Brannian J D, Zhao Y, Burbach J A
Department of Obstetrics and Gynecology, University of South Dakota School of Medicine, Sioux Falls 57105-1570, USA.
Biol Reprod. 1997 Dec;57(6):1461-6. doi: 10.1095/biolreprod57.6.1461.
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.
黄体（CL）内产生的活性氧（ROS）被认为在黄体溶解过程中起着不可或缺的作用。不饱和脂质易受ROS的过氧化作用影响，导致形成有毒醛类，如丙二醛（MDA）和4-羟基壬烯醛（4-HNE），这些醛类会攻击细胞内和细胞外的蛋白质。蛋白质的醛结合对细胞功能可能有多种影响，包括细胞表面受体的交联、酶活性的改变以及脂蛋白的修饰。本研究的目的是：1）通过免疫技术确定在黄体的自然生命周期中是否能原位检测到脂质过氧化衍生的蛋白质表位（即MDA-赖氨酸和4-HNE-赖氨酸）；2）确定这些表位的时空定位。从处于发情周期不同阶段的年轻后备母猪收集新鲜黄体组织：早期（第4 - 7天）、中期（第8 - 12天）和晚期（第14 - 18天）。使用针对MDA-赖氨酸和4-HNE-赖氨酸的单克隆抗体对组织切片进行免疫细胞化学分析。在发情周期的所有阶段，猪黄体中均可见阳性免疫染色。免疫活性主要与类固醇生成细胞相关，最一致地与大黄体细胞相关，并且在退化组织（即晚期）中最为强烈。在用非免疫小鼠血清孵育的切片中或当抗4-HNE-赖氨酸与4-HNE-蛋白质制剂预吸附时，未观察到染色。黄体组织蛋白质提取物的免疫印迹显示出三条主要反应带。这些带中至少有两条与提取的氧化型人低密度脂蛋白（LDL）以及与4-HNE结合的人载脂蛋白B密切相关。这些结果证明了猪黄体中存在脂质过氧化衍生的蛋白质表位，并表明其中一个或多个可能是氧化型LDL部分。这代表了脂质过氧化产物对蛋白质修饰在黄体自然生命周期中发生的首个直接原位证据。其生理后果仍有待确定。
