Roles of antioxidant enzymes in corpus luteum rescue from reactive oxygen species-induced oxidative stress

Reproductive BioMedicine Online (2012) 25, 551– 560

www.sciencedirect.com www.rbmonline.com

REVIEW

Roles of antioxidant enzymes in corpus luteum rescue from reactive oxygen species-induced oxidative stress Kaı¨s H Al-Gubory

a,*

, Catherine Garrel b, Patrice Faure b, Norihiro Sugino

c

a ´veloppement et Reproduction, De ´partement de Institut National de la Recherche Agronomique, UMR 1198 Biologie du De `mes d‘e ´levage, F-78350 Jouy-en-Josas, France; b Centre Hospitalier Universitaire de Grenoble, Physiologie Animale et syste ´ de Biochimie Hormonale et Nutritionnelle, De ´partement de Biologie–Toxicologie–pharmacologie, 38043 Grenoble Unite cedex 9, France; c Yamaguchi University Graduate School of Medicine, Department of Obstetrics and Gynaecology, Minamikogushi 1-1-1, Ube 755-8505, Japan

* Corresponding author. E-mail address: [email protected] (KH Al-Gubory). Kaı¨s H Al-Gubory is a senior scientist at the Department of Animal Physiology and Livestock Systems of the French National Institute for Agricultural Research. His research lies in understanding ovarian, luteal, uterine and placental physiology during pregnancy. He is engaged in the development and use of experimental surgery, animal models and live-cell imaging technology. Currently, his major research interests include periimplantation biology, free radical biology, physiological adaptation to oxidative stress, monitoring oxidative stress biomarkers and apoptosis, antioxidants, maternal peri-conception antioxidant nutrition, prenatal developmental outcomes and fertility.

Abstract Progesterone produced by the corpus luteum (CL) regulates the synthesis of various endometrial proteins required for

embryonic implantation and development. Compromised CL progesterone production is a potential risk factor for prenatal development. Reactive oxygen species (ROS) play diverse roles in mammalian reproductive biology. ROS-induced oxidative damage and subsequent adverse developmental outcomes constitute important issues in reproductive medicine. The CL is considered to be highly exposed to locally produced ROS due to its high blood vasculature and steroidogenic activity. ROS-induced apoptotic cell death is involved in the mechanisms of CL regression that occurs at the end of the non-fertile cycle. Luteal ROS production and propagation depend upon several regulating factors, including luteal antioxidants, steroid hormones and cytokines, and their crosstalk. However, it is unknown which of these factors have the greatest contribution to the maintenance of CL integrity and function during the oestrous/menstrual cycle. There is evidence to suggest that antioxidants play important roles in CL rescue from luteolysis when pregnancy ensues. As luteal phase defect impacts fertility by preventing implantation and early conceptus development in livestock and humans, this review attempts to address the importance of ROS-scavenging antioxidant enzymes in the control of mammalian CL function and integrity. RBMOnline ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: antioxidants, corpus luteum, oxidative stress, progesterone, reactive oxygen species

1472-6483/$ - see front matter ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rbmo.2012.08.004

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Introduction

ROS and ROS-scavenging systems

In mammalian species, the main function of the corpus luteum (CL) is the synthesis of progesterone which is required for the establishment of a uterine environment suitable for the development of peri-implantation conceptus (embryo and associated extra-embryonic membranes) and the successful progression and maintenance of pregnancy (Ryan, 1969). Progesterone acts on the endometrium to regulate the synthesis of growth factors, cytokines, transport and adhesion proteins, protease inhibitors, hormones and enzymes which are primary regulators of conceptus implantation, survival and development (Graham and Clarke, 1997). Thus, compromised CL progesterone production is a potential risk factor for prenatal development and pregnancy outcomes (Arredondo and Noble, 2006; Diskin and Morris, 2008). The periodic CL regression allows initiation of a new reproductive cycle. The demise of the CL at every non-fertile cycle is characterized by the loss of capacity of the luteal cells to produce and secrete progesterone (functional regression) and death of luteal cells (structural regression) (Figure 1). The mammalian CL contains two types of steroidogenic cells, designated large and small luteal cells. Luteal cells are organized between connective tissue and abundant vasculature. The small and large luteal cells are recognizable by light microscopy and have intact and normal appearance in the healthy rescued CL, whereas they are disorganized and exhibit nuclear chromatin condensation and pyknosis in the non-rescued CL (Figure 1). Fluorescent DNA fragments are rarely present in the healthy and functional CL (rescued CL of early pregnancy) whereas they are abundant in the regressed CL (non-rescued CL of the late oestrous cycle) (Figure 1). Although the mechanisms of CL rescue from cell death and maintenance of progesterone production are very complex and vary among mammalian species (Niswender et al., 2000), there is substantial evidence that reactive oxygen species (ROS) are key factors in determining the CL lifespan (Behrman et al., 2001) and that antioxidants play significant roles in CL physiology during the oestrous/menstrual cycle (Al-Gubory et al., 2005, 2006; Sugino, 2006; Sugino et al., 2000a). Luteal ROS production and propagation depends upon several regulating factors, including luteal antioxidants, steroid hormones and cytokines, and their crosstalk. However, it is unknown which of these factors have the greatest contribution to CL function. In addition, the sequence of events leading to the functional and structural luteal regression at the end of the oestrous/menstrual cycle is still not clear. The scarce in-vivo reports studying the CL of rats (Sugino et al., 1993a), women (Sugino et al., 2000a) and sheep (Al-Gubory et al., 2004; Arianmanesh et al., 2011) have shown the importance of antioxidant enzymes in the control of CL function during the peri-implantation period. As a luteal phase defect can impact fertility by preventing implantation and early conceptus development in livestock and humans, this review attempts to address the importance of ROS-scavenging antioxidant enzymes in the control of mammalian CL function and integrity.

