Sex-specific mouse testosterone 16[alpha]-hydroxylase (cytochrome P450) genes : characterization and genetic and hormonal regulations

Abstract

The molecular and genetic backround of sex-specific mouse steroid hydroxylase (cytochrome P450) isozymes was investigated. Mouse testosterone 16 α-hydroxylase activity in liver microsomes is apparently the sum of at least two isozymes: I-P45016α is the female-specific isozyme, while C-P45016α is predominantly expressed in males. Female-specific I-P45016α mRNA and testosterone 16α-hydroxylase activity levels in Balb/cJ male mouse liver are increased by castration from undetectable to levels as high as those seen in normal Balb/cJ females. Moreover, castration does not increase the mRNA or activity levels in the genetic 129/J male variant. The derepression in castrated males exhibits an autosomal codominant inheritance and is regulated by the Ripr locus in crosses between Balb/cJ and 129/J mice. (Noshiro, M. & Negishi, M. Biochemistry 27, 6444-6448, 1988). The genotypical background of Ripr locus was examined by first isolating the cDNA clones associated with I-P45016α - dependent mRNA and activity from Balb/cJ female mouse liver cDNA library. Next, cDNA libraries of castrated Balb/cJ male livers were screened and mouse clones were isolated. The Ripr locus was determined to consist of a cluster of at least three active genes, designated 16αoh-a, 16αoh-b, and 16αoh-d, of which 16αoh-a was always the major type expressed in normal Balb/cJ female liver and in castrated Balb/cJ male liver and was not expressed in 129/J mice. Nuclear run-on assays proved that the "I-P45016α" genes controlled by Ripr locus were transcriptionally regulated. Growth hormone was found to be the direct repressor of the genes in male mouse liver. Several polymorphic clones of the various "I-P45016α" cDNA clones and also of P450cb (one of five members of the C-P45016α gene family) cDNA clones were discovered. The possible role of aberrant splicing of P450 transcripts in P450 regulation was suggested. Next, the genes corresponding to 16αoh-A and 16αoh-B were isolated asmembers of a large multigene family (at least 16 genes) and characterized. Several interesting sequence motifs were discovered. The 16αoh gene family was also analyzed and organized into three different subfamilies according to 1st exon area BamHI fragments. Finally, transgenic mice were generated with a "I-P45016α" (16αoh-a) minigene or with C-P45016α/CAT, a recombinant gene construct containing the 5' -flanking region C-P45016α gene linked to the bacterial gene encoding chloramphenicol acetyltransferase (CAT). Expression of the transgene was detected in only one line carrying the C-P45016α/CAT - recombinant gene. The individual expressing the gene was male and the target organ was liver, indicating that the 5.5 kb flanking region of C-P45016α contains the necessary elements for expression in male liver. A single expressing transgenic among a large number of transgenic mice suggests that testosterone l6a-hydroxylase genes (forms of P450) are possibly sensitive for their chromosomal locations.