| Olive flounder(Paralichthys olivaceus)is an important commercially cultured marine flatfish in China,Korea,and Japan,and female one grows faster than male one.Breeding and culturing all-female population of flounder is a promising approach to boost production.The study on sex control of flounder always attracts researchers` attention.The gonadal differentiation of flounder has been proved to be controlled by both genetic and environmental factors especially water temperature and exogenous hormones.Although,the genetic female(XX)population could be obtained through the combination of gynogenesis and sex-reversal,the sex differentiation of genetic female was unstable that sponstaneous sex-reversal to phenotype male can be induced by environmental factors,especially high water temperature and exogeneous androgens.Therefore,the investigation on the mechanism of flounder sex differentiation not only provides guidance for sex control,but also contributes to enriching the theory of fish sex differentiation.The reported studies about sex study of flounder mainly focused on the cloning of sex-conserved genes,their expression patterns and the dimorphic DNA methylation analysis.More researches are still in process.This study comprehensively clarified the mechanism of gonadal differentiation from the perspective of transcriptome,gene expression,epigenetics,endocrinology,and gene regulation.Firstly,we used RNA-seq technology to investigate gonadal transcriptomes of flounder.The differentially expressed genes between ovary and testis were identified,which could provide candidate genes for further studies of molecular mechanism of flounder sex differentiation.We further comprehensively analyzed the expression levels of cyp19 a and its transcription factors such as foxl2,nr5a2 and nr0b1,and their promoter methylation level variation during gonadal differentiation period.The effect of these genes on gonadal differentiation direction was explored combining the endogenous sex hormone level variation of flounder.The spatial expression patterns of foxl2,nr5a2,nr0b1 in gonads and their regulation on cyp19 a expression were also demonstrated.And the effect of promoter fully methylation on cyp19 a expression was investigated to explore its regulation mechanism.The main results are as follows:1.There were 21,697 annonated contigs in flounder gonadal transriptome.Most of them were involved in signal transduction,cell growth and death,endocrine system,etc.Among them,2,193 contigs were predicted to be differentially expressed in ovary and 887 in testes.KEGG enrichment indicated that differentially expressed genes(DEGs)were enriched in cell growth and death,transcription,translation and energy metabolism,etc.The sex-related genes identified from DEGs were related to sex differentiation and gonadal development,steroid hormone biosynthesis,gametogenesis and formation.The ovarian steroidogenesis pathway was founded in flounder.2.The expression levels of cyp19 a and its transcription factors were detected by real-time quantative RT-PCR(qPCR).The results indicated the expression levels of cyp19 a and foxl2 were consistenly up-regulated during ovarian differentiation.High temperature repressed the expression of them,but contributed to the expression of nr0b1.The expression level of nr5a2 significantly rised up at the early stage of gonadal differentiation with exogenous E2 treatment.During ovarian differentiation,the level of estradiol-17? consistently increased.While,HT group maintained lower levels of estradiol-17? and showed higher levels of androgens during gonadal differentiation period.The DNA methylation levels of CpG islands(CpGI)within promtoter of cyp19 a and its transcription factors were anaylzed.The results indicated the promoter CpGI of cyp19 a was demethylated at the early stage of ovarian differentiation and led to hypomethylation.While,high temperature repressed the demethylation process and maintained hypermethylation.The E2 treatment delayed the demethylation process,but induced de novo methylation to high level during the late stage of gonadal differentiation.The promoter CpGI of foxl2 experienced demethylation at the late stage of ovarian differentiation,while,high temperature was able to repress the demethylation process.However,the methylation level of flounder nr5a2 promoter was not influenced by high temperature and E2 treatment,and maintained hypomethylation during gonadal differentiation.3.The results of in situ hybridization and immunohistochemistry indicated that foxl2,nr5a2 and nr0b1 were expressed in ovarian follicular cell,which is consistent with cyp19 a spatial patterns in ovary.In testis,nr5a2 and nr0b1 were strongly expressed in Sertoli cell and weakly detected in spermatogonia and primary spermatocytes cells.4.The effect of transcription factors on cyp19 a expression regulation was further analyzed by using methods of transient tranfection in HEK 293 T and overexpression in Sertoli cell line of flounder.The results indicated Nr5a2 was able to activate the transcription of cyp19 a.EMSA results provided the evidences of the combination between Nr5a2 and two cis-regulatory elements within cyp19 a promoter.Co-transfection of flounder cyp19 a constructs simultaneous with nr5a2 and nr0b1 expression plasmids showed that nr0b1 significantly repressed nr5a2 stimulation of the cyp19 a promoter.The overexpression of foxl2 in cells of flounder Sertoli cell line dose-dependently increased cyp19 a expression level.Moreover,foxl2 and nr5a2 were able to co-activate cyp19 a gene transcription.During gonadal differentiation,cyp19 a expression level exhibited inverse variation trends with promoter methylation level especially in the control and HT groups.The hypermethylation promoter could repress cyp19 a expression by blocking nr5a2-mediated transactivation activity.The cis-regulatory elements of Nr5a2 are located around the CpG islands of cyp19 a promoter.These evidences strongly indicate that hypermethylation of the cyp19 a promoter may prevent Nr5a2 from binding cyp19 a promoter. |
PDF Full Text Request