The production of adenosine triphosphate is derived from the mitochondrial respiratory chain oxidative phosphorylation, which is the main source of oxygen-free radicals and non-radical ROS. The ROS include superoxide anion (O2), hydroxyl radical (OH), nitric oxide (NO), hydrogen peroxide (H2O2) and peroxynitrite (ONOO). ROS are also produced via enzymic pathways, including the activity of membrane-bound NADH and NADPH oxidases, the activity of xanthine oxidase, the metabolism of arachidonic acid by lipoxygenases and cyclo-oxygenases (COX) and the mitochondrial cytochrome P450 (Bedard and Krause, 2007; Cho et al., 2011; Zangar et al., 2004). Aerobic cells are equipped with antioxidant enzymes that control ROS production and prevent their propagation to toxic ROS (Figure 2). The conversion O2 to H2O2 by superoxide dismutase (SOD) is the first enzymic antioxidative pathway. Two different SOD were identified: copper–zinc-containing SOD (SOD1) is predominantly localized in the cytosol and also found in mitochondria (OkadoMatsumoto and Fridovich, 2001), and manganese-containing SOD (SOD2) occurs in the mitochondrial matrix (Weisiger and Fridovich, 1973). Glutathione peroxidase (GPX) is a group of selenium-containing enzymes that belong to the first antioxidant mechanism preventing the propagation of highly reactive ROS by catalysing the conversion of H2O2 to H2O and O2. NADH and NADPH are key elements in the control of ROS production and maintenance of cellular redox state (Kirsch and De Groot, 2001). The mitochondrial NADP+-dependent isocitrate dehydrogenase generates NADPH via oxidative decarboxylation of isocitrate (Jo et al., 2001).

Role of ROS in corpus luteum steroidogenesis Like any aerobic cells, those of the CL produce ATP through the respiration of O2 with the consequence of luteal ROS production. The rate-limiting step in steroidogenesis in all steroidogenic organs, including the CL (Christenson and Devoto, 2003), is the transfer of cholesterol from the outer to the inner mitochondrial membrane where it is converted into pregnenolone by the enzyme cytochrome P450 side chain cleavage (P450scc). Luteal ROS are generated via enzymic pathways of the mitochondrial cytochrome P450 (Zangar et al., 2004). In the CL, ROS are produced by macrophages (Sugino et al., 1996) and luteal cells (Kato et al., 1997) where they can affect progesterone production. Indeed, there is substantial evidence to indicate that ROS regulate steroid hormone biosynthesis in the CL. The induction of ovarian SOD by LH, which in turn could lead to the production of H2O2, suggests that this action is involved in the mechanism by which LH stimulates progesterone secretion in the rat CL (Laloraya et al., 1988). Carlson et al. (1993) indicated that ROS can function beneficially to control the production of progesterone by luteal cells over the course of the reproductive cycle and inhibit progesterone synthesis at the end of the cycle. The O2 radical is reported to be involved in the mechanism by which LH stimulates progesterone secretion in the rat CL (Sawada and

Roles of antioxidants in corpus luteum rescue

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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 A0 ) or subjected to ex-vivo TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) assay (B and B0 ). Note that fluorescent DNA fragments were detectable in both types of luteal tissues (C and C0 ) 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 = 20 lm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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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+).

Carlson, 1996). It is important to highlight that ovarian NADPH-dependent O2 production appears to be LH inducible (Jain et al., 2000). Ho et al. (1998) first reported that SOD1 deficiency led to reduced fertility due to post-implantation embryonic loss in female mice. A recent study demonstrates that increased intracellular ovarian O2 in SOD1-deficient female mice is associated with increased apoptotic cell death in the CL and impaired luteal formation and progesterone production (Noda et al., 2012). These data indicate that the control of  O2 by SOD1 in the ovary plays a pivotal role in the maintenance of CL function and progesterone secretion. Tightly controlled ROS generation by antioxidants is one of the central elements in the mechanisms involved in cell function, growth, differentiation and death (Valko et al., 2007). It must therefore be presumed that ROS may function as intracellular regulators of steroidogenesis and that the equilibrium between luteal antioxidant capacity and ROS production is crucial for CL integrity and function.

