The production of 11-ketotestosterone (11KT), an important steroid hormone in piscine spermatogenesis, is regulated by the pituitary gonadotropins [Gths: follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh)] and it is synthesized by catalytic reactions involving several steroidogenic enzymes. Among these enzymes, the role of 17β-hydroxysteroid dehydrogenases (Hsd17bs) that exhibited 17-ketosteroid reducing activity (17KSR activity) responsible for 11KT synthesis is still poorly understood. In the present study, for the deeper understanding of testicular 11KT biosynthesis, we first investigated the steroidogenic pathway to produce 11KT in Japanese eel testis. In vitro incubation of the testis with androstenedione (A4) and the subsequent analysis of the metabolites by thin-layer chromatography indicated that 11KT was synthesized from A4 via 11β-hydroxyandrostenedione (11OHA4) and 11-ketoandrostenedione (11KA4), which indicated that the steroidogenic enzyme exhibiting the 17KSR activity responsible for converting 11KA4 to 11KT is crucial for 11KT production. Subsequently, cDNAs encoding three candidate enzymes, Hsd17b type3 (Hsd17b3), Hsd17b type12a (Hsd17b12a), and 20β-hydroxysteroid dehydrogenase type2 (Hsd20b2), potentially with the 17KSR activity were isolated and characterized in the Japanese eel. The isolated hsd17b3, hsd17b12a, and hsd20b2 cDNAs putatively encoded 308, 314, and 327 amino acid residues with high homology to those of other vertebrate counterparts, respectively. The Hsd17b3, Hsd17b12a, and Hsd20b2 expressed either in HEK293T or in Hepa-E1 converted 11KA4 to 11KT. Tissue-distribution analysis by quantitative real time PCR revealed that hsd17b12a and hsd20b2 mRNAs were detected in the testis, while hsd17b3 mRNA was not detectable. Furthermore, we examined the effects of Gths on the 17KSR activity and the expression of the candidate genes in the immature testis. The 17KSR activity was upregulated by administration of Gths. Furthermore, only expression of hsd17b12a among three candidates was upregulated by Gths as well as the 17KSR activity. These findings strongly suggested that Hsd17b12a is one of the enzymes with 17KSR activity responsible for 11KT synthesis in the testis of Japanese eel.

中文翻译：

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17β-羟基类固醇脱氢酶12a型负责日本鳗鳗的睾丸11-酮睾酮合成。

垂体促性腺激素[Gths：促卵泡激素（Fsh）和黄体生成素（Lh）]调节11-酮睾酮（11KT）的生成，这是鱼类精子发生中的重要类固醇激素。类固醇生成酶。在这些酶中，人们对17β-羟基类固醇脱氢酶（Hsd17bs）的作用表现出负责11KT合成的17-酮类固醇还原活性（17KSR活性）的作用仍知之甚少。在本研究中，为了更深入地了解睾丸11KT的生物合成，我们首先研究了在日本鳗鱼睾丸中产生11KT的类固醇生成途径。睾丸与雄烯二酮（A4）的体外温育以及随后通过薄层色谱分析的代谢产物表明，通过11β-羟基雄烯二酮（11OHA4）和11-酮雄烯二酮（11KA4）从A4合成了11KT，这表明类固醇生成表现出负责将11KA4转化为11KT的17KSR活性的酶对于11KT的生产至关重要。随后，在日本鳗鱼中分离了编码三种可能具有17KSR活性的候选酶cDNA，它们分别编码Hsd17b 3型（Hsd17b3），Hsd17b 12a型（Hsd17b12a）和20β-羟基类固醇脱氢酶2型（Hsd20b2）。分离的hsd17b3，hsd17b12a和hsd20b2 cDNA假定分别编码308、314和327个氨基酸残基，与其他脊椎动物对应物具有高度同源性。Hsd17b3，在HEK293T或Hepa-E1中表达的Hsd17b12a和Hsd20b2将11KA4转换为11KT。定量实时PCR的组织分布分析表明，在睾丸中检测到了hsd17b12a和hsd20b2 mRNA，而未检测到hsd17b3 mRNA。此外，我们检查了Gths对17KSR活性和未成熟睾丸中候选基因表达的影响。施用Gths可上调17KSR活性。此外，Gths和17KSR活性仅上调了三个候选基因中hsd17b12a的表达。这些发现强烈表明，Hsd17b12a是具有17KSR活性的酶之一，在日本鳗鱼的睾丸中负责11KT的合成。定量实时PCR的组织分布分析表明，在睾丸中检测到了hsd17b12a和hsd20b2 mRNA，而未检测到hsd17b3 mRNA。此外，我们检查了Gths对17KSR活性和未成熟睾丸中候选基因表达的影响。施用Gths可上调17KSR活性。此外，Gths和17KSR活性仅上调了三个候选基因中hsd17b12a的表达。这些发现强烈表明，Hsd17b12a是具有17KSR活性的酶之一，在日本鳗鱼的睾丸中负责11KT的合成。定量实时PCR的组织分布分析表明，在睾丸中检测到了hsd17b12a和hsd20b2 mRNA，而未检测到hsd17b3 mRNA。此外，我们检查了Gths对17KSR活性和未成熟睾丸中候选基因表达的影响。施用Gths可上调17KSR活性。此外，Gths和17KSR活性仅上调了三个候选基因中hsd17b12a的表达。这些发现强烈表明，Hsd17b12a是具有17KSR活性的酶之一，在日本鳗鱼的睾丸中负责11KT的合成。我们检查了Gths对17KSR活性的影响以及未成熟睾丸中候选基因的表达。施用Gths可上调17KSR活性。此外，Gths和17KSR活性仅上调了三个候选基因中hsd17b12a的表达。这些发现强烈表明，Hsd17b12a是具有17KSR活性的酶之一，在日本鳗鱼的睾丸中负责11KT的合成。我们研究了Gths对17KSR活性和未成熟睾丸中候选基因表达的影响。施用Gths可上调17KSR活性。此外，Gths和17KSR活性仅上调了三个候选基因中hsd17b12a的表达。这些发现强烈表明，Hsd17b12a是具有17KSR活性的酶之一，在日本鳗鱼的睾丸中负责11KT的合成。