| Hyriopsis cumingii is an important breeding shellfish in China,and the performance of male and female individuals is different,among which the male is better than the female.Study on sex determination and gonad development of H.cumingii has important theoretical basis and practical value for unisexual culture of it.Sex hormones play an important role in gonadal development,sex differentiation and functional maintenance.Steroidogenic Acute Regulatory Protein,Cytochrome P450 and Hydroxysteroid dehydrogenase(HSD)can regulate the synthesis of sex hormones.In this study,the StAR3 and Cyp17 a,17β-HSD11 gene fragments located in the upstream and downstream of the sex hormone synthesis pathway were screened from the transcription bank of H.cumingii,and their full-length cDNA sequences were cloned by RACE technique.In order to study the roles of StAR3,Cyp17 a and 17β-HSD11 in gonad development and sexual steroid hormone synthesis of H.cumingii,real-time fluorescence quantitative,in situ hybridization,RNA interference,exogenous hormone immersion and the changes of gene expression levels after exogenous hormone injection were used to study.1.Full length cDNA and sequence analysis of 3 genesThe full length of HC-StAR3 gene was 2072 bp,including 5’ UTR 460 bp,3 ’UTR 193 bp,open reading frame 1419 bp.It encoded 472 amino acids.There was a START domain in amino acid sequence 264～466.The full length of HC-Cyp17 a gene was 3115 bp,including5’ UTR 95 bp,open reading frame 1256 bp,3’ UTR1762 bp.It encoded 502 amino acids.Sequence analysis showed that Ono sequence,Ozols subpeptide region and heme binding region were three functional conserved regions.The gene had hydroxylase activity and lyase activity.The full length of HC-17β-HSD11 gene was 1134 bp,including 5’ UTR 40 bp,3’UTR 171 bp and open reading frame 923 bp.It encoded 307 amino acids.The cofactor binding domain(TG×××G×G),catalytic active site(Y××SK)and structure stability domain(NNAG)were found in the encoded amino acids.2.Expression characteristics of 3 genesStAR3,Cyp17 a and 17β-HSD11 genes were expressed in both sexes of H.cumingii,and the expression level in different age also had certain difference.The expression level of StAR3 was highest in the gonads,and the male gonads were significantly higher than the female.In young mussel individuals,StAR3 gene was expressed at all months,and the expression level at 2 months old was significantly higher than other months.During the development of gonads in 12/24/32 month old H.cumingii,StAR3 gene was significantly higher in male than in female,and showed an upward trend with the increase of age.The expression level of StAR3 gene was the highest in the 36-month-old H.cumingii.Cyp17 a was highly expressed in liver,mantle and gonads,and the female gonads were significantly higher than the male ones.In 1～5-month-old mussel individuals,the expression of HCCyp17 a was relatively high and gradually maintained at a stable level.During the development of gonads in 12/24/32 month old H.cumingii,the expression of Hc-Cyp17 was significantly higher in female than in male.The expression level was highest in liver,and female was significantly higher than male.In gonads,the expression of 17β-HSD11 gene was significantly higher in ovary than in testis.At 6 months of age,the expression level of HC-17β-HSD11 was significantly higher than that of other months.At the same time,in the gonads of H.cumingii from the 12/24/32 month old mussel,the expression level of ovary was significantly higher than that of testis,and the expression level of ovary was the highest in the 24-month-old mussel.In situ hybridization showed that there was hybridization signal on the follicular membrane of male gonads.In situ hybridization showed that StAR3 gene was hybridization signal on the follicular wall cells of male gonads,Cyp17 a and 17β-HSD11 were significantly signaled in the follicular wall cells and oocyte,including in the cytoplasm and nucleus of ovary.3.Functional study of 3 genesTo investigate the role of StAR3 gene in H.cumingii sex hormone synthesis,which was upstream of the sex hormone pathway,RNA interference was performed to detect the relative expression levels of downstream Cyp17 a and 17β-HSD11 genes and the corresponding hormone content.The expression of Cyp17 a decreased by 56.49% in males and 60.24% in females.Meanwhile,the 17β-HSD11 decreased by 90.34% in males and 43.58% in females,respectively.The contents of pregnenolone,testosterone and estradiol in gonads were declined compared with the blank group.It was involved in the synthesis of pregnenolone,testosterone and in estradiol H.cumingii.It was further speculated that H.cumingii may have a similar pathway of sex hormone synthesis with vertebrates.The functions of Cyp17 a and 17β-HSD11 genes were further studied by exogenous hormone immersion.After exposing 10 months of age mussels to cultured waters containing17β-Estradiol(E2)and 17α-Methyltestosterone(MT)for 28 days,both E2 and MT at 50ng/L and 200 ng/L could affect the transcription level of Cyp17 a and 17β-HSD11.They were inhibited initially ant the early stage of exposing and increased or even upregulated in the later stage.According to the experimental results,E2 and MT can directly target the sex hormone signaling pathway and affect the activities of Cyp17 a and 17β-HSD11 in gonads,thus indirectly affecting steroid production.There may be a sex hormone synthesis pathway in H.cumingii similar to that in vertebrates.The function of 17β-HSD11 gene was studied by exogenous estrogen injection.The relative expression of 17β-HSD11 gene was inhibited in both male and female gonads by injecting different concentrations of 17β-estradiol into24-month-old mussel.The inhibition effect of high concentration injection group was more obvious than low concentration injection group.Therefore,17β-HSD11 may be related to estrogen synthesis in H.cumingii.In conclusion,StAR3 gene was highly expressed in male gonads in H.cumingii,while Cyp17 a and 17β-HSD11 genes were highly expressed in female gonads.It also played an important role in the development of male and female gonads.These genes were involved in gonadal development and sex determination through their roles in sex hormone synthesis pathways. |