Impact of hypoxia on luteal vascularization and steroidogenesis The CL has an intense vasculature where the contact and exchange between steroidogenic luteal cells and capillaries are crucial for steroidogenesis and progesterone release into the general circulation (Reynolds et al., 2000). The CL is therefore considered to be highly exposed to locally

produced ROS due to its high blood vasculature and steroidogenic activity. The ROS generated in and around the vascular endothelium play an important role in normal cellular signalling mechanisms controlling the CL lifespan and function (Agarwal et al., 2005). Oxygen deficiency or hypoxia is important for the control of cellular O2 homeostasis (Iyer et al., 1998). The family of hypoxia-inducible factors (HIF) transcription proteins has been identified to be regulated by the intracellular redox state and their expression and activity are tightly controlled by cellular ROS concentration (Chandel et al., 1998). Hypoxia-inducible factor 1a (HIF-1a) regulates many genes which are crucial for cell survival under hypoxia, including erythropoietin, vascular endothelial growth factor, glucose transporter 1 and glycolytic enzymes (Semenza, 1999). Hypoxia decreases progesterone synthesis by attenuating P450scc activity in bovine luteal cells (Nishimura et al., 2006). Since hypoxia suppresses luteal progesterone production, Nishimura et al. (2008) suggest that prolonged oxygen deficiency caused by a decreasing blood supply in bovine CL contributes to functional and structural luteolysis. The expression of HIF-1a was reported to occur in porcine (Boonyaprakob et al., 2005), bovine (Nishimura and Okuda, 2010), non-human primate (Duncan et al., 2008) and rodent (Tam et al., 2010) luteal cells of the developing CL. The expression of HIF-1a is hormonally regulated in human (van den Driesche et al., 2008) and rodent (Tam et al., 2010) luteal cells. Taken together, these results suggest that HIF-1a may play a role in the regulation of luteal function during

Roles of antioxidants in corpus luteum rescue CL formation and development. The newly formed blood vessels (angiogenesis), and the consequent increase in the blood flow in the CL, supply nutrients, luteotropic hormones and other systemic factors to the luteal tissue to promote CL growth, development and steroidogenesis (Robinson et al., 2009). In the ovulated bovine follicle, the initial hypoxic environment triggers the development of the vascular system which is necessary for luteal formation and development (Nishimura and Okuda, 2010).

Role of ROS in corpus luteum apoptosis Currently, the sequence of initial events leading to the functional and structural luteal regression is still not clear. There is substantial evidence to indicate that ROS play an important role in the regression of rat CL (Behrman and Aten, 1991; Gatzuli et al., 1991; Musicki et al., 1994; Riley and Behrman, 1991; Sawada and Carlson, 1989). Uncontrolled ROS generation leads to irreversible alteration of biomacromolecules, including lipids, proteins and nucleic acids, and ultimately causes mitochondrial dysfunction, mitochondrial ROS-induced ROS release and cell death by apoptosis (Zorov et al., 2006). Luteal cell death by apoptosis, occurring at the end of the CL lifespan (Figure 1), is a complex process which involves in-situ changes in ROS and steroid hormone production, a number of immune cells and cytokine expression (Kato et al., 1997; Pate and Landis Keyes, 2001). Cytokines of different types secreted from various immune cells interact with steroidogenesis, influencing CL development and function (Bornstein et al., 2004). Pro-inflammatory cytokines (Sugino et al., 1998a) and luteotropic hormones (Sugino et al., 1998b) are likely candidates for the control of luteal ROS generation and function via the regulation of SOD1 and SOD2 expression. Human chorionic gonadotrophin prolongs the lifespan of the human CL by increasing the expression of BCL2 and decreasing that of BAX during early pregnancy (Sugino et al., 2000b). Although mammalian species have different apoptotic mechanisms for controlling luteal function (Sugino and Okuda, 2007), evidence indicates that ROS-induced apoptosis is of fundamental importance for determining the CL lifespan (Behrman et al., 2001; Garrel et al., 2007; Kato et al., 1997; Tanaka et al., 2000). There is in-vivo evidence that progesterone promotes luteal cell survival in various species. This includes: (i) induction of apoptosis concomitant with the rapid decline in luteal progesterone production during spontaneous luteolysis in cattle (Juengel et al., 1993), humans (Shikone et al., 1996; Sugino et al., 2000b), hamsters (McCormack et al., 1998), rats (Bruce et al., 2001) and sheep (Al-Gubory et al., 2005); (ii) inhibition of luteal progesterone secretion (Garrel et al., 2007) precedes DNA fragmentation and cell death in the ovine CL; (iii) induction of functional luteolysis and luteal apoptosis in late pregnant rats with progesterone antagonists (Telleria et al., 2001); (iv) progesterone antagonists promote apoptotic cell death in bovine luteal cells (Okuda et al., 2004; Rueda et al., 2000); and (v) the low number of luteal cells which undergo apoptosis in CL of rats (Goyeneche et al., 2003; Takiguchi et al., 2004) and sheep (Al-Gubory et al., 2004) throughout pregnancy correlates with sustained high concentrations of circulating progester-

