The effect of fixatives and/or air-drying on the plasma and acrosomal membranes of human sperm.

It has been hypothesized that some fixatives and conditions of slide preparation expose internal sperm antigens and thus are not suitable for demonstrating surface-specific antigens by immunocytochemical assays. This study examined the ability of five fixatives (glutaraldehyde, acetone, methanol, paraformaldehyde, and periodate-lysine-paraformaldehyde [PLP]) and two conditions of slide preparation (air-drying or maintaining sperm in a liquid phase) to maintain the integrity of human spermatozoal membranes at the ultrastructural level as monitored by transmission electron microscopy. Regardless of the fixative employed, air-drying was detrimental to plasma and acrosomal membrane integrity. Acetone and methanol completely disrupted the plasma and acrosomal membranes over the entire sperm surface whether air-drying or liquid phase conditions were employed. Fixation with glutaraldehyde and paraformaldehyde (to a lesser degree) maintained the morphologic integrity of acrosomal and plasma membranes provided the sperm were maintained in a liquid phase. However, glutaraldehyde and paraformaldehyde fixation increased the postfixation binding of immunoglobulins to the sperm surface. We concluded that immunocytochemistry at the light microscopy level may imply erroneous interpretations regarding antigen/antibody interactions on the sperm plasma membrane surface unless care is taken to verify that the antibodies of interest are binding to the antigenically intact plasma or acrosomal membrane.