Xi Wu

Background: Membraneless RNA granules play important roles in germ cell development, yet their molecular composition and developmental dynamics remain poorly understood in fish. In this study, we investigated the Balbiani body (BB) and a nuage-associated nucleus-containing region (NNR) in oocytes of leopard coral grouper using laser capture microdissection (LCM), followed by integrated Smart-seq2 and DIA proteomics analyses. Because nuage is difficult to isolate reliably as a standalone compartment, the nucleus was co-dissected together with the surrounding nuage to generate the NNR samples. Results: Ultrastructural observations showed that nuage appeared in oogonia and persisted throughout oocyte development, whereas the BB formed in primary oocytes and gradually expanded and dispersed into the cytoplasm. Integrative analysis of Smart-seq2 and DIA proteomics revealed relatively limited transcriptional variation among BB stages but more pronounced protein-level changes, while the NNR exhibited stage-associated molecular variation. The compartment-biased expression of established marker molecules was consistent with the known molecular characteristics of the BB and the presence of nuage-associated components in the NNR. However, functional signals detected in the NNR were interpreted as exploratory because of the contribution of nuclear molecules. Furthermore, Ddx6 and Igf2bp3 were identified and validated as candidate BB-associated markers. Their mRNA and protein signals corresponded to the BB region, and their stage-dependent localization closely paralleled BB formation, expansion, and dispersion during oocyte development. Conclusions: These findings expand the candidate marker repertoire for the BB and provide a framework for investigating its developmental dynamics in fish oocytes. The molecular characteristics of the NNR provide preliminary information on the nuage-associated region, but their interpretation should remain exploratory because the dissected region contains the nucleus. Further studies using more specific approaches to isolate nuage will be required to clarify its molecular composition and its possible relationship with the BB.

Background: Sex reversal is an intricate and fascinating process orchestrated by a finely organized network. The orange-spotted grouper ( Epinephelus coioides ) is a protogynous hermaphroditic fish, but the regulatory mechanism of its sex reversal is not yet well understood. Results: Here, single-cell transcriptome sequencing (scRNA-seq) of the gonads was performed during sex reversal induced by 17α-methyltestosterone (MT). Based on the scRNA-seq data, various gonadal cell types and subtypes, including eight types of germ cells and nine types of somatic cells, were characterized and identified. Moreover, a single-cell atlas of dynamic changes in gene expression during sex reversal in orange-spotted grouper was constructed. We outlined the precise trajectories of germ stem cells (GSCs) and male somatic cells, which revealed that new male germ cells were originated from a subcluster of GSCs with increased levels of spermatogenesis genes. Moreover, we revealed that piwil1 and dmrt1 were important for the male-oriented differentiation of GSCs. Additionally, oocytes exhibited abnormal and significant apoptosis and active proliferation during the early stage of sex reversal. Conclusions: Overall, these results address the questions of how male germ cells are generated and how female germ cells disappear during sex reversal in orange-spotted grouper. These findings profile a comprehensive dynamic map of gonadal cells during sex reversal, which can provide new insights into sex change in lower vertebrates.

Sex change, a manifestation of sexual plasticity, is widespread among fish and can occur spontaneously or in response to environmental stimuli. Despite its prevalence, the molecular regulatory mechanisms underlying sex change, particularly those involving miRNAs, remain largely unresolved. Orange-spotted grouper (Epinephelus coioides), a hermaphroditic species exhibiting natural female-to-male sex reversal, serves as an excellent model for investigating these processes. In this study, we established a reversible sex change model in E. coioides by inducing precocious female-to-male transition through 17α-methyltestosterone implantation, followed by spontaneous male-to-female reversion. Comprehensive transcriptome analyses were conducted during both transitions to identify differentially expressed miRNAs and their putative target mRNAs. Regulatory networks were constructed, revealing dynamic miRNA-mRNA interactions associated with sex differentiation. Key miRNAs were found to be inversely correlated with sex-related gene expression, suggesting their involvement in the regulation of sex reversal. Functional validation further supported the existence of targeted negative regulatory relationships between specific miRNAs and sex-related genes. Collectively, these findings demonstrate that miRNAs play essential roles in orchestrating bidirectional sex change in orange-spotted grouper, providing new insights into the molecular basis of sex determination and differentiation in teleosts.

