A chromosome-level genome assembly of Hong Kong catfish (Clarias fuscus) uncovers a sex-determining region.

BACKGROUND: Hong Kong catfish (Clarias fuscus) is an ecologically and economically important species that is widely distributed in freshwater regions of southern China. Hong Kong catfish has significant sexual growth dimorphism. The genome assembly of the Hong Kong catfish would facilitate study of the sex determination and evolution mechanism of the species. RESULTS: The first high-quality chromosome-level genome of the Hong Kong catfish was constructed. The total genome was 933.4 Mb, with 416 contigs and a contig N50 length of 8.52 Mb. Using high-throughput chromosome conformation capture (Hi-C) data, the genome assembly was divided into 28 chromosomes with a scaffold N50 length of 36.68 Mb. A total of 23,345 protein-coding genes were predicted in the genome, and 94.28% of the genes were functionally annotated in public databases. Phylogenetic analysis indicated that C. fuscus and Clarias magur diverged approximately 63.7 million years ago. The comparative genome results showed that a total of 60 unique, 353 expanded and 851 contracted gene families were identified in Hong Kong catfish. A sex-linked quantitative trait locus identified in a previous study was located in a sex-determining region of 30.26 Mb (0.02 to 30.28 Mb) on chromosome 13 (Chr13), the predicted Y chromosome. This QTL region contained 785 genes, of which 18 were identified as sex-related genes. CONCLUSIONS: This study is the first to report the chromosome-level genome assembly of Hong Kong catfish. The study provides an excellent genetic resource that will facilitate future studies of sex determination mechanisms and evolution in fish.

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

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

Comparative transcriptome analysis of male and female gonads reveals sex-biased genes in spotted scat (Scatophagus argus).

Scatophagus argus is a new emerging aquaculture fish in East and Southeast Asia. To date, research on reproductive development and regulation in S. argus is lacking. Additionally, genetic and genomic information about reproduction, such as gonadal transcriptome data, is also lacking. Herein, we report the first gonadal transcriptomes of S. argus and identify genes potentially involved in reproduction and gonadal development. A total of 136,561 unigenes were obtained by sequencing of testes (n = 3) and ovaries (n = 3) at stage III. Genes upregulated in males and females known to be involved in gonadal development and gametogenesis were identified, including male-biased dmrt1, amh, gsdf, wt1a, sox9b, and nanos2, and female-biased foxl2, gdf9, bmp15, sox3, zar1, and figla. Serum estradiol-17ß and 11-ketotestosterone levels were biased in female and male fish, respectively. Sexual dimorphism of serum steroid hormone levels were interpreted after expression analysis of 20 steroidogenesis-related genes, including cyp19a1a and cyp11b2. This gonadal transcript dataset will help investigate functional genes related to reproduction in S. argus.

Expression and transcriptional regulation of gsdf in spotted scat (Scatophagus argus).

Gonadal soma-derived factor (Gsdf) is critical for testicular differentiation and early germ cell development in teleosts. The spotted scat (Scatophagus argus), with a stable XX-XY sex-determination system and the candidate sex determination gene dmrt1, provides a good model for understanding the mechanism of sex determination and differentiation in teleosts. In this study, we analyzed spotted scat gsdf tissue distribution and gene expression patterns in gonads, as well as further analysis of transcriptional regulation. Tissue distribution analysis showed that gsdf was only expressed in testis and ovary. Real-time PCR showed that both gsdf and dmrt1 were expressed significantly higher in testes at different phases (phase III, IV and V) compared to ovaries at phase II, III and IV, while gsdf was expressed significantly higher in phase II ovaries than those of phase III and IV. Western blot analysis also showed that Gsdf was more highly expressed in the testis than ovary. Immunohistochemistry analysis showed that Gsdf was expressed in Sertoli cells surrounding spermatogonia in the testis, while it was expressed in the somatic cells surrounding the oogonia of the ovary. Approximately 2.7 kb of the 5' upstream region of gsdf was cloned from the spotted scat genomic DNA and in silico promoter analysis revealed the putative transcription factor binding sites of Dmrt1 and Sf1. The luciferase reporter assay, using the human embryonic kidney cells, demonstrated that Dmrt1 activated gsdf expression in a dose-dependent manner in the presence of Sf1 in spotted scat. These results suggest that Gsdf could play a role in regulating the development of spermatogonia and oogonia, and also participate in male sex differentiation by acting as a downstream gene of Dmrt1 in spotted scat.

Phoenixin: Expression at different ovarian development stages and effects on genes ralated to reproduction in spotted scat, Scatophagus argus.

Phoenixin (Pnx), a recently discovered neuropeptide, has been implicated in reproduction. Pnx mainly exists in two active isoforms, phoenixin-14 (Pnx-14) and phoenixin-20 (Pnx-20). However, little is known about the functions of Pnx in teleosts. To determine the roles of Pnx in the regulation of reproduction in Scatophagus argus, the physiological characterization of the Pnx was analyzed. During ovary development, the expression of pnx in phase IV was higher than in phase II and III in the hypothalamus. In the pituitary, pnx expression was highest in phase IV, moderate in phase III, and lowest in phase II. When hypothalamus and pituitary fragments were cultured in vitro with Pnx-14 and Pnx-20 (10 nM and 100 nM) for 6 h, the expression of GnRHR (gonadotropin releasing hormone receptor), lh (luteinizing hormone) and fsh (follicular stimulating hormone) in the pituitary increased significantly, except GnRH (gonadotropin releasing hormone) in the hypothalamus. Similarly, the expression of GnRHR, lh and fsh in the pituitary increased significantly after injecting S. argus with Pnx-14 and Pnx-20 (10 ng/g and 100 ng/g body weight (bw)), except GnRHR and fsh treated with 10 ng/gbw Pnx-20 in the pituitary and GnRHs in the hypothalamus. These results indicate that Pnx may not only stimulate the reproduction of the S. argus through the hypothalamic-pituitary-gonadal (HPG) axis, but also directly through the pituitary.

Phoenixin participated in regulation of food intake and growth in spotted scat, Scatophagus argus.

Phoenixin (Pnx) is an endogenous peptide known to be involved in reproduction and food intake in rats, with two active isoforms, phoenixin-14 (Pnx-14) and phoenixin-20 (Pnx-20). However, little is known about the functions of Pnx in teleost. Here, pnx was cloned and was detected in all tissues of both male and female in spotted scat (Scatophagus argus), including growth axis, hypothalamus, pituitary, and liver. Real-time PCR analysis showed that pnx in the hypothalamus increased significantly after 2 d and 7 d fasting, while reduced significantly after re-feeding (P < 0.05). When pituitary and liver fragments were cultured in vitro with Pnx-14 and Pnx-20 (10 nM and 100 nM) for 6 h, the expression of ghrhr (growth hormone-releasing hormone receptor) and gh (growth hormone) in the pituitary, and ghr1 (growth hormone receptor 1) in the liver increased significantly, except ghr2 (growth hormone receptor 2) incubated with 10 nM and 100 nM Pnx-20 and ghr1 incubated with 10 nM Pnx-20. Similarly, the expression of ghrhr and gh in the pituitary, as well as ghr1 and ghr2 in the liver, increased significantly after injecting S. argus with Pnx-14 and Pnx-20 (10 ng/g and 100 ng/g body weight). These results indicate that Pnx is likely to be involved in the regulation of food intake, and also regulates the growth of S. argus by increasing ghrhr and gh expression in the pituitary, ghr1 and ghr2 in the liver, and ghr1 directly in the liver.