555 one. Inhibition of progesterone synthesis by uncontrolled luteal ROS production may be one of the key pathways involved in CL demise at the end of the oestrous/menstrual cycle. Oestradiol is produced by CL of mammalian species, including humans (Shutt et al., 1975), rats (Elbaum and Keyes, 1976), pigs (Gregoraszczuk and Oblonczyk, 1996) and cattle (Okuda et al., 2001). These results indicate that luteal oestradiol may have some role as a paracrine and/or autocrine regulator of CL function during the oestrous/ menstrual cycle. Luteal oestradiol is thought to be luteotropic in rodents (Stocco et al., 2007). Indeed, this hormone enhances cholesterol supply by stimulating the uptake of cholesterol from circulation and intracellular cholesterol mobilization. Nevertheless, there is evidence to indicate that oestradiol can induce luteal cell death by stimulating the synthesis pathway of NO, ONOO and eventually other luteolytic pathways. NO acts as signalling regulator of many physiological processes including ovarian steroidogenesis. NO interacts with the iron-containing enzymes, such as cytochrome P-450 enzymes, and inhibits ovarian oestradiol synthesis (Stadler et al., 1994; Wink et al., 1993). NO donors inhibit aromatase activity and oestradiol secretion, whereas inhibition of endogenous NOS enhances oestradiol secretion from human (Van Voorhis et al., 1994), rat (Olson et al., 1996) and porcine (Masuda et al., 1997) granulosa cells. Phospholipases A2 (PLA2) hydrolyse membrane phospholipids to release arachidonic acid that can be utilized as substrate for prostaglandins (PG) synthesis, including prostaglandin F2a (PGF2a). It has been proposed that luteal PGF2a acts via a paracrine and/or autocrine mechanism to induce luteolysis in many species, including non-human primates, women, sows, sheep, cattle and rodents (Auletta and Flint, 1988). The regression of CL is considered to be an ischaemic process and it involves O2 and/or H2O2mediated PLA2 activation (Wu et al., 1992). Prostaglandin G/H synthases or COX catalyse the rate-limiting step in PG biosynthesis. COX1 and COX2 convert arachidonic acid into PGH2, the common metabolite for PG synthesis, including PGF2a (Smith et al., 1996). Locally produced cytokines may play important roles in functional and structural luteal regression by activation of luteal ROS and/or PGF2a generation pathways. The recruitment of immune cells from the systemic circulation and their infiltration into the CL and the expression of proinflammatory cytokines have been shown to increase markedly around the time of luteolysis (Pate and Landis Keyes, 2001). Substantial evidence indicates that PGF2a is implicated in luteal regression via the generation of O2 (Aten et al., 1998; Sawada and Carlson, 1991). PGF2a-induced H2O2 generation has been suggested to be a key mediator of leukocyte accumulation during the CL regressing (Minegishi et al., 2002). PGF2a has been shown to decrease the expression of ROS scavenger proteins and hence facilitate the accumulation of ROS, which in turn initiate a cascade of events that cause luteal cell death in the mouse CL during luteal regression (Foyouzi et al., 2005). PGF2a stimulates COX2 expression via ROS generation in rat CL in vivo (Taniguchi et al., 2010). PGF2a is an important regulator of functional regression and cell death by apoptosis (Al-Gubory, 2005; Arosh et al., 2004; Garrel et al., 2007; Kurusu et al., 2007).

556 The free radical NO has an anti-steroidogenic action in human (Vega et al., 1998), rabbit (Gobbetti et al., 1999) and bovine (Skarzynski and Okuda, 2000) luteal cells in vitro. NO is a mediator of lipid peroxidation induced in rat CL by PGF2a during luteolysis concomitantly with inhibition of progesterone production (Motta et al., 2001). Infusion of an inhibitor of NOS prolongs the duration of the oestrous cycle (Jaroszewski and Hansel, 2000) through the prevention of PGF2a-induced luteolysis in cattle (Skarzynski et al., 2003). NO through its interaction with components of the electron-transport chain can enhance mitochondrial ROS generation and thereby trigger mechanisms of cell survival or death (Moncada and Erusalimsky, 2002). The results of the above studies lead to the proposal of a model of regulation of luteal progesterone synthesis and apoptosis by ROS and antioxidant networks (Figure 3). The ROS, O2, H2O2, OH, ONOO and, in association with

KH Al-Gubory et al. the NOS-NO system, proinflammatory cytokines and the PG synthetic pathway, play critical roles in the mechanisms of functional and structural luteal regression. Locally produced ROS mediate apoptosis by at least three potential pathways (indicated in the figure with pointers). (Pointer 1) The O2 radical is the precursor of most ROS, including H2O2 and OH, and could be a mediator in oxidative chain reactions and apoptosis. The free radical OH is formed by the Fenton reaction in which a reduced transition metal, such as Fe2+, reduces H2O2 to form OH. (Pointer 2) High concentrations of O2 and NO may induce activation of COX resulting in the enhancement of PGH2 and PGF2a production. This pathway leads to the inhibition of progesterone synthesis and activation of the signalling pathway for cell death by apoptosis. (Pointer 3) Elevated concentrations of O2 and NO radicals, generated in response to pro-inflammatory cytokines and/or oestrogen, could lead

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 F2a (PGF2a). Locally produced O2, NO and H2O2 can induce activation of PLA2 and/or COX, which in turn enhances the luteal production of PGF2a. Intraluteal PGF2a 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).

