Roles of antioxidant enzymes in corpus luteum rescue from reactive oxygen species- induced oxidative stress  Kaïs H. Al-Gubory, Catherine Garrel, Patrice Faure, Norihiro Sugino  Reproductive BioMedicine Online  Volume 25, Issue 6, Pages 551-560 (December 2012) DOI: 10.1016/j.rbmo.2012.08.004 Copyright © 2012 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 Histological representation of the luteal tissues and in-situ identification of apoptotic nuclei by fluorescence labelling of nicked DNA in sheep corpus luteum (CL) at day 16 of the oestrous cycle (C16, left panels) and day 16 of pregnancy (P16, right panels). Sections were stained with haematoxylin and eosin (A and A′) or subjected to ex-vivo TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) assay (B and B′). Note that fluorescent DNA fragments were detectable in both types of luteal tissues (C and C′) by using the fibred confocal fluorescence microscopy (FCM1000) with Cellvizio technology. In haematoxylin and eosin micrographs of the rescued day-16 pregnant CL, large luteal cells (arrow) can be distinguished from small luteal cells (arrowhead) by size and nuclear morphology. Note cellular disorganization, nuclear chromatin condensation and densely staining bodies in luteal tissue of the non-rescued of day-16 CL of the oestrous cycle. In TUNEL micrographs, non-fragmented nuclei are stained red (propidium iodide) whereas apoptotic nuclei with fragmented DNA are stained red and yellow. TUNEL and FCM1000 in-situ detection of apoptosis both revealed that luteal tissue of late luteal phase of the oestrous cycle (C16) displayed a higher frequency of apoptotic nuclei compared with healthy luteal tissue of early pregnancy (P16). (D) The CL of the late oestrous cycle secretes relatively low progesterone compared with the CL of early pregnancy. Bar=20μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Reproductive BioMedicine Online 2012 25, 551-560DOI: (10.1016/j.rbmo.2012.08.004) Copyright © 2012 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 A schematic overview of the control of reactive oxygen species (ROS) production by cellular antioxidants. The generation of superoxide anion (O2−) by a single electron donation to molecular oxygen (O2) is the first step in the formation of most ROS. Nitric oxide (NO), generated from l-arginine in a reaction catalysed by NO synthases (NOS), interact with O2− and consequently promote the formation of peroxynitrite (ONOO−). In the presence of free iron ions, O2− and hydrogen peroxide (H2O2) interact in a Haber–Weiss reaction to generate hydroxyl radical (OH). Copper–zinc-containing superoxide dismutase (Cu,Zn-SOD or SOD1) and manganese-containing SOD (Mn-SOD or SOD2) catalyse the dismutation of O2− into H2O2. Glutathione peroxidase (GPX) and catalase (CAT) both convert H2O2 to water (H2O) and O2. GPX catalyses the conversion of H2O2 to H2O through the oxidation of reduced glutathione (GSH). Glutathione reductase (GSR) catalyses the reduction of the oxidized form of glutathione (GSSG) to GSH with reduced nicotinamide adenine dinucleotide phosphate (NADPH) as the reducing agent. Glucose-6-phosphate dehydrogenase and isocitrate dehydrogenases both generate NADPH from the oxidized nicotinamide adenine dinucleotide phosphate (NADP+). Reproductive BioMedicine Online 2012 25, 551-560DOI: (10.1016/j.rbmo.2012.08.004) Copyright © 2012 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 A schematic overview of the potential pathways involved in reactive oxygen species (ROS) production by luteal cells and ROS-mediated inhibition of progesterone production, DNA strand breaks and luteolysis. The intraluteal ROS concentration is controlled by copper–zinc-containing superoxide dismutase (Cu,Zn-SOD or SOD1), manganese-containing SOD (Mn-SOD or SOD2), glutathione peroxidases (GPX) and catalase (CAT). The superoxide radical (O2−) is the starting point of the generation and propagation of other ROS. After the dismutation of O2− to hydrogen peroxide (H2O2) by SOD, the next step is generally the conversion of H2O2 to water (H2O) by GPX and CAT. The iron-catalysed Fenton reaction can lead to the generation of hydroxyl radical (OH) from H2O2. Nitric oxide (NO) derived from l-arginine in a reaction catalysed by NO synthases (NOS) react with O2− to produce peroxynitrite (ONOO−). OH and ONOO− are highly reactive ROS and can react with lipids, proteins and DNA, leading to cell membrane lipid peroxidation, DNA damage and apoptotic cell death. Arachidonic acid (AA), a precursor of eicosanoids including prostaglandins (PG), is released from membrane phospholipids via the activity of cytosolic phospholipase A2 (cPLA2) and is subsequently metabolized by cyclo-oxygenase (COX) to generate prostaglandin H2 (PGH2) and prostaglandin F2α (PGF2α). Locally produced O2−, NO and H2O2 can induce activation of PLA2 and/or COX, which in turn enhances the luteal production of PGF2α. Intraluteal PGF2α produced by both pathways lead to the inhibition of progesterone synthesis and activation of signalling pathway for apoptotic cell death. Locally produced progesterone promotes luteal cell survival. Luteotropic hormones are necessary to sustain luteal progesterone production, which in turn inhibits DNA strand breaks and cell death. Pointers 1 and 3 indicate where locally produced ROS mediate apoptosis (see text for details). Reproductive BioMedicine Online 2012 25, 551-560DOI: (10.1016/j.rbmo.2012.08.004) Copyright © 2012 Reproductive Healthcare Ltd. Terms and Conditions