Androgen-binding protein (ABP) is a Serotoli cell product though to be involved in spermatogenesis, the acquisition of fertilizing ability by spermatozoa, and/or the regulation of epididymal function. However, none of these putative roles has been clearly demonstrated. The long-term goal of this research is to determine if ABP is involved in these processes. As initial steps in achieving this goal, six Specific Aims are proposed: 1) to determine if there are cellular receptors for ABP. This will be done be examining in vivo uptake of (125I)ABP and in vitro binding of {125I)ABP to tissue homogenates and membrane fractions. 2) to determine the oligosaccharide composition of serum and epididymal ABP. Serial lectin chromatography will be used to separate various glycosylated forms of ABP. Following enzyme treatment to release the oligosaccharides, their structure will be determined by mass spectrometry alone or in combination with NMR. 3) to determine the functional significance of the microheterogeneity of ABP by testing various glycosylated forms of ABP for their ability to bind to receptors and to bind androgens. 4) to study the biosynthesis and secretion of ABP by examining the incorporation of (3H)mannose and (3H)glucosamine into Asn- and O-linked oligosaccharides on immunoprecipitated ABP from cultured Sertoli cells. Incorporation of 35S and 32P into threonine, serine, or oligosaccharide residues on APB will also be studied. 5) to develop a library of monoclonal antibodies to ABP to map its structural and functional domains. Monoclonal antibodies will be screened for their ability to react with various glycosylated forms of ABP and its binding-site peptide. Antibodies recognizing various domains will be identified and tested for their ability to interfere with ABP in tissues and fluids and to determine its possible function. This will be accomplished by RIA and immunochemical localization of ABP and by passive immunization studies. The effects of ABP neutralization on target tissue histology and on the fertilizing ability of spermatozoa will be assessed. Knowledge gained will be important for diagnosing and possibly treating cases of male infertility arising from defects in ABP function and for developing contraceptive agents that will interfere with it.
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