Testosterone-Binding Protein in Reproductive Tracts of Fetal Rats

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Abstract
Testosterone-binding proteins may mediate the induction of Wolffian duct differentiation by testicular testosterone. The presence of such protein(s) was sought in reproductive tracts of 14.5-21.5 day old fetal rats. Supernatant fractions (127,000 .times. g) were equilibrated with [3H]testosterone ([3H]T) .+-. radioinert testosterone in Tris .cntdot. HCl:EDTA buffer, pH 7.4, (.apprx. 0.1 mg protein/0.5 ml at 4.degree. C for 16 h. Bound and free [3H]T were separated by charcoal-dextran absorption or Sephadex G-100 gel filtration. The results with 14.5-15.0 day old tracts were: specific binding to protein was saturated with increasing [3H]T concentration; scatchard plot analysis indicated the presence of a single class of binding sites with high affinity (apparent Kd = 2 nM) and limited capacity (.apprx. 16 femtomol/mg protein) for [3H]T; specific uptake was limited to [3H]T and [3H]5.alpha.-dihydrotestosterone; [3H]T uptake by the tract supernatant was tissue-specific; pronase treatment abolished binding capacity for [3H]T; bound radioactivity consisted solely of [3H]T and the mesonephric and ductal segment of the genital tract specifically binds [3H]T. The data demonstrate binding protein(s), specific for testosterone and possibly dihydrotestosterone, in the genital ducts of 14.5-15 day old fetal rats. [3H]T binding to genital duct supernatants from male but not from female fetuses increased about 5-fold between 14.5-20.5 days of gestation. Upon Sephadex G-100 gel filtration, radioactivity was confined to the macromolecular fraction appearing in the void volume. Nuclear fractions, obtained from intact ducts incubated with [3H]T at 30.degree. C but not at 0.degree. C contained radioactivity. These observations are compatible with the existence of a cytoplasmic testosterone receptor or carrier protein aggregate. Testosterone-binding proteins are present in the genital ducts of rat fetuses and, in the male, their concentrations increase with progressive Wolffian duct differentiation.