Immunological identification of the protein-producing masculine differentiation of the Wolffian duct in the fetal mouse: western blot analysis and determination of the biological activity.

Abstract

Recently, we identified a protein in the developing male reproductive tract of the fetal mouse which induced Wolffian duct differentiation in vitro in the absence of testis or testosterone. In this paper, we further evaluated the masculinizing role of this protein using an immunological approach. Thus, we purified a 72K protein from the 18-day-old male fetal reproductive tracts, prepared an antibody against the protein, and determined the role of this protein in producing masculine differentiation of the Wolffian duct using the polyclonal antibody against the protein. We demonstrate that the antibody reacted with the purified 72K protein, producing only one immunoreactive band around the 72K region in Western blot analysis. However, it reacted with 72K and 63K proteins present in the 18-day-old male reproductive tract. Both of these immunoreactive bands disappeared when the antibody was pretreated with the purified antigen. Nonimmune immunoglobulin G (IgG) produced no reactive bands with the extract of the male reproductive tracts. The IgG preparation of the immune serum specifically prevented Wolffian duct differentiation when tested in organ culture containing 13-day-old fetal male or female reproductive tracts in the presence of testis or testosterone (1 micrograms/ml) in a dose-dependent manner. No effect was found on the development of other organs, namely testis, Mullerian duct, and urogenital sinus, that were included in the organ culture. Nonimmune IgG, as expected, produced no effect on Wolffian duct differentiation. Western blot analysis of male and female reproductive tracts indicated a difference in their reactivities with this antibody. In females, the reactivity to the proteins around the 72K and 63K regions was very weak compared to that found with male proteins in those regions. Both of the above bands of the female reproductive tract disappeared when the antibody was pretreated with an excess of female reproductive tract extract, but in males, only the 63K band disappeared, whereas its 72K band remained. In conclusion, it appears that a 72K protein of the male reproductive tract plays a role in the Wolffian duct differentiation of the fetal mouse.