Only low levels of spermadhesin AWN are detectable on the surface of live ejaculated boar spermatozoa

The zona-binding protein family of spermadhesins are constituents of boar s eminal plasma that are generally believed to attach to the acrosomal region of spermatozoa and thereby assist sperm interaction with the zona pellucid a at fertilization. However, previous studies have yielded conflicting resu lts with respect to amounts of adhesin bound to ejaculated cells, to the di stribution of bound adhesin within the sperm Population, and the regionaliz ation of binding on the sperm surface. In the present study, spermadhesin A WN in unfixed living suspensions of boar spermatozoa was assessed by means of flow cytometry and immunocytochemistry, using a polyclonal antibody rais ed in chicken. Direct probing with an Oregon Green conjugate of the antibod y was compared with indirect probing using Alexa Fluor-conjugated goat anti -chicken IgG as second antibody. Regardless of staining procedure, the live sub-population showed homogeneously low levels of staining, whereas the de ad sub-population showed high (more than 5-fold greater) levels of staining . The live cells were stained about 2-fold more intensely by anti-AWN than by preimmune immunoglobulin. indicating the presence of small amounts of AW N. Immunocytochemistry showed the live cells to be faintly stained all over their surface, whereas staining of the dead cells was largely localized to the acrosomal region. This latter staining was non-specific, preimmune imm unoglobulin resulting in as much bound fluorescence as anti-AWN. Attempts t o block non-specific staining with appropriate pretreatment with chicken or goat serum (as compared with routine use of BSA) met with variable and inc omplete success, and did not increase staining by anti-AWN relative to prei mmune serum in either live or dead cells. It is concluded that limited amou nts of spermadhesin AWN bind tightly over the whole surface of live ejacula ted boar sperm. However, the acrosomal region of disrupted sperm has an ala rming tendency to bind fluoro-conjugates of immunoglobulins non-specificall y.