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

L. Y. Zhou, D. S. Wang, B. Senthilkumaran, M. Yoshikuni, Y. Shibata, T. Kobayashi, C. C. Sudhakumari, Y. Nagahama

Research output: Contribution to journalArticle

36 Citations (Scopus)

Abstract

In order to elucidate the roles of 17β-HSDs in fish gonadal steroidogenesis, three types of 17β-HSDs (17β-HSD1, 17β-HSD8 and putative 17β-HSD12) were cloned and characterized from the Nile tilapia, Oreochromis niloticus. The cloned cDNAs of 17β-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 17β-HSD1 was dominantly expressed in the ovary, while the putative 17β-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, 17β-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 17β-HSDs were performed using recombinant proteins expressed in E. coli or HEK 293 cells. Tilapia 17β-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 17β-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 17β-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 17β-HSDs in the gonadal steroidogenesis of teleosts.

Original languageEnglish
Pages (from-to)103-116
Number of pages14
JournalJournal of Molecular Endocrinology
Volume35
Issue number1
DOIs
Publication statusPublished - Aug 1 2005
Externally publishedYes

Fingerprint

17-Hydroxysteroid Dehydrogenases
Cichlids
HEK293 Cells
Tilapia
Organism Cloning
Androstenedione
Estrone
Tissue Distribution
Escherichia coli
Recombinant Proteins
Testosterone
Estradiol
Fishes
Polymerase Chain Reaction
Gonads
Enzyme Assays
Enzymes
NADP
Northern Blotting
Open Reading Frames

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Endocrinology

Cite this

Cloning, expression and characterization of three types of 17β-hydroxysteroid dehydrogenases from the Nile tilapia, Oreochromis niloticus. / Zhou, L. Y.; Wang, D. S.; Senthilkumaran, B.; Yoshikuni, M.; Shibata, Y.; Kobayashi, T.; Sudhakumari, C. C.; Nagahama, Y.

In: Journal of Molecular Endocrinology, Vol. 35, No. 1, 01.08.2005, p. 103-116.

Research output: Contribution to journalArticle

Zhou, L. Y. ; Wang, D. S. ; Senthilkumaran, B. ; Yoshikuni, M. ; Shibata, Y. ; Kobayashi, T. ; Sudhakumari, C. C. ; Nagahama, Y. / Cloning, expression and characterization of three types of 17β-hydroxysteroid dehydrogenases from the Nile tilapia, Oreochromis niloticus. In: Journal of Molecular Endocrinology. 2005 ; Vol. 35, No. 1. pp. 103-116.
@article{56c96864eb25416bbc6bcc548b6dbed7,
title = "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 17β-HSDs in fish gonadal steroidogenesis, three types of 17β-HSDs (17β-HSD1, 17β-HSD8 and putative 17β-HSD12) were cloned and characterized from the Nile tilapia, Oreochromis niloticus. The cloned cDNAs of 17β-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 17β-HSD1 was dominantly expressed in the ovary, while the putative 17β-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, 17β-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 17β-HSDs were performed using recombinant proteins expressed in E. coli or HEK 293 cells. Tilapia 17β-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 17β-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 17β-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 17β-HSDs in the gonadal steroidogenesis of teleosts.",
author = "Zhou, {L. Y.} and Wang, {D. S.} and B. Senthilkumaran and M. Yoshikuni and Y. Shibata and T. Kobayashi and Sudhakumari, {C. C.} and Y. Nagahama",
year = "2005",
month = "8",
day = "1",
doi = "10.1677/jme.1.01801",
language = "English",
volume = "35",
pages = "103--116",
journal = "Journal of Molecular Endocrinology",
issn = "0952-5041",
publisher = "Society for Endocrinology",
number = "1",

}

TY - JOUR

T1 - Cloning, expression and characterization of three types of 17β-hydroxysteroid dehydrogenases from the Nile tilapia, Oreochromis niloticus

AU - Zhou, L. Y.

AU - Wang, D. S.

AU - Senthilkumaran, B.

AU - Yoshikuni, M.

AU - Shibata, Y.

AU - Kobayashi, T.

AU - Sudhakumari, C. C.

AU - Nagahama, Y.

PY - 2005/8/1

Y1 - 2005/8/1

N2 - In order to elucidate the roles of 17β-HSDs in fish gonadal steroidogenesis, three types of 17β-HSDs (17β-HSD1, 17β-HSD8 and putative 17β-HSD12) were cloned and characterized from the Nile tilapia, Oreochromis niloticus. The cloned cDNAs of 17β-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 17β-HSD1 was dominantly expressed in the ovary, while the putative 17β-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, 17β-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 17β-HSDs were performed using recombinant proteins expressed in E. coli or HEK 293 cells. Tilapia 17β-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 17β-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 17β-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 17β-HSDs in the gonadal steroidogenesis of teleosts.

AB - In order to elucidate the roles of 17β-HSDs in fish gonadal steroidogenesis, three types of 17β-HSDs (17β-HSD1, 17β-HSD8 and putative 17β-HSD12) were cloned and characterized from the Nile tilapia, Oreochromis niloticus. The cloned cDNAs of 17β-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 17β-HSD1 was dominantly expressed in the ovary, while the putative 17β-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, 17β-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 17β-HSDs were performed using recombinant proteins expressed in E. coli or HEK 293 cells. Tilapia 17β-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 17β-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 17β-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 17β-HSDs in the gonadal steroidogenesis of teleosts.

UR - http://www.scopus.com/inward/record.url?scp=23844448838&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=23844448838&partnerID=8YFLogxK

U2 - 10.1677/jme.1.01801

DO - 10.1677/jme.1.01801

M3 - Article

C2 - 16087725

AN - SCOPUS:23844448838

VL - 35

SP - 103

EP - 116

JO - Journal of Molecular Endocrinology

JF - Journal of Molecular Endocrinology

SN - 0952-5041

IS - 1

ER -