Protein expression patterns and metal metabolites in a protogynous hermaphrodite fish, the ricefield eel (Monopterus albus).

BACKGROUND: The ricefield eel Monopterus albus undergoes a natural sex change from female to male during its life cycle, and previous studies have shown the potential mechanisms of this transition at the transcriptional and protein levels. However, the changes in protein levels have not been fully explored, especially in the intersexual stage. RESULTS: In the present study, the protein expression patterns in the gonadal tissues from five different periods, the ovary (OV), early intersexual stage gonad (IE), middle intersexual stage gonad (IM), late intersexual stage gonad (IL), and testis (TE), were determined by untargeted proteomics sequencing. A total of 5125 proteins and 394 differentially expressed proteins (DEPs) were detected in the gonadal tissues. Of the 394 DEPs, there were 136 between the OV and IE groups, 20 between the IM and IE groups, 179 between the IL and IM groups, and 59 between the TE and IL groups. Three candidate proteins, insulin-like growth factor 2 mRNA-binding protein 3 isoform X1 (Igf2bp3), triosephosphate isomerase (Tpi), and Cu-Zn superoxide dismutase isoform X1 [(Cu-Zn) Sod1], were validated by western blotting to verify the reliability of the data. Furthermore, metal metabolite-related proteins were enriched in the IL vs. IM groups and TE vs. IL groups, which had close relationships with sex change, including Cu2+-, Ca2+-, Zn2+- and Fe2+/Fe3+-related proteins. Analysis of the combined transcriptome data revealed consistent protein/mRNA expression trends for two metal metabolite-related proteins/genes [LOC109953912 and calcium Binding Protein 39 Like (cab39l)]. Notably, we detected significantly higher levels of Cu2+ during the sex change process, suggesting that Cu2+ is a male-related metal metabolite that may have an important function in male reproductive development. CONCLUSIONS: In summary, we analyzed the protein profiles of ricefield eel gonadal tissues in five sexual stages (OV, IE, IM, IL, and TE) and verified the plausibility of the data. After preforming the functional enrichment of metal metabolite-related DEPs, we detected the contents of the metal metabolites Zn2+, Cu2+, Ca2+, and Fe2+/Fe3+ at these five stages and screened for (Cu-Zn) Sod1 and Mmp-9 as possible key proteins in the sex reversal process.

Identification, characterization and functional analysis of gonadal long noncoding RNAs in a protogynous hermaphroditic teleost fish, the ricefield eel (Monopterus albus).

BACKGROUND: An increasing number of long noncoding RNAs (lncRNAs) have been found to play important roles in sex differentiation and gonad development by regulating gene expression at the epigenetic, transcriptional and posttranscriptional levels. The ricefield eel, Monopterus albus, is a protogynous hermaphroditic fish that undergoes a sequential sex change from female to male. However, the roles of lncRNA in the sex change is unclear. RESULTS: Herein, we performed RNA sequencing to analyse lncRNA expression patterns in five different stages of M. albus development to investigate the roles of lncRNAs in the sex change process. A total of 12,746 lncRNAs (1503 known lncRNAs and 11,243 new lncRNAs) and 2901 differentially expressed lncRNAs (DE-lncRNAs) were identified in the gonads. The target genes of the DE-lncRNAs included foxo1, foxm1, smad3, foxr1, camk4, ar and tgfb3, which were mainly enriched in signalling pathways related to gonadal development, such as the insulin signalling pathway, MAPK signalling pathway, and calcium signalling pathway. We selected 5 highly expressed DE-lncRNAs (LOC109952131, LOC109953466, LOC109954337, LOC109954360 and LOC109958454) for full length amplification and expression pattern verification. They were all expressed at higher levels in ovaries and intersex gonads than in testes, and exhibited specific time-dependent expression in ovarian tissue incubated with follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG). The results of quantitative real-time PCR (qRT-PCR) analysis and a dual-luciferase assay showed that znf207, as the gene targeted by LOC109958454, was expressed in multiple tissues and gonadal developmental stages of M. albus, and its expression was also inhibited by the hormones FSH and hCG. CONCLUSIONS: These results provide new insights into the role of lncRNAs in gonad development, especially regarding natural sex changes in fish, which will be useful for enhancing our understanding of sequential hermaphroditism and sex changes in the ricefield eel (M. albus) and other teleosts.

Circular RNA expression profiles and CircSnd1-miR-135b/c-foxl2 axis analysis in gonadal differentiation of protogynous hermaphroditic ricefield eel Monopterus albus.

