Abstract:
The zona-binding protein family of spermadhesins are constituents of boarseminal plasma that are generally believed to attach to the acrosomal regionof spermatozoa and thereby assist sperm interaction with the zona pellucida atfertilization. However, previous studies have yielded conflicting results withrespect to amounts of adhesin bound to ejaculated cells, to the distributionof bound adhesin within the sperm population, and the regionalization ofbinding on the sperm surface. In the present study, spermadhesin AWN inunfixed living suspensions of boar spermatozoa was assessed by means of flowcytometry and immunocytochemistry, using a polyclonal antibody raised inchicken. Direct probing with an Oregon Green conjugate of the antibody wascompared with indirect probing using Alexa Fluor-conjugated goat anti-chickenIgG as second antibody. Regardless of staining procedure, the livesub-population showed homogeneously low levels of staining, whereas the deadsub-population showed high (more than 5-fold greater) levels of staining. Thelive cells were stained about 2-fold more intensely by anti-AWN than bypreimmune immunoglobulin, indicating the presence of small amounts of AWN.Immunocytochemistry showed the live cells to be faintly stained all over theirsurface, whereas staining of the dead cells was largely localized to theacrosomal region. This latter staining was non-specific, preimmuneimmmunoglobulin resulting in as much bound fluorescence as anti-AWN. Attemptsto block non-specific staining with appropriate pretreatment with chicken orgoat serum (as compared with routine use of BSA) met with variable andincomplete success, and did not increase staining by anti-AWN relative topreimmune serum in either live or dead cells. It is concluded that limitedamounts of spermadhesin AWN bind tightly over the whole surface of liveejaculated boar sperm. However, the acrosomal region of disrupted sperm has analarming tendency to bind fluoro-conjugates of immunoglobulinsnon-specifically.