STIMULATION OF EPIDERMAL GROWTH-FACTOR GENE-EXPRESSION DURING THE FETAL MOUSE REPRODUCTIVE-TRACT DIFFERENTIATION - ROLE OF ANDROGEN AND ITSRECEPTOR

We have shown previously that epidermal growth factor (EGF) plays a ro le in testosterone-dependent fetal Wolffian duct differentiation. To f urther assess the role for EGF, we determined whether EGF gene express ion was modulated in response to male reproductive tract differentiati on. The expression of EGF messenger RNA (mRNA) was measured by an RT-P CR assay using primer pairs spanning the coding sequence of 3228 nucle otide (nt) to 3401 nt. Using the RT-PCR reaction, an amplimer of the e xpected size, 173 bp, was detected in the fetal male reproductive trac t. The amplimer hybridized with a radioactive probe representing an in ternal sequence of the amplified product and was digested by the restr iction enzyme HaeIII, which has an unique cleavage site at 3365 nt. Th e level of EGF mRNA at different stages of sexual differentiation was measured by a newly developed quantitative competitive RNA PCR (QCPCR) assay for EGF mRNA. The assay was sensitive and reproducible within a linear range of amplification with 5 x 10(4) to 140 x 10(4) copies of mRNA. Using the quantitative competitive RNA PCR we found that the le vel of EGF mRNA was higher in the male reproductive tract than that in the female reproductive tract. Exposure to testosterone (40 mg/kg . d ay) during days 13-17 of gestation induced the Wolffian duct in the fe male fetuses and resulted in stimulation of EGF-mRNA expression. Simil arly, an antiandrogen receptor, flutamide (100 mg/kg . day) exposure d uring days 13-17 of gestation inhibited male reproductive tract differ entiation and resulted in inhibition of EGF-mRNA expression. Moreover, during the differentiation of the male reproductive tract there was a biphasic increase in the level of EGF-mRNA, first at day 14 of gestat ion, the period of onset of testicular activity and Wolffian duct morp hogenesis, and second at day 18 of gestation, corresponding to onset o f differentiation of the urogenital sinus, epididymal duct, and semina l vesicle. Thus, it appears that testosterone-induced male sexual diff erentiation is accompanied by an increase in EGF gene expression.