BACKGROUND: The expression and biological functions of circular RNAs (circRNAs) in reproductive organs have been extensively reported. However, it is still unclear whether circRNAs are involved in sex change. To this end, RNA sequencing (RNA-seq) was performed in gonads at 5 sexual stages (ovary, early intersexual stage gonad, middle intersexual stage gonad, late intersexual stage gonad, and testis) of ricefield eel, and the expression profiles and potential functions of circRNAs were studied. RESULTS: Seven hundred twenty-one circRNAs were identified, and the expression levels of 10 circRNAs were verified by quantitative real-time PCR (qRT-PCR) and found to be in accordance with the RNA-seq data, suggesting that the RNA-seq data were reliable. Then, the sequence length, category, sequence composition and the relationship between the parent genes of the circRNAs were explored. A total of 147 circRNAs were differentially expressed in the sex change process, and GO and KEGG analyses revealed that some differentially expressed (such as novel_circ_0000659, novel_circ_0004005 and novel_circ_0005865) circRNAs were closely involved in sex change. Furthermore, expression pattern analysis demonstrated that both circSnd1 and foxl2 were downregulated in the process of sex change, which was contrary to mal-miR-135b. Finally, dual-luciferase reporter assay and RNA immunoprecipitation showed that circSnd1 and foxl2 can combine with mal-miR-135b and mal-miR-135c. These data revealed that circSnd1 regulates foxl2 expression in the sex change of ricefield eel by acting as a sponge of mal-miR-135b/c. CONCLUSION: Our results are the first to demonstrate that circRNAs have potential effects on sex change in ricefield eel; and circSnd1 could regulate foxl2 expression in the sex change of ricefield eel by acting as a sponge of mal-miR-135b/c. These data will be useful for enhancing our understanding of sequential hermaphroditism and sex change in ricefield eel or other teleosts.

Molecular characterization, expression, and apoptosis regulation of siva1 in protogynous hermaphrodite fish ricefield eel (Monopterus albus).

Siva1, which induces extensive apoptosis, has been well characterized. To elucidate the molecular function of Siva1 in ricefield eel, molecular characterization and phylogenetic analysis were performed, and the mRNA expression in the ovary at different developmental stages and ovary tissues exposed to H2O2 and Z-VAD-FMK in vitro were also evaluated. The results indicated that ricefield eel Siva1 was highly conserved and contains three conserved motifs, despite 83 amino acid differences upstream of the initiation codon. Phylogenetic analysis demonstrated that ricefield eel Siva1 clusters together with the Siva1 protein of the other fish, with high sequence homology with that of Lates calcarifer. Quantitative real-time polymerase chain reaction analysis showed high siva1 expression levels in the ovary and low expression levels in the liver. The higher mRNA levels of siva1 were detected in the IE and IM, and the lower siva1 mRNA levels were found in the OM, IL, and TE during gonadal development. Additionally, siva1 expression levels in the ovarian tissues were significantly increased at 1 h post incubation (hpi) with H2O2 and then significantly decreased at 2 hpi; however, siva1 expression was upregulated significantly at 4 and 8 hpi, similar to the patterns observed with caspase3, which was used as a molecular marker of apoptosis. Moreover, the siva1 mRNAs were elevated significantly than that in control groups at 1 hpi, but the expression of siva1 was down-regulated dramatically at 2, 4, and 8 hpi, which were similar with that of caspase3 expression profiles after Z-VAD-FMK incubation. What's more, Pearson's correlation coefficients showed strongly positive relationships between siva1 and caspase3. These findings suggest that Siva1 plays an important apoptosis role in gonadal development of ricefield eel.

Expression and regulation of Smad2 by gonadotropins in the protogynous hermaphroditic ricefield eel (Monopterus albus).

Smad2, a receptor-activated Smad, plays a critical role in regulating gametogenesis. In this study, a smad2 homologue was identified and sequenced from ricefield eel ovary cDNA, and its mRNA and protein expression levels were analysed during oocyte development. The cDNA sequence of ricefield eel smad2 consisted of 1863 bp encoding a 467-amino acid protein that had high sequence homology with Smad proteins in other teleosts, especially in Poeciliopsis prolifica. The results of real-time quantitative PCR (RT-qPCR) analysis revealed that smad2 is expressed in the ovary during gonad development, increased continuously until the early vitellogenic stage in the ovaries, and then decreased with ovary maturation. Smad2 protein immunoreactivity was localized in the cytoplasm of follicular cells, oogonia, and primary growth stage oocytes. In vitro experiments revealed that follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG) promoted smad2 expression in ovary tissue in a time- and dose-dependent manner, respectively. In summary, Smad2 plays a potentially vital role in ricefield eel ovary development.

Fish Community Structure and Biomass Particle-Size Spectrum in the Upper Reaches of the Jinsha River (China).

