MOLECULAR REPRODUCTION AND DEVELOPMENT 62:456–469 (2002) Molecular Cloning and Expression During Spermatogenesis of a cDNA Encoding Testicular 11b-Hydroxylase (P45011b) in Rainbow Trout (Oncorhynchus mykiss)

Cytochrome P450 1b-hydroxylase (P45011b) is an important steroidogenic enzyme for glucocorticoid and mineralocorticoid production in vertebrates. In teleosts, P45011b also plays a role in the production of 11-ketotestosterone (11-KT), the predominant androgen in male fish. In this study, we cloned a cDNA encoding rainbow trout (Oncorhynchus mykiss) testicular P45011b. The cDNA contains 1,740 nucleotides that encode a protein of 551 amino acids which shares 65.2%homology with testicular P45011b from Japanese eel, and 33–45% homology with adrenal P45011b from rat, human, and frog. HEK293 cells transfected with an expression vector containing the rainbow trout P45011b cDNA open reading frame showed 11b-hydroxylating activity in the presence of exogenous testosterone. Analysis of tissue distribution by RT-PCR showed great abundance of P45011bmRNA in testis and head kidney. In order to clarify the sites of P45011b gene expression, cRNA in situ hybridization analysis was performed. Hybridization signals were detected in Leydig cells and head kidney inter-renal cells. The results of Northern blot analysis indicated a single 1.8-kb transcript encoding P45011b in testis and in head kidney, suggesting that the testicular form of P45011b may also be involved in cortisol production by inter-renal cells. Seasonal changes in total P45011b mRNA levels in testes during spermatogenesis showed a pattern similar to that of plasma androgens. These results suggest that androgen production in male rainbow trout is partially regulated by changes in abundance of P45011b mRNA. Mol. Reprod. Dev. 62: 456–469, 2002. 2002 Wiley-Liss, Inc.