaemolyticus spreads through the blood circulation system primary as an extracellular pathogen. Meanwhile, liver is an important target organ when infected by V. parahaemolyticus . miR-29b in liver was involved in the progress of liver steatosis during V. parahaemolyticus infection. Moreover, V. parahaemolyticus infection in vivo may have an effect of immunosuppression on host.

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.

The yellowtail clownfish (Amphiprion clarkii) is a hermaphrodite fish, whose sex differentiation and gonad development are closely related to its social status. The kisspeptin/KissR system is regarded as a key factor mediating social stress on reproductive regulation. In order to understand the effects of social rank stress on the yellowtail clownfish gonadal differentiation, full-length cDNAs of two paralogous genes encoding kisspeptin (kiss1 and kiss2) and KissR (kissr2 and kissr3) were cloned and characterized. The results of real-time PCR showed that kiss1 was primarily expressed in the hypothalamus, and kiss2/kissr2 were abundantly expressed in the liver, while kissr3 was almost exclusively concentrated in the cerebellum and pituitary. Moreover, both Kiss1-10 and Kiss2-10 peptides could initiate downstream signaling pathways by interacting with cognate receptors expressed in eukaryotic cells. Among the three social status groups, the mRNA levels of kiss2 in the hypothalamus and pituitary as well as kissr2 in the pituitary were significantly higher in subordinate individuals (nonbreeders) than dominate individuals (females and males); while the mRNA levels of kissr3 in the hypothalamus and gonad were low in subordinate individuals. Furthermore, the plasma estradiol (E2) and testosterone (T) levels were higher in subordinate than dominate individuals. This study shows that kiss2 is involved in the regulation of social stress on the gonad development in the yellowtail clownfish, but not kiss1.

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.

Estrogens exert their biological functions through the estrogen receptors (ERs). In zebrafish, three nuclear estrogen receptors (nERs) named ERα, ERβ1 and ERβ2 and one membrane-bound G protein-coupled estrogen receptor (GPER) are identified. Vitellogenin (Vtg) is predominantly expressed in liver and strongly response to the stimulation of estrogen. It has been proposed that all three nERs are functionally involved in vitellogenesis and ERα may act as the major mediator in teleost. However, the role of GPER and its interaction with nERs in this process are not yet defined in teleost species. In the present study, we provide genetic evidence for the functional significance of ERα that the expression of Vtg genes (vtg1, vtg2, vtg3) and their response to estradiol stimulation were significantly decreased in esr1 mutant zebrafish. Activation of ERβ1 and ERβ2 induced Vtg expression through ERα. Moreover, the involvement of GPER in vitellogenesis and its interaction with nERs in zebrafish were firstly proposed in this work. Activation of GPER induced Vtg genes expression while inhibition of GPER significantly attenuated the estrogenic effect on Vtg. Both treatments altered the expression levels of nERs, suggesting GPER acts interactively with nERs. Collectively, the involvement of both nERs and GPER in regulation of vitellogenesis is demonstrated. ERα is the central factor, acting interactively with ERβ1, ERβ2 and GPER, and GPER regulates vitellogenesis directly and interactively with nERs.

Vibrio parahaemolyticus is the major pathogen of vibriosis in aquatic animals and causes inflammation that may be related to tissue damage. Here, we have established a V. parahaemolyticus flagellin stimulation model using grouper spleen (GS) cell line. Purified V. parahaemolyticus flagellin was used to stimulate GS cells. Our results showed that the mRNA levels of orange-spotted grouper (Epinephelus coioides) toll-like receptor 5M (EcTLR5M), EcTLR5S and downstream cytokines, such as interferon-γ2 (IFN-γ2), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), were all significantly increased after stimulation with V. parahaemolyticus flagellin in GS cells. Gene silencing of the EcTLR5M and EcTLR5S in GS cells by using small interfering RNA resulted in suppression of the V. parahaemolyticus flagellin-induced cytokines expression. We further demonstrated that activation of both mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) were required for cytokines expression. We observed that the phosphorylation of NF-κB inhibitor-α (IκBα), extracellular signal-regulated kinase (ERK) and p38 were induced following treatment with flagellin. Additionally, most of p65, a NF-κB subunit, was found to translocate to the nucleus after 60 min stimulation. Overall, our results suggest that V. parahaemolyticus flagellin influences cytokines expression, such as IFN-γ2, IL-6 and TNF-α, via EcTLR5s recognition and MAPKs/NF-κB signaling pathway activation in GS cells.

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.

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.