Molecular cloning of two isoforms of 11beta-hydroxylase and their expressions in the Nile tilapia, Oreochromis niloticus.
P450 11beta-hydroxylase, encoded by P450(11beta) gene, is a key mitochondrial enzyme to produce 11beta-hydroxy testosterone, substrate for the production of 11-ketotestosterone (11-KT), which has been shown to be potent androgen in several fish species. In the present work, two alternative splicing isoforms i.e. P450(11beta)-1 and P450(11beta)-2 cDNAs were cloned from the Nile tilapia, Oreochromis niloticus. They were 1614 and 1227bp in length with open reading frames encoding proteins of 537 and 408 amino acids, respectively. In contrast to P450(11beta)-1, which derived from 9 exons of the P450(11beta) gene, the 7th and 8th exons were absent in P450(11beta)-2. Tilapia P450(11beta)-1 shares the highest homology with that of medaka, Oryzias latipes. Expressions of P450(11beta)-1 and -2 were detected in the kidney and head kidney of both sexes, and in the testis but not in the ovary, with P450(11beta)-2 lower than P450(11beta)-1. Ontogenic expressions of both isoforms were detected in testis from 50dah onwards. P450(11beta)-1 and -2 were strongly expressed in sex reversed XX testis after fadrozole and tamoxifen treatment, but completely inhibited in 17beta-estradiol induced XY ovary. The existence of two alternatively spliced isoforms and the sexual dimorphic expression of P450(11beta)s were further confirmed by Northern blot. Strong expression signals in Leydig cells and weak signals in spermatogonia were detected by in situ hybridization and immunohistochemistry. Taken together, our data suggest a role for P450(11beta) in the spermatogenesis of tilapia through the production of 11-KT in testis, in addition to cortisol production in head kidney.
Zhang
Wei-Li
WL
Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), Key Laboratory of Aquatic Science of Chongqing, School of Life Science, Southwest University, Chongqing, China.
Zhou
Lin-Yan
LY
Senthilkumaran
Balasubramanian
B
Huang
Bao-Feng
BF
Sudhakumari
Cheni Chery
CC
Kobayashi
Tohru
T
Nagahama
Yoshitaka
Y
Wang
De-Shou
DS
D017398:Alternative Splicing / Q000235:genetics; D000818:Animals; D015152:Blotting, Northern; D023681:Cichlids / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D007668:Kidney / Q000201:enzymology; D008297:Male; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013250:Steroid Hydroxylases / Q000235:genetics* / Q000378:metabolism*; D013737:Testis / Q000201:enzymology; D013739:Testosterone / Q000031:analogs & derivatives / Q000378:metabolism
D020033:Protein Isoforms; D013739:Testosterone; D013250:Steroid Hydroxylases; C074514:steroid 11-alpha-hydroxylase; C003600:11-ketotestosterone