Study On SNP And DNA Methylation Of Genes Involved In Steroi D Hormone Biosynthesis Correlated To The Reproductive Endocrine Of Female Japanese Founder (Paralichthys Olivaceus)

1. Polymorphism in exons CpG rich regions of the cyp17-Ⅱ gene affecting itsmRNAexpression and reproductive endocrine levels in female Japanese founder(Paralichthys olivaceus)Cytochrome P450c17-II (cyp17-Ⅱ) gene is an important factor affecting thegrowth, gonad differentiation and development, and other reproductive traits of fsh.There are three CpG rich regions in the coding region of cyp17-Ⅱ gene in Japanesefounder (Paralichthys olivaceus). The aim of this study was to understand whethermutations in exons of the cyp17-Ⅱ gene occured at CpG sites, and mutations andmethylation status of those CpG sites were involved in regulation of the expressionlevel of cyp17-Ⅱ gene and the reproductive endocrine of Japanese founder. Theresults showed that three single nucleotide polymorphisms (SNPs) were identifed.SNP1[(c. G594A (p.Gly188Arg)] located in exon4of L1locus, and SNP2(c.A939G)and SNP3(c.C975T) of L2locus located in CpG rich region of the exon6of cyp17-Ⅱ gene. Furthermore, the A to G transition at939bp position added a newmethylation site to the cyp17-Ⅱ coding region. According to multiple-comparisonanalysis, two loci (L1and L2) were signifcantly associated with serum testosterone(T) level (P<0.05) and the expression of cyp17-Ⅱ in ovary (P<0.01). Intriguingly,individuals with GG genotype of L2locus containing eight CpG methylation sites hadsignifcantly lower serum testosterone level and cyp17-Ⅱ mRNA expression thanthose with AA genotype containing seven CpG methylation sites. Moreover, the CpGsite was highly methylated (P≥77.8%) at938bp position of individuals with GG genotype of L2locus. These implied that the mutation and methylation status of thecoding region of cyp17-Ⅱ could infuence the gene expression and the reproductiveendocrine levels in female Japanese founder and L2locus could be regarded as acandidate genetic or epigenetic marker for Japanese founder breeding programs.2DNA methylation status of cyp17-Ⅱ gene correlated with its expression patternand reproductive endocrinology during ovarian development stages of Japaneseflounder (Paralichthys olivaceus)Cytochrome P450c17-Ⅱ (cyp17-Ⅱ,17α-hydroxylase) is responsible for theproduction of steroid hormones during oocyte maturation in vertebrates. Thecomparative expression pattern of cyp17-Ⅱ gene during the gonadal developmentstages will provide important insights into its function of gonadal development. Inaddition, epigenetic modification especially DNA methylation plays a vital role inregulation of gene expression. The adult female Japanese flounder at different ovariandevelopment stage (from stage Ⅱ to Ⅴ) was obtained in this experiment. Theexpression of cyp17-Ⅱ gene in the ovary of Japanese flounder during the gonadaldevelopment stages was measured by quantitative PCR. Reproductive traits includedgonadosomatic index (GSI), plasma estradiol-17(E2) and testosterone (T) were alsomeasured. Moreover, whole CpG dinucleotides methylation status of the two CpGrich regions in cyp17-Ⅱ coding region was detected by bisulfate sequencing. In theovary, the cyp17-Ⅱ gene had the lowest mRNA expression at the early ovariandevelopment stage, but then increased afterward. The variation trends of T and E2level were consistent with the cyp17-Ⅱ expression pattern in ovary. In contrast, thewhole methylation levels of each CpG rich region (exon4and6) in cyp17-Ⅱ codingregion were declined from stage Ⅱ to Ⅳ, and then increased at stage Ⅴ. Themethylation levels of whole CpG sites in each CpG rich region were inversecorrelation with the values of ovarian cyp17-Ⅱ gene expression, T and E2level, andGSI. Based on the present study, we proposed that cyp17-Ⅱ may regulate the level ofsteroid hormone，then stimulate the oocyte growth and maturation; The cyp17-Ⅱ genetranscriptional activity was possibly affected by the methylation level of CpG richregions in coding region. These findings will help to study the molecular mechanism of fish reproduction and endocrine physiology.3DNA methylation status of Cyp19a and Foxl2gene correlated with itsexpression pattern and reproductive endocrinology during ovarian developmentstages of Japanese flounder (Paralichthys olivaceus)The Foxl2and Cyp19a gene are crucial for the ovarian development, and theFOXL2could play a direct regulatory role on Cyp19transcription. In this study, weaimed to explain the molecular mechanism of the regulation of Foxl2on Cyp19a genetranscription from the point of view of epigenetic. The relative expression level ofCyp19a and Foxl2gene in the ovary of Japanese flounder during the gonadaldevelopment stages was measured by quantitative PCR. The results showed that theCyp19a and Foxl2were low expressed in stage Ⅱ and Ⅴ, but had highestexpression in stage Ⅳ. Moreover, the CpG dinucleotides methylation status of theCpG rich regions in promoter and coding region of Cyp19a and Foxl2was detectedby bisulfate sequencing. The variation trend of the methylation level of the nine CpGsites in promoter of Cyp19a gene was negatively associated with its expression levelduring the ovarian development. In addition, The CpG site at-223bp and-141bp werealso in the conserved P450c17-specific region and the Ad4protein binding sequence,respectively. There were eleven CpG sites in exon1of the coding region of Cyp19agene; meanwhile seven of them were on the putative transcription factors bindingsequence. The total methylation level of the eleven CpG sites in exon1had negitavecorrelation with the gene expression of Cyp19a. According to the report about thegene transcription level can be pressed by DNA methylation. We hypothesized thatthe DNA methylation pattern of the CpG sites in the cis-acting element andtrans-acting factors recognition sequence of Cyp19a gene could alter the efficiency oftranscription factor binding, affecting efficiency of gene expression finally. The DNAmethylation pattern of three CpG rich regions (namely F1, F2and F3) in codingregion of Foxl2gene was also detected. The total methylation level was negativelycorrelated to the expression of Foxl2during ovarian development stages. The resultimplied that the efficiency of Foxl2transcription could influenced by the DNAmethylation. Furthermore, the forkhead domain which was important for Foxl2 binding to its target gene was in the F1and F2region. These results suggest thatFoxl2could plays an important role in promoting gonadal differentiation toward thefemale pathway, either by binding directly to the promoter of Cyp19a or interactingwith Ad4BP/SF-1to enhance the Cyp19a transcription and thereby estrogenproduction specifically in the female Japanese founder gonad.In this study, we detected the SNP of the coding region of ovarian cyp17-Ⅱgene of Japanese flounder, analyzed the methylation pattern of CpG rich region incyp17-Ⅱ，Cyp19a and Foxl2gene at different ovarian development stages. Theresearch results may have certain reference significance to the study of molecularmechanism of fish growth and development.