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.

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.

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.

Acute hypoxia is a common abiotic stress in commercial aquaculture and has significant effects on fish physiology and metabolism. Due to its large size and rapid growth, the greater amberjack (Seriola dumerili) is an economically important fish with high farming value. This species is intolerant to hypoxia, which makes it susceptible to mass mortality and hinders the progress of amberjack cultivation. Based on a comparative analysis of the liver transcriptome between acute hypoxia-tolerant (HT) and -intolerant (HS) groups, this study first explored the molecular mechanisms of acute hypoxia in greater amberjack. By simulating the acute hypoxic environment and using RNA sequencing (RNA-Seq), the differences in liver transcriptional changes between the acute hypoxia-tolerant (HT) and hypoxia-intolerant (HS) groups of greater amberjack were probed. Based on differential expression analysis, 829 differentially expressed genes (DEGs) were screened in both groups. Relative to the HS group, 374 DEGs were upregulated and 455 were downregulated in the HT group. Compared with the HS group, genes such as slc2a5 and prkaa2 related to promoting sugar transport and inhibiting lipid syntheses were upregulated, while genes that inhibit gluconeogenesis and promote lipid syntheses, such as pgp and aacs, were downregulated. The expression of odc1 was significantly and relatively downregulated in the HT group, which would lead to the inhibition of intracellular antioxidant activity and decreased scavenging of ROS. The NF-kB pathway was also promoted to some extent in individuals in the HT group relative to the HS group to resist apoptosis. In addition, the relative downregulation of apoptosis and autophagy-related genes, such as endog, hm13, and casp6, was also detected in the HT group. The present findings first reported the regulation mechanism by which liver tissue coped with the acute hypoxia stress in greater amberjack, which will provide important technical support for preventing acute hypoxia-induced death in advance and reducing economic losses.

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.

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.