CYTOCHROME-P-450 17-ALPHA-HYDROXYLASE PROTEIN AND MESSENGER-RNA 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 17alpha-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 17alpha-hydroxylase cytochrome P-450 (P-450(17alpha)) protein and mRNA levels in the testes of Tfm mice. Levels of P-450(17alpha) 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(17alpha) mRNA relative to beta-actin mRNA. In extracts of Tfm testes P-450(17alpha) protein was undetectable using antiserum against porcine P-450(17alpha). In contrast, a protein of around 54 kDa was clearly detectable in extracts of control cryptorchid testes. Using RT-PCR, P-450(17alpha) mRNA was detectable in both control and Tfm testes but, expressed in terms of beta-actin mRNA, levels of P-450(17alpha) 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(17alpha) mRNA levels per testis were up to 400-fold higher in control testes. These results show that the reduced level of 17alpha-hydroxylase activity in Tfm testes is related to reduced protein synthesis. Previous results have shown that androgens reduce P-450(17alpha) mRNA levels in cultured Leydig cells. Results from this study suggest, however, that androgens are required to induce normal levels of P-450(17alpha) mRNA in Leydig cells.