Roles of antioxidants in corpus luteum rescue to increased ONOO production and apoptosis. In this cellular model, an anti-apoptotic pathway (Pointer 4), counteracting the three apoptotic pathways mentioned above, is the stimulation of progesterone production by luteotropic hormones. Overall, the net luteal ROS and progesterone production depends upon several factors and it is unknown which of these factors have the greatest contribution to the inhibition of progesterone synthesis and activation of the signalling pathway for CL apoptosis and regression.

Roles of antioxidant enzymes in corpus luteum rescue and establishment of pregnancy The inhibition of apoptosis in cultured rabbit CL by SOD, CAT or a putative stimulator of endogenous GPX (Dharmarajan et al., 1999), the induction of apoptosis in bovine luteal cells by inhibition of GPX expression (Nakamura et al., 2001) and by treatment of bovine luteal cells with an inhibitor of GPX (Nakamura and Sakamoto, 2001) suggest that antioxidant enzymes protect luteal cells against ROS-induced oxidative damage and apoptosis. In addition, the inhibition of SOD1 activity by antisense oligonucleotide induces functional luteal regression of rat CL (Sugino et al., 1999). There is evidence to suggest that ROS-scavenging antioxidant enzymes play important roles in CL rescue from luteolysis when pregnancy ensues in various mammalian species. During early pregnancy, the high concentrations of SOD in mouse ovaries (Laloraya et al., 1989), the increased activity of SOD1 and SOD2 in the rat CL (Sugino et al., 1993a) the high expression of SOD2 and CAT in the bovine CL (Rueda et al., 1995), the high expression and activity of SOD1 in the human CL (Sugino et al., 2000a), the increased activities of SOD1, GPX and GST in sheep CL (Al-Gubory et al., 2004) and the increased expression of GSTA1 protein in sheep CL (Arianmanesh et al., 2011) are sound arguments supporting this proposal. The ability of SOD1 and CAT to stimulate the secretion of progesterone in vivo by rat CL of late pregnancy (Sugino et al., 1993b) and the presence of SOD1 protein in sheep CL of mid-pregnancy (Al-Gubory et al., 2003) suggest that ROS scavenging antioxidants enzymes play a role in regulating luteal function during pregnancy. Maintained concentrations of SOD1, SOD2, GPX, GSR and GST in sheep CL throughout pregnancy may be linked to luteal ROS and may be involved in the maintenance of luteal steroidogenic activity and cellular integrity (Al-Gubory et al., 2004). Uncontrolled ROS production plays a role in embryonic implantation-related disorders and prenatal developmental defects in human and domestic animals (Agarwal et al., 2008; Al-Gubory et al., 2010). Further studies are therefore still required to determine the role of antioxidant enzymes in CL function and integrity during the peri-implantation period.

Conclusions The mechanisms involved in the formation, development and regression of the CL are complex and varied in different mammalian species. Nevertheless, there is a start to understanding the roles of ROS-scavenging antioxidant enzymes in the regulation of CL progesterone synthesis and CL rescue in harmony with many autocrine, paracrine and endocrine

557 factors during the luteal phase of the menstrual/oestrous cycle. The high metabolic rate of the CL results in great consumption of oxygen and energy substrates, with a consequence of high production of ROS as well as greater exposure of the luteal cells to high ROS concentrations. It is important to stress that the control of physiological concentrations of luteal ROS by antioxidant enzymes is a key element for CL progesterone production, whereas the uncontrolled ROS generation due to imbalance between ROS and the antioxidant systems is detrimental for the demise of the CL at the end of non-fertile reproductive cycle. Fundamental knowledge of the contribution of antioxidant enzymic pathways in the synthesis of progesterone and in the maintenance of luteal cell integrity in relation to the establishment of pregnancy is still relatively limited. Recent studies have begun to unravel the complexity of regulation of the CL formation and steroidogenic activity during early pregnancy using the tools of molecular histology and proteomics (Arianmanesh et al., 2011; Gonza ´lezFerna ´ndez et al., 2008). Proteomics will provide physicians and biologists with an improved understanding of the underlying biological processes involved in the maintenance of structural integrity and functional activity of the CL during the peri-implantation period. Elucidation of antioxidant mechanisms involved in CL rescue from luteolysis when pregnancy ensues may help in the design of strategies for promoting successful conceptus implantation and for prevention or management of female subfertility and pregnancy disorders associated with CL defect and progesterone insufficiency in livestock and humans.

