Study On Steroidgenesis Important Genes’ Expression And Regulation In The Olive Flounder Paralichthys Olivaceus

Sex hormones,including estrogen and testosterone,are indispensable to the gonadal differentiation and development.In the sex steroid synthesis process,many proteins and enzymes play important roles.Among them,steroidogenic acute regulatory protein 2(St AR2)is a key protein in transporting cholesterol from the outer mitochondria membrane to the inner mitochondria membrane for steroid synthesis.Cyp11a(or P450 side-chain cleavage enzyme,P450scc)encoded by cyp11 a gene,locating on the mitochondrial inner membrane in the adrenal gland and gonads,is a main enzyme for the initial and rate-limiting reaction(i.e.conversion of cholesterol to pregnenolone)in the synthesis of steroid hormones.And17β-hydroxysteroid dehydrogenase 1(17β-HSD1)plays an important role in the process of estrone conversion into estradiol.The olive flounder Paralichthys olivaceus is an important marine aquaculture species in Korea,Japan and China.Flounder sex control and the mechanism have been paid attention many years.In this study,the open reading frame(ORF)sequences or genomic sequences of StAR2,cyp11 a and 17β-HSD1 were cloned or gained from flounder gonadal transcriptome data.And expression patterns in the flounder adult tissues and gonads during gonadal differentiation period were analyzed.The effects of cAMP,NR5a2 and NR0b1 on these genes expressions were further studied.The related results were shown as below.StAR2,cyp11 a and 17β-HSD1 were cloned and verified based on the flounder gonadal transcriptome data.The cDNA sequences of flounder StAR2a(KF032071.1)and StAR2b(KF032076.1)were 843 bp and 657 bp,encoding 280 and 218 amino acids,respectively.The ORF of cyp11 a was 825 bp,encoding 275 amino acids.And the ORF of 17β-HSD1 was 873 bp,encoding 290 amino acids.Phylogenetic trees showed these three genes of flounder were clustered with their proteins from other teleost fish species.The expression levels of StAR2,cyp11 a and 17β-HSD1 genes were analyzed by using RT-PCR,in situ hybridization(ISH)or immunohistochemistry methods.The RT-PCR results in the tissues of wild-type adult male and female flounder,of which the developmental stages of ovary and testis were respectively at stage Ⅲ and stageⅡ,indicated that these three genes were mainly expressed in gonads.StAR2 and cyp11 a expression levels in testis were higher than in ovary,while 17β-HSD1 was higher in ovary than in testis.The results of ISH and immunohistochemistry showed that StAR2 a and cyp11 a were mainly expressed in the theca cells and oocytes of ovary,and Leydig cells of testis.Further study presented that StAR2 a protein was in the cytoplasm of Hela cells and flounder testicular cells by using transfection or immunofluorescence,respectively.StAR2,cyp11 a and 17β-HSD1 expression levels were detected during flounder gonadal differentiation period by using qPCR.And the levels of flounder cholesterol and pregnenolone in this period were also analyzed.In the gynogenetic control group,StAR2 and cyp11 a expression levels were relatively stable,and 17β-HSD1 expression level reduced during the period.In the 17β-estradiol treatment group(5 ppm),StAR2 and cyp11a genes’ expression levels were high in differentiating ovary,and then significantly decreased in differentiated ovary(P<0.05).And the pregnenolone level also decreased in the differentiation ovary.17β-HSD1 expression level kept stable during ovarian differentiation period,increased in the differentiated ovary,and then little reduced to keep the higher level.In the testosterone treatment group(5 ppm),these three genes’ expressions were all high before testicular differentiation,and then significantly down-regulated after differentiation(P<0.05).In the high temperature(28℃)treatment group,StAR2 and cyp11 a genes’ expression levels were stable before testicular differentiation and significantly up-regulated during differentiation(P<0.05).After testicular differentiation,both genes expression levels were lower.Moreover,HT group cholesterol was at high level,but pregnenolone level was higher than control group.And cholesterol and pregnenolone were high in T group.17β-HSD1 expression pattern was similar to that in the control group,but its expression level wasrelative low.The second messenger(cAMP)and transcription factors(NR5a2 or NR0b1)were used to study their function for expressions of StAR2,cyp11 a and 17β-HSD1 genes by transfecting the cultured flounder primary testis cells.The results showed that cAMP could regulate the expressions of StAR2 and cyp11 a,and the regulations were all dose-dependent.Lower dose of cAMP down-regulated their expression,while higher dose of cAMP up-regulated their expression.cAMP also significantly down-regulated 17β-HSD1 expression(P<0.05).Moreover,StAR2 and cyp11 a expressions were significantly up-regulated by transfecting the cultured primary testis cells with pcDNA3.1-NR5a2 or pcDNA3.1-NR0b1.The regulations were also all dose-dependent.Higher dose of NR5a2 or NR0b1 all weakened the effect of up-regulation.17β-HSD1 expression was significantly down-regulated by transfecting the cultured primary testis cells with 2 μg,3 μg pcDNA3.1-NR5a2 or 1 μg pcDNA3.1-NR0b1,and up-regulated with 2 μg pcDNA3.1-NR0b1(P<0.05).The regulations were also all dose-dependent.It is implied that all these three genes involve in flounder gonadal differentiation and their expressions are respectively regulated by cAMP,NR5a2 or NR0b1,which may provide useful information for the study on fish gonadal differentiation and development.