Efficient Creation Of High Quality XX Neomale Yellow Catfish And Identification Of Differentially Expressed Genes Between X And Y Sperms

Yellow catfish(Pelteobagrus fulvidraco)is one of the economically important freshwater fish species in China due to its highly nutritional and economic values.The annual output of yellow catfish in China was about 565 thousand tons in 2020(China Fishery Statistical Yearbook,2021).There is a great demand for female broodfish of yellow catfish.At present,the female broodfish are usually purchased from the farmers or collected from rivers.Since the all-male yellow catfish and infertile hybrid yellow catfish(Pelteobagrus fulvidraco♀×Pelteobaggrus vachelli♂)have excellent growth performance,farmers do not want to culture the normal yellow catfish,resulting in the serious lack and increasing price(as high as ￥80 per kilogram)of female broodfish in the past two years.In addition,most of the female broodfish have not been systematically selected and are very confused.Therefore,building all-female yellow catfish population will conducive to long-term development of yellow catfish industry,while the creation of high quality XX-neomales become a critical step.In this study,we explored different biotechnological manipulation of sex reversal on yellow catfish and assessed the sperm quality and reproductive performance of XX neomale yellow catfish.Furthermore,we obtained X and Y sperms from XX neomale and YY super-male yellow catfish respectively,and performed comparative analysis of transcriptome and DNA methylome in X and Y sperms to screen specific molecular markers,which provide theoretical basis for rapid separation of X sperm and Y sperm and innovation of sex control breeding technology.1.Letrozole induced sex-reversal leads to transient transition from genetic to temperature-dependent sex determination in yellow catfish.First,we investigated the effects of temperature on the sex differentiation of yellow catfish.The results showed that gonad development of XX yellow catfish in high temperature group(treated from 7 dph to 60 dph)were delayed comparing with room temperature group.After transferred to ambient temperature,the ovary development of high temperature group gradually recovered.The above results indicated that high temperature treatment resulted in the delay of ovarian development in yellow catfish,but could not induce sex reversal.Aromatase inhibitor treatment is a common and effective method to obtain the neomale fish,so we examined the effects of Letrozole(LZ),an aromatase inhibitor on the sex differentiation of yellow catfish.As a result,39% XX yellow catfish had been sex reversed(designated as XXm-LZ neomale yellow catfish)after treated with LZ1000(1000 μg/kg)at room temperature,in which the lobular structure of testis was short and not as sharp as the XY males.When the reared temperature was elevated to 34℃,the proportion of XX neomales was improved to 78% in 34℃+LZ1000 group,but their testis phenotype was similar to XXm-LZ.Interestingly,the offspring produced by mating XXm-LZ and XX female,developed into all-female fish at room temperature,whereas developed into neomale fish after treated with high temperature.The ratios of XX neomales were gradually increased when the temperature and incubation time were elevated,and 99% XX fish were sex-reversed into neomale fish after high temperature treatment(designated as XXm-HT neomale yellow catfish),and its testis phenotype was similar to that of XY male.Therefore,artificially induced sex-reversal leads to transition from genetic to temperature-dependent sex determination in yellow catfish.We further investigated whether the transition from genetic to temperature-dependent sex determination in yellow catfish could be permanently inherited.The offspring of XXm-HT and normal XX female were treated with high temperature.Surprisingly,the gonad morphology of the offspring of XXm-HT with high temperature treatment was abnormal,such as ovary-like structure and no seminiferous lobule.Histological results showed that germ cells were undifferentiated in most of the gonads at 90 dph.Immunofluorescence analysis indicated that Dmrt1 and Pfpdz1 proteins were highly expressed in the gonad of XYm and the offspring of XXm-HT treated high temperature.Vasa-positive signals were also located in the germ cells of the offspring of XXm-HT.However,the meiosis marker,Sycp3 was not detected in the germ cell of the offspring of XXm-HT of high temperature group,although it was enriched in spermatocytes of XY tests.The above results indicated that high temperature treatment led to the masculinization of the germ cell in the gonads of the offspring of XXm-HT,but the meiosis possess had been prevented.2.Comparison of testis quality and reproductive performances in XX neo-male yellow catfish created by different sex reversal methodsHow about the quality of XXm-LZ and XXm-HT? Comparative analysis of sperm motility parameters and the breeding performance were performed on XXm-LZ,XXm-HT and XYm yellow catfish.We found that the fertilization rate,hatching rate and survival rate of XXm-HT were similar to those of XYm yellow catfish,and were significantly higher than those of XXm-LZ.Meanwhile,the malformation rate of fries generated by XXm-HT(10.2%)was significantly lower than XXm-LZ(30.71%).Sperm motility parameters of XXm-HT were also similar to those of XYm yellow catfish and significantly higher than XXm-LZ,including the percentages of motile spermatozoa,VSL(straight line velocity),VCL(curvilinear velocity)and VAP(average path velocity).The expression levels mi R-200 family including mi R-141/-200a/-429 b that were negatively correlated with sperm quality,and we detected lower expression in the testis of XXm-HT and XYm compared with the expression in XXm-LZ yellow catfish.In addition,luciferase reporter assays showed that dmrt1 was a target gene of mi R-429 b in yellow catfish.Significantly down-regulated expression of dmrt1 and pfpdz1,male sex differentiation related gene,were observed in the testis of XXm-LZ compared with XXm-HT and XYm yellow catfish.These data suggest that the sperm quality of XXm-HT were better than XXm-LZ yellow catfish.3.Comparative analysis of transcriptome and DNA methylation between X and Y sperms in yellow catfishThe conventional sex control technologies have some problems,such as incomplete sex reversal and long breeding cycle.Recently,some scientists have screened X and Y sperms by using the flow cytometry with specific antigen or activation of TLR7/8 alters sperms motility,whereas there was no report in fish.In orded to screen the specific molecular markers of X and Y sperms,we collected mature X and Y sperms from XX male and YY male by percoll gradient-centrifuged method.Then,RNA-seq and WGBS analysis were carried out on X and Y sperms.We found that 480 and 2574 genes had higher expression in X sperms and Y sperms,respectively.In addition,ccdc173,spink1,kcnip3,clec4 m and four Lnc RNA specifically expressed in X sperms,while mrc1,rt1,ca10,apela and two Lnc RNA were only presented in Y sperms.GO and KEGG enrichment analysis revealed that highly expressed genes in X sperms were enriched in the ion channel signaling and highly expressed genes in Y sperms were enriched in the response to molecule of bacterial origin.However,there were no significant expression differences for most members of Toll-like receptor family,aside from TLR5 as its expression in Y sperms was 10 times higher than in X sperms.DNA methylation analysis showed that hypermethylation level of X sperms was higher than Y sperms,suggesting that Y sperms was more active than X sperms.Through combined analysis of differentially expressed genes and differentially methylated genes,we found that 12.51% highly expressed genes in Y sperms(322 genes),and 11.25% highly expressed genes(58genes)in X sperms were hypo-mathylated.However,no methylated regions were indentified in the specifically expressed genes and Lnc RNAs,as well as TLR5 in X and Y sperms,suggesting that they might be regulated by other mechanisms of transcriptional regulation.In conclusion,precise application of the transition from genetic to temperature-dependent sex determination could produce the highest quality neomale yellow catfish with better sperms quality and breeding performance,after comparing different approaches.Furthermore,after analysis the transcriptome and DNA methylome between X and Y sperms,we screened out specifically expressed genes and Lnc RNAs,as well as differentially expressed gene TLR5 in X and Y sperms,which provide the theoretical basis for the separation of X and Y sperms in fish species,as well as the innovation of sex control breeding technology.