Acknowledgements The authors thank the staff of the animal sheds of Broue ¨ssy (INRA) for their outstanding technical help and animal management that permitted the conduct of this study group’s research on the corpus luteum over the last 15 years. They thank the Region d’Iˆle-de-France and INRA for grants awarded that equipped the INRA Research Centre of Jouyen-Josas with new technology for cellular imaging in live animals, the fibred confocal fluorescence microscopy (FCM1000) with Cellvizio technology, to advance this research of biological processes in vivo. The authors would like to thank the anonymous reviewers for their close examination of this review article and their useful comments.

References Agarwal, A., Gupta, S., Sharma, R.K., 2005. Role of oxidative stress in female reproduction. Reprod. Biol. Endocrinol. 3, 28. Agarwal, A., Gupta, S., Sekhon, L., Shah, R., 2008. Redox considerations in female reproductive function and assisted reproduction: from molecular mechanisms to health implications. Antiox. Redox Signal. 10, 1375–1403. Al-Gubory, K.H., 2005. Fibered confocal fluorescence microscopy for imaging apoptotic DNA fragmentation at the single-cell level in vivo. Exp. Cell Res. 310, 474–481. Al-Gubory, K.H., Huet, J.C., Pernollet, J.C., Martal, J., Locatelli, A., 2003. Corpus luteum derived copper, zinc dismutase serves as a luteinizing hormone-release inhibiting factor in sheep. Mol. Cell. Endocrinol. 199, 1–9.

558 Al-Gubory, K.H., Bolifraud, P., Germain, G., Nicole, A., Ceballos-Picot, I., 2004. Antioxidant enzymatic defence systems in sheep corpus luteum throughout pregnancy. Reproduction 128, 767–774. Al-Gubory, K.H., Ceballos-Picot, I., Nicole, A., Bolifraud, P., Germain, G., Michaud, M., Mayeur, C., Blachier, F., 2005. Changes in activities of superoxide dismutase, nitric oxide synthase, glutathione-dependent enzymes and the incidence of apoptosis in sheep corpus luteum during the estrous cycle. Biochem. Biophys. Acta 1725, 348–357. Al-Gubory, K.H., Camous, S., Germain, G., Bolifraud, P., Nicole, A., Ceballos-Picot, I., 2006. Reconsideration of the proposed luteotropic and luteoprotective actions of ovine placental lactogen in sheep: in vivo and in vitro studies. J. Endocrinol. 188, 559–568. Al-Gubory, K.H., Fowler, P.A., Garrel, C., 2010. The roles of cellular reactive oxygen species, oxidative stress and antioxidants in pregnancy outcomes. Int. J. Biochem. Cell Biol. 42, 1634–1650. Arianmanesh, M., McIntosh, R., Lea, R.G., Fowler, P.A., AlGubory, K.H., 2011. Ovine corpus luteum proteins, with functions including oxidative stress and lipid metabolism, show complex alterations during implantation. J. Endocrinol. 210, 47–58. Arosh, J.A., Banu, S.K., Chapdelaine, P., Madore, E., Sirois, J., Fortier, M.A., 2004. Prostaglandin biosynthesis, transport, and signaling in corpus luteum: a basis for autoregulation of luteal function. Endocrinology 145, 2551–2560. Arredondo, F., Noble, L.S., 2006. Endocrinology of recurrent pregnancy loss. Semin. Reprod. Med. 24, 33–39. Aten, R.F., Kolodecik, T.R., Rossi, M.J., Debusscher, C., Behrman, H.R., 1998. Prostaglandin f2alpha treatment in vivo, but not in vitro, stimulates protein kinase C-activated superoxide production by nonsteroidogenic cells of the rat corpus luteum. Biol. Reprod. 59, 1069–1076. Auletta, F.J., Flint, A.P.F., 1988. Mechanisms controlling corpus luteum function in sheep, cows, non-human primates, and women, especially in relation to the time of luteolysis. Endocr. Rev. 9, 88–105. Bedard, K., Krause, K.H., 2007. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol. Rev. 87, 245–313. Behrman, H.R., Aten, R.F., 1991. Evidence that hydrogen peroxide blocks hormone-sensitive cholesterol transport into mitochondria of rat luteal cells. Endocrinology 128, 2958–2966. Behrman, H.R., Kodaman, P.H., Preston, S.L., Gao, S., 2001. Oxidative stress and the ovary. J. Soc. Gynecol. Investig. 8, S40–S42. Boonyaprakob, U., Gadsby, J.E., Hedgpeth, V., Routh, P.A., Almond, G.W., 2005. Expression and localization of hypoxia inducible factor-1a mRNA in the porcine ovary. Can. J. Vet. Res. 69, 215–222. Bornstein, S.R., Rutkowski, H., Vrezas, I., 2004. Cytokines and steroidogenesis. Mol. Cell. Endocrinol. 215, 135–141. Bruce, N.W., Hisheh, S., Dharmarajan, A.M., 2001. Patterns of apoptosis in the corpora lutea of the rat during the oestrous cycle, pregnancy and in vitro culture. Reprod. Fert. Dev. 13, 105–109. Carlson, J.C., Wu, X.M., Sawada, M., 1993. Oxygen radicals and the control of ovarian corpus luteum function. Free Radic. Biol. Med. 14, 79–84. Chandel, N.S., Maltepe, E., Goldwasser, E., Mathieu, C.E., Simon, M.C., Schumacker, P.T., 1998. Mitochondrial reactive oxygen species trigger hypoxia-induced transcription. Proc. Natl. Acad. Sci. USA 95, 11715–11720. Christenson, L.K., Devoto, L., 2003. Cholesterol transport and steroidogenesis by the corpus luteum. Reprod. Biol. Endocrinol. 1, 1–9.

