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 seminal plasma that are generally believed to attach to the acrosomal region of spermatozoa and thereby assist sperm interaction with the zona pellucida at fertilization. However, previous studies have yielded conflicting results with respect to amounts of adhesin bound to ejaculated cells, to the distribution of bound adhesin within the sperm population, and the regionalization of binding on the sperm surface. In the present study, spermadhesin AWN in unfixed living suspensions of boar spermatozoa was assessed by means of flow cytometry and immunocytochemistry, using a polyclonal antibody raised in chicken. Direct probing with an Oregon Green conjugate of the antibody 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 dead 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 AWN. 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 immmunoglobulin resulting in as much bound fluorescence as anti-AWN. Attempts to block non-specific staining with appropriate pretreatment with chicken or goat serum (as compared with routine use of BSA) met with variable and incomplete success, and did not increase staining by anti-AWN relative to preimmune serum in either live or dead cells. It is concluded that limited amounts of spermadhesin AWN bind tightly over the whole surface of live ejaculated boar sperm. However, the acrosomal region of disrupted sperm has an alarming tendency to bind fluoro-conjugates of immunoglobulins non-specifically.