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Abstract 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.

Abstract 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.

Abstract 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.

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.

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.

The proteins P53 and Mdm2 (murine double minute 2) play a prominent role in preventing the induction of uncontrolled apoptosis in numerous cell types. To better understand their potential roles during gonadal development in Monopterus albus, p53 and mdm2 homologues were isolated and characterized, and their mRNA expression patterns in developing gonadal and ovarian tissue incubated with H2O2 in vitro were analysed. The open reading frames (ORFs) of p53 and mdm2 consisted of 1125 and 1497 base pairs (bp), respectively. A multiple amino acid residue alignment analysis showed that p53 and Mdm2 harboured the typical conserved domains of their gene families. Phylogenetic analysis also demonstrated that the Mdm2 of M. albus exhibited high sequence similarity with Lates calcarifer; however, the p53 sequences of the Neoteleostei, including M. albus, were distinct from those of the Euteleostei and other vertebrates. Quantitative real-time PCR revealed that p53 and mdm2 were mainly expressed in the ovary; their expression was significantly increased in the previtellogenic stage, was maintained at the increased level thereafter, showed a second increase in the early intersexual stage, and then decreased with the natural sex change from female to male. After incubation with H2O2, the expression patterns of p53 and mdm2 in ovarian fragments were similar to that of the apoptotic molecular marker caspase3, showing a significant increase at 1 h post incubation (hpi), a decrease after 2 hpi, and then a return to significantly upregulated levels at 4 hpi and 8 hpi. In addition, p53, mdm2 and caspase3 were strongly correlated. These results will be useful for further studies on the function and regulation of p53 and Mdm2 during gonadal development in Monopterus albus, particularly in the ovary.

TGF-β receptors play important roles in mediating TGF-β signals during gonadal development. To identify the functions of TGF-β 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.