Effect of lipid peroxidation on the immunocytochemical detection of a leukocyte antigenic determinant in fresh and cryopreserved bovine spermatozoa

Abstract

Studies on different species, including rats, monkeys and humans, have shown the presence of leukocyte differentiationantigens in the spermatozoa. In some case the expression of these molecules is related to a specific functional state ofthe sperm cell, as was found for the CD 46 antigen, that in humans can be used as a marker of the acrosome reaction.The aim of the present study was to assess wether promoted lipid peroxidation of the spermatozoa induces any variationsin their immunoreactivity with ILA 147 antibody that, in bull spermatozoa, recognizes bovine leukocyte antigens.Freshly ejaculated bovine spermatozoa and cryopreserved semen were tested for ILA 147 reactivity by standardimmunoperoxidase staining, before and after promoted lipid peroxidation. Staining intensity was assessed in the individualcells using the microdensitometric method to measure integrated optical density (IOD), overcoming the disadvantageof an operator’s subjective interpretation of the results. After the lipid peroxidation there was significantlydecreased staining intensity in the fresh spermatozoa, but not in the cryopreserved cells. Furthermore, in the preincubationconditions, the cryopreserved spermatozoa had a lower mean I.O.D. value than the fresh sperm, showing that thefreezing and thawing processes induced an alteration in the antigen exposure. However the mean immunoreactivity ofthe cryopreserved cells was not significantly influenced by lipid peroxidation. The absorbance value maps, made followingimmunoperoxidase staining by the examined antibody, showed that the reaction sites in the fresh and cryopreservedspermatozoa fell mainly within the periacrosomal region. Moreover, after induced lipid peroxidation there were fewerreaction sites in this domain. The present research has confirmed the presence of the examined leukocyte antigenicdeterminant in the bull spermatozoa, and suggests that promoted lipid peroxidation and the freezing and thawing of spermatozoacan produce membrane damage, leading to reduced ILA 147 antigenic site exposure.

Keywords

• Bovine spermatozoa, Sperm antigens, Lipid peroxidation, Immunocytochemistry