KH Al-Gubory et al. Cho, K.J., Seo, J.M., Kim, J.H., 2011. Bioactive lipoxygenase metabolites stimulation of NADPH oxidases and reactive oxygen species. Mol. Cells 2, 1–5. Dharmarajan, A.M., Hisheh, S., Singh, B., Parkinson, S., Tilly, K.I., Tilly, J.L., 1999. Antioxidants mimic the ability of chorionic gonadotropin to suppress apoptosis in the rabbit corpus luteum in vitro: a novel role for superoxide dismutase in regulating bax expression. Endocrinology 140, 2555–2561. Diskin, M.G., Morris, D.G., 2008. Embryonic and early foetal losses in cattle and other ruminants. Reprod. Domest. Anim. 43, 260–267. Duncan, W.C., van den Driesche, S., Fraser, H.M., 2008. Inhibition of vascular endothelial growth factor in the primate ovary up-regulates hypoxia-inducible factor-1alpha in the follicle and corpus luteum. Endocrinology 149, 3313–3320. Elbaum, D.J., Keyes, P.L., 1976. Synthesis of 17b-estradiol by isolated ovarian tissues of the pregnant rat: aromatization in the corpus luteum. Endocrinology 99, 573–579. Foyouzi, N., Cai, Z., Sugimoto, Y., Stocco, C., 2005. Changes in the expression of steroidogenic and antioxidant genes in the mouse corpus luteum during luteolysis. Biol. Reprod. 72, 1134–1141. Garrel, C., Ceballos-Picot, I., Germain, G., Al-Gubory, K.H., 2007. Oxidative stress-inducible antioxidant adaptive response during prostaglandin F2alpha-induced luteal cell death in vivo. Free Radic. Res. 41, 251–259. Gatzuli, E., Aten, R.F., Behrman, H.R., 1991. Inhibition of gondatropic action and progesterone synthesis by xanthine oxidase in rat luteal cells. Endocrinology 128, 2253–2258. Gobbetti, A., Boiti, C., Canali, C., Zerani, M., 1999. Nitric oxide synthase actuely regulates progesterone production by in vitro cultured rabbit corpora lutea. J. Endocrinol. 160, 275–283. Gonza ´lez-Ferna ´ndez, R., Martı´nez-Galisteo, E., Gayta ´n, F., Ba ´rcena, J.A., Sa ´nchez-Criado, J.E., 2008. Changes in the proteome of functional and regressing corpus luteum during pregnancy and lactation in the rat. Biol. Reprod. 79, 100–114. Goyeneche, A.A., Deis, R.P., Gibori, G., Telleria, C.M., 2003. Progesterone promotes survival of the rat corpus luteum in the absence of cognate receptors. Biol. Reprod. 68, 151–158. Graham, J.D., Clarke, C.L., 1997. Physiological action of progesterone in target tissues. Endocr. Rev. 18, 502–519. Gregoraszczuk, E.L., Oblonczyk, K., 1996. Effect of aspecific aromatase inhibitor on oestradiol secretion by porcine corpora lutea at various stages of the luteal phase. Reprod. Nutr. Dev. 36, 65–72. Ho, Y.S., Gargano, M., Cao, J., Bronson, R.T., Heimler, I., Hutz, R.J., 1998. Reduced fertility in female mice lacking copper–zinc superoxide dismutase. J. Biol. Chem. 273, 7765–7769. Iyer, N.V., Kotch, L.E., Agani, F., Leung, S.W., Laughner, E., Wenger, R.H., Gassmann, M., Gearhart, J.D., Lawler, A.M., Yu, A.Y., Semenza, G.L., 1998. Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1 alpha. Genes Dev. 12, 149–162. Jain, S., Saxena, D., Kumar, G.P., Laloraya, M., 2000. NADPH dependent superoxide generation in the ovary and uterus of mice during estrous cycle and early pregnancy. Life Sci. 66, 1139–1146. Jaroszewski, J.J., Hansel, W., 2000. Intraluteal administration of a nitric oxide synthase blocker stimulates progesterone and oxytocin secretion and prolongs the life span of the bovine corpus luteum. Proc. Soc. Exp. Biol. Med. 224, 50–55. Jo, S.H., Son, M.K., Koh, H.J., Lee, S.M., Song, I.H., Kim, Y.O., Lee, Y.S., Jeong, K.S., Kim, W.B., Park, J.W., Song, B.J., Huh, T.L., 2001. Control of mitochondrial redox balance and cellular defense against oxidative damage by mitochondrial NADP+-dependent isocitrate dehydrogenase. J. Biol. Chem. 276, 16168–16176. Juengel, J.L., Garverick, H.A., Johnson, A.L., Youngquist, R.S., Smith, M.F., 1993. Apoptosis during luteal regression in cattle. Endocrinology 132, 249–254.