The Hulong hybrid grouper was bred from the brown-marbled grouper (Epinephelus fuscoguttatus) ♀ and the giant grouper (E. lanceolatus) ♂, combining the advantageous traits of both parents. Possessing an excellent performance, this hybrid’s cultivation promotes the development of the grouper industry. Its male parent, the giant grouper, possesses the fastest growth and the largest body size among all coral-reef-dwelling fish. This species is not only an economically important species in marine aquaculture, but it is also an ideal male parent in the interspecific crossing of grouper species. In the present study, a high-quality chromosome-level genome of the giant grouper was constructed with a total length of 1.06 Gb, consisting of 24 chromosomes and 69 scaffolds. To analyze the genetic differences between the parents of the Hulong hybrid grouper, the structural variations (SVs) between both parental genomes were detected, and a total of 46,643 SVs were obtained. High-quality SNPs were identified from resequencing data. There were significant differences between the two genomes, and the average F_ST reached 0.685. A total of 234 highly differentiated regions were detected with an F_ST > 0.9. The protein-coding genes involved in SVs and highly differentiated regions were significantly enriched in metabolic pathways, including fatty metabolism, carbohydrate metabolism, amino acid metabolism and the TCA cycle. These genes may be related to the differences in feeding preferences and the ability to digest carbohydrates between the two grouper species under natural conditions. In addition, protein-coding genes related to the cell cycle and p53-signaling pathway were also detected. These genes may play important roles in the regulation of body size and growth performance. This research provides genomic resources for further breeding works and evolutionary analyses.

Brown-marbled grouper is one of the most important mariculture species in China and is widely used as a crossbreeding parent in the grouper industry. Enhancing growth rates is a key target in fish breeding, and gaining insight into the underlying mechanisms responsible for growth differences between individuals can aid in the improvement of grouper growth rates. However, the mechanism behind growth differences in this fish remains unclear. We analyzed the transcriptome profiles of muscle tissues between fast- and slow-growing brown-marbled grouper using RNA-seq and identified 77 significantly up-regulated genes and 92 significantly down-regulated genes in the extreme growth groups. Our findings suggest that up-regulated genes such as ghr and tnni2, as well as down-regulated genes such as stc2 and pdp1, are associated with growth advantages in brown-marbled grouper. We used differentially expressed genes (DEGs) for GO and KEGG enrichment analyses. The results of the GO enrichments showed that the significantly up-regulated genes in the fast-growing group were involved in protein folding, the actin cytoskeleton, the myosin complex, and other processes. The results of the KEGG enrichments showed that the significantly up-regulated genes in the fast-growing group were involved in various pathways such as glycolysis/gluconeogenesis, adipocytokine signaling, MAPK signaling, carbon metabolism, and PI3K-Akt signaling. Additionally, gene set enrichment analysis (GSEA) showed that the PI3K/AKT/mTOR pathway was up-regulated in the fast-growing group, which may be responsible for the higher nutrient absorption efficiency and muscle growth in these fish. Our results contribute to a better understanding of the molecular mechanisms and regulatory pathways underlying fast growth in brown-marbled grouper. However, further studies are necessary to fully elucidate the mechanisms behind growth differences between individuals.

Brown-marbled grouper is one of the most important mariculture species in China, which is used as an important crossbreeding parent in grouper industry. Enhancing growth rates is a key target in fish breeding, and gaining insight into the underlying mechanisms responsible for growth differences among individuals can aid in the improvement of grouper growth rates. However, the mechanism behind this difference in growth in this fish is unclear. The difference of transcriptome profiles of muscle tissue between fast- and slow-growing brown-marbled grouper was analyzed by RNA-Seq. 77 significantly up-regulated genes and 92 significantly down-regulated genes were identified in the growth extreme groups. The up-regulated of ghr and tnni2 and the down-regulated of stc2 led to the growth advantages of brown-marbled grouper. The differently expressed genes (DEGs) were used for GO and KEGG enrichment analysis. The results of GO enrichments indicated that the significantly upregulated genes in the fast-growing group were involved in protein folding, actin cytoskeleton, myosin complex, etc. The results of KEGG enrichments indicated that the significantly upregulated genes in the fast-growing group were involved in glycolysis/ gluconeogenesis, adipocytokine signaling pathway, MAPK signaling pathway, carbon metabolism, PI3K-Akt signaling pathway, etc. To analysis the difference gene sets between fast- and slow-growing group, the RNA-seq data were used by gene set enrichment analysis (GSEA). The results showed that the PI3K/AKT/mTOR pathway was up-regulated in the fast-growing group. The up-regulated of this pathway could lead to higher nutrient absorption efficiency and lead to muscle growth in the fast-growing group. These results contribute to understanding of the molecular mechanisms of fast growth and regulative pathways regulating growth in brown-marbled grouper.

