Localization of lipid peroxidation-derived protein epitopes in the porcine corpus luteum

Reactive oxygen species (ROS) generated within the corpus luteum (CL) are believed to play an integral role in luteolysis. Unsaturated lipids are susceptible to peroxidation by ROS, leading to the formation of toxic aldehydes such as malondialdehyde (MDA) and 4-hydroxy-nonenal (4-HNE) that can attack cellular and extracellular proteins. Aldehyde conjugation of proteins can have diverse effects on cell function, including cross-linking of cell surface receptors, alteration of enzyme activities, and modification of lipoproteins. The objectives of the present study were to 1) determine by immunological techniques whether lipid peroxidation-derived protein epitopes (i.e., MDA- and 4-HNE-lysine) could be detected in situ during the natural life span of the CL, and 2) determine the temporal and spatial localization of these epitopes. Fresh luteal tissue was collected from young gilts at distinct phases of the estrous cycle: early (Days 4-7), mid (Days 8-12), and late (Days 14-18). Immunocytochemistry was performed on tissue sections using monoclonal antibodies against MDA-lysine and 4-HNE-lysine. Positive immunostaining was evident in porcine CL at all stages of the estrous cycle. Immunoactivity was associated primarily with the steroidogenic cells, most consistently with the large luteal cells, and was most intense in regressing tissue (i.e., late). No staining was observed in sections incubated with nonimmune mouse serum or when anti-4-HNE-lysine was preadsorbed with 4-HNE-protein preparations. Immunoblotting of protein extracts from luteal tissue revealed three major reactive bands. At least two of these bands were closely associated with extracted oxidized human low-density lipoprotein (LDL) and with 4-HNE-conjugated human apoprotein B. These results demonstrate the presence of lipid peroxidation-derived protein epitopes within the porcine CL and show that one or more of these may be an oxidized-LDL moiety. This represents the first direct in situ evidence that protein modification by lipid peroxidation products occurs during the natural luteal life span. The physiological consequences remain to be determined.