| The Nile tilapia(Oreochromis niloticus)displays GSD+TE sex determination,in which temperature during embryonic development determines the sexual fate of the individual.However,the molecular mechanisms governing this process remain a mystery,including influences of environmental temperature on the comprehensive gonadal gene expression patterns underlying GSD+TE.In our study,we developed three all-female families by hybridization of wild females and sex reversed males.And proportions of males in high temperature treatment group were identified,and the highest male ratio of Nile tilapia among 3 families after high temperature treatment was 90.4%,the others were 86%and 74% respectively.Furthermore,gonadal histology showed that the gonads of sex-reversal males were not different from that of wild males.But an irregular shape and loose arrangement of oocytes were found in the gonads of high-temperature treated females,compared with that in control group.And our characterization of gonadal transcriptome of control and high temperature treatment group allowed us to identify novel candidate genes involved in sex reversal caused by high temperature treatment.RNA-seq yielded 149,624,210 Raw Reads and124,515,460 Clean Reads,and average 80.09% of which were mapped to the Nile tilapia genome,and used to characterize differential expression genes.Changes in the transcriptome occurring as a function of both steroidogenesis and sexual differentiation were extensively profiled.Overall,CF/TF showed 79 DEGs,with 34 up-regulated genes and 45 down-regulated genes.In CF/IM,there are 11,117 DEGs,of which 5,779 genes were up-regulated and 5,338 genes were down-regulated.TF/IM showed 11,000 DEGs,with5,607 up-regulated genes and 5,393 down-regulated genes.In addition,as shown in the Venn diagram,among 79 DEGs in the CF/TF,54(68.4%)were included in the list of DEGs in the CF/IM,and 44(55.7%)in the TF/IM,and among 11,000 DEGs in the TF/IM,there were 9,471 DEGs displaying expression in the CF/IM.And the analysis generated 5 CF/TF specific DEGs,and there were 1,636 and 1,529 DEGs specific to CF/IM and TF/IMrespectively.Furthermore,we analyzed fold change distribution of differentially expressed genes in CF/TF,CF/IM and TF/IM.And we found there are many genes with fold change >7 in CF/TF.And we identified genes involved in sex determination,steroidgenesis,response to high temperature,including genes such as cyp19a1 a,3?-hsd,cdc42,mrpl28,and ten sex-related genes involved in high temperature treatment sex reversal were validated by qRT-PCR.Our results showed that estrogen synthesis-related genes like cyp19a1 a were downregulated,in contrast,androgen synthesis-related genes like hsd17b7 and 3?-hsd were upregulated.Moreover,the lasting effects of high temperature on cyp19a1 a,pycr,mrpl28,cdc42 and ccnh were found in Nile tilapia gonad.For further study of the lasting effects of high temperature on cyp19a1 a gene,Western blot was performed,revealing that cyp19a1 a protein expression levels declined significantly after high temperature treatment and reached a level comparable to that of the wild males.In conclusion,our study showed that an irregular shape and loose arrangement of oocytes were found in TF,compared with XX gonads in control group.And gonadal global gene expression during high temperature treatment sex reversal has been extensively profiled for the first time in Nile tilapia.This data showed that last downregulated effect of high temperature on estrogen synthesis-related genes such as cyp19a1 a,and response to high temperature-related genes such as mrpl28 and nuclear receptors in Nile tilapia gonad was readily observed.These findings provide insights into the genetic framework underlying GSD+TE,and pave the way for further study of the molecular mechanisms in Nile tilapia sex reversal after high temperature treatment. |
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