| Sex steroid hormones play an important role in fish sex differentiation,gonadal development and secondary sexual characteristics.Their synthesis involves a series of catalyzing enzymes,such as cytochrome P450(CYPs),hydroxysteroid dehydrogenase(HSDs)and so on.Among them,17β-hydroxysteroid dehydrogenase(Hsd17b)is a key class of enzyme that acts in downstream part of the steroidogenesis pathway and possesses the capacity for oxidation and reduction between estrone and estradiol and between androstenedione and testosterone.The olive flounder Paralichthys olivaceus,a commercially valuable marine aquaculture fish,was studied in this study.A total of10 flounder Hsd17 b family genes were obtained.Combined with the Hsd17b1,one gene published previously in our laboratory,we analyzed their genetic structure and the protein biochemical characteristics.The expression profiles of Hsd17 b family genes were detected in 12 different tissues of adult female and male flounders and in gonads at I-V gonadal development stages.Hsd17b1 and-3 were used as examples to further study their function in the flounder steroidogenesis pathway.These results could contribute to our understanding of the regulation of sex steroid hormone synthesis in fish gonadal development and provide a reference for the monoculture of Paralichthys olivaceus and other fishes.1.Hsd17b3,-4,-7,-8,-9,-10,-12 a,-12 b,-14 and-15 gene sequences were screened within the flounder genome and transcriptome data.After cloning and sequencing,ORF regions were obtained.Hsd17b9 and-15 have never been reported in a fish.According to the domain and motif analyses,they all belong to the short-chain dehydrogenase/reductase(SDR)superfamily and contain conserved motifs,such as the cofactor binding site TGxxx Gx G,reaction direction determining motif PGxxx T,structural stable motif NNAG and active center motif Yxxx K.Analysis of amino acid sequences predicted that Hsd17b1,-4,-7,-12 a and-14 were hydrophilic proteins,and the remaining proteins were hydrophobic.The stability of Hsd17b1,-3 and-12 b proteins was predicted to be low.Chromosome distribution results showed that the flounder Hsd17 b genes were distributed on eight chromosomes(chr 1,7,10,17,19,22,23 and 24),of which Hsd17b12 a and-12 b were located on chromosomes 19 and 7,respectively.It was demonstrated that they were two genes and had just highly similarity rather than different transcripts derived from the same gene.2.Real-time quantitative PCR(q PCR)was used to detect the expression patterns of the various Hsd17 b family genes in different tissues of adult flounder and in gonads at I-V gonadal developmental stages.The results showed that expression of most genes in tissues was widely distributed.Hsd17b9,-10,-12 a and-12 b were differently expressed in the testis and ovary,and among them Hsd17b10,-12 a and-12 b were highly expressed in the ovary,and Hsd17b9 was highly expressed in the testis than that in the ovary.Moreover,throughout gonadal development,the expression of Hsd17b3 and-9 was higher in the testis than that of the ovary,and Hsd17b1,-4,-8,-10,-12 a and-12 b expression was higher in the ovary than that of the testis,suggesting that they might play an important role in androgen and estrogen synthesis in the flounder gonadal development.3.Functional studies on Hsd17b1 and-3 were carried out.Firstly,the expression levels of Hsd17b3 in the gonadal differentiation were detected by q PCR.The results showed that in the control,high temperature(28 °C ± 0.3 °C,HT)and estrogen receptor inhibitors(tamoxifen,100 ppm,TM)groups,the expression level of Hsd17b3 was highest when the flounder was at 2 cm total length(TL),and compared with the control group,the expression of Hsd17b3 was decreased in the HT and TM groups.Transfection analysis in the HEK 293T(human embryonic kidney 293T)cells showed that Hsd17b1 and-3 were located in the cytoplasm and nucleus.Then,the results of enzyme specific activity determination showed that the activities of Hsd17b1 at stages I-V were higher in the testis than those in the ovary,and except for the stage IV,the difference between testis and ovary was extremely significant(P <0.01).Similar to the gene expression pattern,the specific activities of Hsd17b3 at stages I-V was significantly higher in the testis than that of the ovary(P < 0.05,P <0.01).At the same time,the in vitro intraperitoneal injection on the flounder was performed with estrogen tamoxifen(10 mg/m L)and androgen receptor inhibitor(flutamide,50 mg/m L).The results showed that after challenge with tamoxifen,the expression levels of Hsd17b1 in the testis and the ovary were no significantly difference.The Hsd17b3 expression level in the testis decreased significantly(P <0.01),and in the ovary no significant expression change was observed.Moreover,after challenge with flutamide,the expression levels of Hsd17b1 in the ovary and testis were up-regulated,and in the ovary,the increase was distinct(P < 0.05).Hsd17b3 expression was down-regulated in the testis and ovary without significant difference. |
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