Cloning, expression and characterization of three types of 17β-hydroxysteroid dehydrogenases from the Nile tilapia, Oreochromis niloticus

Abstract

In order to elucidate the roles of 17 -HSDs in fish gonadal steroidogenesis, three types of 17 -HSDs (17 -HSD1, 17 -HSD8 and putative 17 -HSD12) were cloned and characterized from the Nile tilapia, Oreochromis niloticus. The cloned cDNAs of 17 -HSD type 1, 8 and 12 were 1504, 1006 and 1930 bp long, with open reading frames encoding proteins of 289, 256 and 314 aminoacids, respectively. Tissue distribution pattern analyzed by RT-PCR and Northern blot showed that 17 -HSD1 was dominantly expressed in the ovary, while the putative 17 -HSD12, one of the two duplicates found in fish, is a male specific enzyme and expressed exclusively in testis (detected by RT-PCR only). On the other hand, 17 -HSD8 was expressed in the brain, gill, heart, liver, intestine, gonad, kidney and muscle of both male and female. Enzymatic assays of the three types of 17 -HSDs were performed using recombinant proteins expressed in E. coli or HEK 293 cells. Tilapia 17 -HSD1 expressed in E. coli had the preference for NADP(H) as cofactor and could catalyze the inter-conversion between estrone and estradiol efficiently as well as the inter-conversion between androstenedione and testosterone, but less efficiently. Tilapia 17 -HSD8 recombinant protein expressed in HEK 293 cells could catalyze the conversion of testosterone to androstenedione, as well as the inter-conversion between estrone and estradiol. However, the putative 17 -HSD12 expressed in E. coli or in HEK 293 cells showed no conversion to any of the four substrates tested in this study. Based on enzyme characterization and tissue distribution, it is plausible to attribute crucial roles to 17 -HSDs in the gonadal steroidogenesis of teleosts.

Steroidogenic acute regulatory (StAR) protein and cholesterol side-chain cleavage (P450scc)-regulated steroidogenesis as an organ-specific molecular and cellular target for endocrine disrupting chemicals in fish

“…5), while StAR mRNA was not quantifiable. Several studies have reported the expression of steroidogenic enzymes and proteins in fish hepatic tissues (von Hofsten et al 2002;Wang and Ge 2004;Zhou et al 2005). To our knowledge, our studies represent the first reports of the parallel expression of aromatase gene isoforms, StAR, CYP11β and 3β-HSD in fish liver and the subsequent modulation by an estrogen mimic (Mortensen and Arukwe 2007).…”

Ovarian steroidogenesis and the role of sex steroid hormones on ovarian growth and maturation of the Japanese eel

“…2). Gaining a better understanding of the regulation of E2 synthesis is reliant on studies on extragonadal metabolism of androgens and estrogens, and molecular characterization of multiple forms of 17␤-HSD in fish, although such reports [31,44,[49][50][51][52] have been restricted to date.…”

Transcriptional regulation of the human type 8 17β-hydroxysteroid dehydrogenase gene by C/EBPβ

“…Multiple different mammalian 17␤-HSD have been cloned and characterized to date. Human 17␤-HSDs differ in nucleotide cofactor and substrate specificities, subcellular compartmentalization and tissue-specific expression patterns [1][2][3][4][5][6]. One of these enzymes, the type 8 17␤-HSD (HSD17B8, also known as ke6) was primarily characterized as a protein whose abnormal gene regulation predominantly expressed in the liver and pancreas and moderately present in the skeletal muscle and kidney [13].…”