Cytochrome P-450 17 alpha-hydroxylase protein and mRNA in the testis of the testicular feminized (Tfm) mouse.
The testicular feminized (Tfm) mouse lacks functional androgen receptors and develops with a female external phenotype and internal testes. The testes of these animals contain normal, or close to normal, numbers of Leydig cells but secrete very low amounts of androgen due to a lack of 17 alpha-hydroxylase activity. To determine whether this loss of activity is due to a lack of enzyme synthesis or a change in catalytic activity we have examined 17 alpha-hydroxylase cytochrome P-450 (P-450(17 alpha)) protein and mRNA levels in the testes of Tfm mice. Levels of P-450(17 alpha) protein were measured by immunoblotting, while mRNA was measured following reverse transcription (RT) and amplification by the polymerase chain reaction (PCR). Conditions for RT-PCR were determined which allowed semiquantification of P-450(17 alpha) mRNA relative to beta-actin mRNA. In extracts of Tfm testes P-450(17 alpha) protein was undetectable using antiserum against porcine P-450(17 alpha). In contrast, a protein of around 54 kDa was clearly detectable in extracts of control cryptorchid testes. Using RT-PCR, P-450(17 alpha) mRNA was detectable in both control and Tfm testes but, expressed in terms of beta-actin mRNA, levels of P-450(17 alpha) mRNA in control testes were 40-fold higher than those in Tfm testes. If the total amount of RNA extracted from each testis is taken into account then P-450(17 alpha) mRNA levels per testis were up to 400-fold higher in control testes. These results show that the reduced level of 17 alpha-hydroxylase activity in Tfm testes is related to reduced protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
O'Shaughnessy
P J
PJ
Department of Veterinary Physiology, Veterinary School, University of Glasgow, UK.
Murphy
L
L
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D018076:DNA, Complementary / Q000032:analysis; D005262:Feminization / Q000201:enzymology* / Q000235:genetics*; D015151:Immunoblotting; D007985:Leydig Cells / Q000201:enzymology; D008297:Male; D051379:Mice; D008817:Mice, Mutant Strains; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism*; D013254:Steroid 17-alpha-Hydroxylase / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D013737:Testis / Q000201:enzymology*
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D013254:Steroid 17-alpha-Hydroxylase