Spermatogonial stem cells (SSCs) are unique adult stem cells capable of self-renewal and differentiation into sperm. Grouper is a protogynous hermaphroditic fish farmed widely in the tropical and subtropical seas. In this study, we established an SSC line derived from adult testis of orange-spotted grouper, Epinephelus coioides. In the presence of basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF), the cells could be maintained with proliferation and self-renewal over 20 months and 120 passages under in vitro culture conditions. The cells exhibited strong alkaline phosphatase activity and the characteristics of SSCs with the expression of germ cell markers, including Vasa, Dazl, and Plzf, as well as the stem cell markers Nanog, Oct4, and Ssea1. Furthermore, the cultured cells could be induced by 11-ketotestosterone treatment to highly express the meiotic markers Rec8, Sycp3, and Dmc1, and produce some spherical cells, and even sperm-like cells with a tail. The findings of this study suggested that the cultured grouper SSC line would serve as an excellent tool to study the molecular mechanisms behind SSCs self-renewal and differentiation, meiosis during spermatogenesis, and sex reversal in hermaphroditic vertebrates. Moreover, this SSC line has great application value in grouper fish aquaculture, such as germ cell transplantation, genetic manipulation, and disease research.

Pluripotency markers Pou5f1 and Nanog are core transcription factors regulating early embryonic development and maintaining the pluripotency and self-renewal of stem cells. Pou5f1 and Nanog also play important roles in germ cell development and gametogenesis. In this study, Pou5f1 (EcPou5f1) and Nanog (EcNanog) were cloned from orange-spotted grouper, Epinephelus coioides. The full-length cDNAs of EcPou5f1 and EcNanog were 2790 and 1820 bp, and encoded 475 and 432 amino acids, respectively. EcPou5f1 exhibited a specific expression in gonads, whereas EcNanog was expressed highly in gonads and weakly in some somatic tissues. In situ hybridization analyses showed that the mRNA signals of EcNanog and EcPou5f1 were exclusively restricted to germ cells in gonads. Likewise, immunohistofluorescence staining revealed that EcNanog protein was limited to germ cells. Moreover, both EcPou5f1 and EcNanog mRNAs were discovered to be co-localized with Vasa mRNA, a well-known germ cell maker, in male and female germ cells. These results implied that EcPou5f1 and EcNanog could be also regarded as reliable germ cell marker genes. Therefore, the findings of this study would pave the way for elucidating the mechanism whereby EcPou5f1 and EcNanog regulate germ cell development and gametogenesis in grouper fish, and even in other protogynous hermaphroditic species.

The humpback grouper (Cromileptes altivelis), an Epinephelidae species, is patchily distributed in the reef habitats of Western Pacific water. This grouper possesses a remarkably different body shape and notably low growth rate compared with closely related grouper species. For promoting further research of the grouper, in the present study, a high-quality chromosome-level genome of humpback grouper was assembled using PacBio sequencing and high-throughput chromatin conformation capture (Hi-C) technology. The assembled genome was 1.013 Gb in size with 283 contigs, of which, a total of 143 contigs with 1.011 Gb in size were correctly anchored into 24 chromosomes. Moreover, a total of 26,037 protein-coding genes were predicted, of them, 25,243 (96.95%) genes could be functionally annotated. The high-quality chromosome-level genome assembly will provide pivotal genomic information for future research of the speciation, evolution and molecular-assisted breeding in humpback groupers. In addition, phylogenetic analysis based on shared single-copy orthologues of the grouper species showed that the humpback grouper is included in the Epinephelus genus and clustered with the giant grouper in one clade with a divergence time of 9.86 Myr. In addition, based on the results of collinearity analysis, a gap in chromosome 6 of the humpback grouper was detected; the missed genes were mainly associated with immunity, substance metabolism and the MAPK signal pathway. The loss of the parts of genes involved in these biological processes might affect the disease resistance, stress tolerance and growth traits in humpback groupers. The present research will provide new insight into the evolution and origin of the humpback grouper.

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.