To understand the characteristics of the fish community structure and biomass particle-size spectrum in the upper reaches of the Jinsha River, fish and environmental surveys were conducted in 21 segments of the upper reaches of the Jinsha River in September 2019 and June 2020. A total of 4062 fish belonging to 2 orders, 5 families, 18 genera, and 28 species were collected. Among them, Cyprinidae fish were the most abundant (14 species), accounting for 50.00%. The Shannon index and Pielou evenness index values varied from 0.402-1.770 and 0.254-0.680, respectively. The dominant species of fish were Triplophysa stenura, Schizothorax wangchiachii, and Schizopygopsis malacanthus. Redundancy analysis (RDA) was used to analyse the relationship between the fish community and environmental factors. Velocity, altitude, and dissolved oxygen were the main influencing factors of fish community structure differences in the upper reaches of the Jinsha River. The abundance/biomass curves showed that the fish communities in the upper reaches of the Jinsha River were moderately or severely disturbed. The standardized biomass particle-size spectrum of fish showed that the degree of disturbance of fish in tributaries was much lower than that in the main stream. Compared with the historical data, the fish community structure in the Jinsha River has changed significantly, with the number of exotic species increasing, and the individual fish showing miniaturization and younger ages. It is suggested that habitat conservation strategies be adopted in the upper tributaries of the Jinsha River to provide a reference for the restoration of fishery resources and the conservation of fish diversity in the Yangtze River.

Genetic Variation in Schizothorax kozlovi Nikolsky in the Upper Reaches of the Chinese Yangtze River Based on Genotyping for Simplified Genome Sequencing.

Schizothorax kozlovi Nikolsky is a unique cold-water fish in the upper reaches of the Yangtze River in China and has high economic value. In our study, genetic diversity and population structure analyses were performed on seven wild populations (originating from the Jinsha River, Yalong River, and Wujiang River) in the upper reaches of the Yangtze River by genotyping by sequencing (GBS). The results indicated that a total of 303,970 single-nucleotide polymorphisms (SNPs) were identified from the seven wild populations. Lower genetic diversity was exhibited among the intrapopulations of the three tributaries, and the Wujiang River population had significant genetic differentiation when compared to the Jinsha River and Yalong River populations. Furthermore, the selected SNPs were enriched in cellular processes, environmental adaptation, signal transduction, and related metabolic processes between the Wujiang population and the other two populations. The above results indicate that the populations of S. kozlovi have different degrees of tolerance and selection pressure in response to temperature and altitude. The Wujiang intrapopulation has greater genetic diversity and differentiation than the Jinsha River and Yalong River populations, which demonstrates that the Jinsha and Yalong populations require more attention and resources for their protection. The results of this study will increase our understanding of the diversity of S. kozlovi in the upper reaches of the Yangtze River and provide a basis for the conservation and utilization of wild resources.

Estradiol Upregulates the Expression of the TGF-ß Receptors ALK5 and BMPR2 during the Gonadal Development of Schizothorax prenanti.

TGF-ß receptors play important roles in mediating TGF-ß signals during gonadal development. To identify the functions of TGF-ß receptors, including the type I receptor (activin receptor-like kinase 5, ALK5) and type II receptor (bone morphogenetic protein receptor 2, BMPR2), during the gonadal development of S. prenanti, the full-length cDNA sequences of ALK5 and BMPR2 were isolated and characterized. Their expression patterns in developing gonads and in the gonads of exogenous estradiol (E2) -fed fish were analyzed. The cDNAs of ALK5 and BMPR2 were 1925 bp and 3704 bp in length and encoded 501 and 1070 amino acid residues, respectively. ALK5 and BMPR2 were mostly expressed in gonads, particularly in cortical alveoli stage ovaries and mid-spermatogenic stage testes; however, the overall level of BMPR2 mRNA was higher than that of ALK5 during gonadal development. Furthermore, immunohistochemical signals of ALK5 and BMPR2 were mostly detected at chromatin nucleolar oocytes and perinuclear oocytes in ovaries and at spermatocytes and spermatogonia in testes. Exogenous E2 induces the gonadal expression of ALK5 and BMPR2, and BMPR2 is more responsive to E2 than ALK5. These results suggest that ALK5 and BMPR2 might play a potentially vital role in both folliculogenesis and spermatogenesis in S. prenanti.

Expression patterns and oestradiol regulation of growth differentiation factor 9 in Schizothorax prenanti.

Growth differentiation factor 9 (GDF9) plays pivotal roles in regulating follicular development in many mammalian species. In the present study, the full-length Schizothorax prenanti gdf9 cDNA sequence was isolated and characterized, and its expression pattern in developing gonads and in the gonads of exogenous oestradiol (E2)-fed fish were analysed. The S. prenanti gdf9 cDNA sequence consisted of 1958 base pairs (bp), encoded a 413 amino acid, and showed high sequence similarity with Carassius gibelio and Cyprinus carpio. The quantitative real-time PCR analysis revealed that gdf9 was mainly expressed in the gonads, with particularly high expression in the cortical alveoli stage ovary and late-spermatogenic stage testis. Immunohistochemical signals for Gdf9 were mainly detected in chromatin nucleolar oocytes, spermatogonia and spermatocytes. Furthermore, the gonadal expression of gdf9 induced by exogenous E2 was related to the feeding time and dose. Taken together, these findings were helpful to gain a better understanding of the role of Gdf9 in both folliculogenesis and spermatogenesis in S. prenanti.