| Steroid hormones are involved in various physiological functions like development, growth, reproduction and stress-homeostasis in vertebrates. Biosynthesis of steroid hormones from cholesterol involves a cascade of steroidogenic enzymes. Among these,3β-HSD (3β-hydroxysteroid dehydrogenase) and StAR (Steroidogenic acute regulatory protein) are the rate-limiting steroidogenic genes in the biosynthesis of steroid hormones. The progressively completed sequencing of fish genomes and transcriptome databases provided a platform and chance for further study on the molecular mechanism of steroidogenesis.In the present study, we isolated two isoforms of3β-HSD and StAR genes from Nile tilapia. There are obvious differences in the gene structure between3/3-HSD-Ⅰ and3β-HSD-Ⅱ. The phylogenetic analysis of3β-HSDs in vertebrates showed that3β-HSD-Ⅰ and-Ⅱ clustered into different clades. On the other hand, the two StAR genes structures showed higher similarity. The phylogenetic tree showed that fish StAR-Ⅰ was clustered into one clade with other vertebrates StARs, while StAR-Ⅱ formed a distinct fish specific clade. Furthermore, the phylogenetic tree demonstrated clearly that this duplication is a phenomenon unique to teleosts. This might due to that the genomes of most bony fishes have another round (the3round) duplication during evolution of modern fishes.Through both Real-Time PCR and ISH, we analyzed the expression profiles of the two types of3β-HSDs and StARs in different tissues, during the ontogenetic development, the spawning cycle and the letrozole induced sex reversal. We found that they displayed different expression profiles during all the processes, so we speculate that they might have different roles in the steroidogenesis.Firstly, they show different tissue distribution patterns. Real-Time PCR showed that3β-HSD-Ⅰ dominantly expressed in the head kidney and the testis, while3β-HSD-Ⅱ dominantly expressed in the ovary and the testis. Similarly, StAR-Ⅰ showed higher expression level in the head kidney, kidney and the testis. However, StAR-Ⅱ was exclusively expressed in the gonads. All these were further confirmed by ISH, that is the signals of3β3-HSD-Ⅰ and StAR-Ⅰ were observed in the interrenal cells of the head kidney and the interstitial cells of the testis. And the expression of StAR-Ⅰ and3/3-HSD-Ⅱ was also detected in the oocyte of the ovary. However, the signal of StAR-Ⅱ was only detected in the cells around the blood vessel in the ovary and interstitial cells of the testis. We speculate that one copy of the duplicated steroidogenic genes were specifically expressed in the interregnal cells and3β-HSD-Ⅰ and StAR-Ⅰ might play key roles in the production of cortisol in the head kidney.Secondly, the expression patterns of3/3-HSDs and StARs showed sexual and spatiotemporal differences in ontogenetic process. Real-Time PCR demonstrated that the expression of3β-HSD-Ⅰ was first observed from5dah in both the XX and XY gonad. In the XX gonad, the expression level was increased from5dah and peaked at30dah, then declined dramatically. However, in the XY gonad the level of it kept at an increasing trend from5dah. During the early gonadal developmental stage,3β-HSD-Ⅱ was only expressed in XX gonads and in the ovary it was sharply up-regulated from60dah. However, in the XY gonad, the level of3β-HSD-Ⅱ was kept very low during the early gonadal developmental stage, but it could be only detected from adult stage in the testis. The expression of StAR-Ⅰ was increased sharply from30dah in the XY gonads and was kept high expression level continuously. Whereas in XX gonads, it was kept at a minimal expression level throughout all the stages. The expression of StAR-Ⅱ declined from30dah in XX gonads, it turned out to be a male dominated gene in XY gonads from around60dah. By ISH analysis, we could detect the signal of3β-HSD-Ⅰ in somatic cells of both gonads from10dah. But we could not detect the signal of3β-HSD-Ⅱ during the early gonadal developmental stage. The expression of StAR-Ⅱ was first observed from5dah in the XX gonad and30dah in the XY gonad, and it indicated that StAR-Ⅱ might be involved in the ovarian development.In the spawning cycle,3β-HSD-Ⅰ was abundantly expressed in the spawning cycle, so it might be involved in the production and maturation of oocytes. However, the expression level of3β-HSD-Ⅱ was relatively low during all the stages. The expression level of StAR-Ⅰ was found to be gradually up-regulated from day1to8, peaked at day10, and then declined steadily. High expression level during day8to12indicated its possible role in the steroidogenesis, especially in oocyte maturation. On the contrary, expression of StAR-II remained at a barely changed profile during the spawning cycle.Long term of letrozole (aromatase inhibitor) treatment on3month old XX tilapia resulted in ovotestis and further testis formation. And during the phenotype from female to male, the genotype did not change. Real-Time PCR analysis revealed that3β-HSD-Ⅰ was significantly up-regulated during sex reversal. However, the expression level of3β-HSD-Ⅱ increased first and then decreased, which may be caused by a compensatory increase. Real-Time PCR analysis also revealed that both StARs were significantly up-regulated during sex reversal. ISH analysis revealed that the signal of3β-HSD-Ⅰ and-Ⅱ were detected in the testicular region and the degenerating ovarian portion of the letrozole treated XX gonad, respectively. The expression of both StAR-Ⅰ and-Ⅱ was specifically detected in interstitial cells of the testicular region. In short, the expression of3β-HSDs and StARs has closely associated with the phenotype.Promoter analysis showed that the transcription factors CREB-O and CREB-T could increase the expression level of3β-HSD-Ⅰ,3β-HSD-Ⅱ, StAR-Ⅰ and StAR-Ⅱ, which showed a dose dependence manner.In summary, in the present study, we isolated two types of3/3-HSD and StAR genes from Nile tilapia, respectively. Sequence homology and phylogenetic analysis showed that StAR (fish StAR-Ⅰ) are more conservative throughout the vertebrates, while StAR-Ⅱ is a teleost specific gene, which might due to the fish specific genome duplication. And all the vertebrates have3β-HSD duplication phenomenon, so the duplication may occur in the first two vertebrate genome duplication process. It was found that they display different roles at different gonadal development stages, sex reversed course and cortisol synthesis in the head kidney. Particularly,3β-HSD-Ⅰ may be involved in the testis development and the production of androgen; And3/3-HSD-Ⅱ exclusively expressed in the adultovary, therefore it is probably required for estrogen synthesis and ovarian maintenance. StAR-Ⅰ might be essential for cortisol production in the head kidney, DHP production during oocyte maturation and androgen production in the testis, while StAR-Ⅱ is probably required for early steroid synthesis in the XX gonad and androgen production in the testis. The transcription factor CREB-O and CREB-T were specifically expressed in the ovary and testis, repectively, but both could stimulate the expression of3/3-HSD and StAR genes, so we speculated that fish steroidogenesis was regulated by cAMP signaling pathway, moreover, the factors involved in the regulation between female and male are different. |
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