Roles of antioxidants in corpus luteum rescue Kato, H., Sugino, N., Takiguchi, S., Kashida, S., Nakamura, Y., 1997. Roles of reactive oxygen species in the regulation of luteal function. Rev. Reprod. 2, 81–83. Kirsch, M., De Groot, H., 2001. NAD(P)H, a directly operating antioxidant? FASEB J. 15, 1569–1574. Kurusu, S., Sakaguchi, S., Kawaminami, M., 2007. Regulation of luteal prostaglandin F(2 alpha) production and its relevance to cell death: an in vitro study using rat dispersed luteal cells. Prostaglandins Other Lipid Mediat. 83, 250–256. Laloraya, M., Pradeep, K.G., Laloraya, M.M., 1988. Changes in the levels of superoxide anion radical and superoxide dismutase during the estrous cycle of Rattus norvegicus and induction of superoxide dismutase in rat ovary by lutropin. Biochem. Biophys. Res. Commun. 157, 146–153. Laloraya, M., Kumar, G.P., Laloraya, M.M., 1989. A possible role of superoxide anion radical in the process of blastocyst implantation in Mus musculus. Biochem. Biophys. Res. Commun. 161, 762–770. Masuda, M., Kubota, T., Karnada, S., Aso, T., 1997. Nitric oxide inhibits steroidogenesis in cultured porcine granulosa cells. Mol. Hum. Reprod. 3, 285–292. McCormack, J.T., Friederichs, M.G., Limback, S.D., Greenwald, G.S., 1998. Apoptosis during spontaneous luteolysis in the cyclic golden hamster: biochemical and morphological evidence. Biol. Reprod. 58, 255–260. Minegishi, K., Tanaka, M., Nishimura, O., Tanigaki, S., Miyakoshi, K., Ishimoto, H., Yoshimura, Y., 2002. Reactive oxygen species mediate leukocyte–endothelium interactions in prostaglandin F2alpha-induced luteolysis in rats. Am. J. Physiol. Endocrinol. Metab. 283, E1308–E1315. Moncada, S., Erusalimsky, J.D., 2002. Does nitric oxide modulate mitochondrial energy generation and apoptosis? Nat. Rev. Mol. Cell. Biol. 3, 214–220. Motta, A.B., Estevez, A., Franchi, A., Perez-Martinez, S., Farina, M., Ribeiro, M.L., Lasserre, A., Gimeno, M.F., 2001. Regulation of lipid peroxidation by nitric oxide and PGF2a during luteal regression in rats. Reproduction 121, 631–637. Musicki, B., Aten, R.E., Behrman, H.R., 1994. Inhibition of protein synthesis and hormone-sensitive steroidogenesis in response to hydrogen peroxide in rat luteal cells. Endocrinology 134, 588–595. Nakamura, T., Ishigami, T., Makino, N., Sakamoto, K., 2001. The downregulation of glutathione peroxidase causes bovine luteal cell apoptosis during structural luteolysis. J. Biochem. 129, 937–942. Nakamura, T., Sakamoto, K., 2001. Reactive oxygen species up-regulates cyclooxygenase-2, p53 and Bax mRNA expression in bovine luteal cells. Biochem. Biophys. Res. Commun. 284, 203–210. Nishimura, R., Okuda, K., 2010. Hypoxia is important for establishing vascularization during corpus luteum formation in cattle. J. Reprod. Dev. 56, 110–116. Nishimura, R., Sakumoto, R., Tatsukawa, Y., Acosta, T.J., Okuda, K., 2006. Oxygen concentration is an important factor for modulating progesterone synthesis in bovine corpus luteum. Endocrinology 147, 4273–4280. Nishimura, R., Komiyama, J., Tasaki, Y., Acosta, T.J., Okuda, K., 2008. Hypoxia promotes luteal cell death in bovine corpus luteum. Biol. Reprod. 78, 529–536. Niswender, G.D., Juengel, J.L., Silva, P.J., Rollyson, M.K., McIntush, E.W., 2000. Mechanisms controlling the function and life span of the corpus luteum. Physiol. Rev. 80, 1–29. Noda, Y., Ota, K., Shirasawa, T., Shimizu, T., 2012. Copper/zinc superoxide dismutase insufficiency impairs progesterone secretion and fertility in female mice. Biol. Reprod. 86, 1–8. Okado-Matsumoto, A., Fridovich, I., 2001. Subcellular distribution of superoxide dismutases (SOD) in rat liver: Cu, Zn-SOD in mitochondria. J. Biol. Chem. 276, 38388–38393.

559 Okuda, K., Uenoyama, Y., Berisha, B., Lange, I.G., Taniguchi, H., Kobayashi, S., Kobayashi, S., Miyamoto, A., Schams, D., 2001. Estradiol-17beta is produced in bovine corpus luteum. Biol. Reprod. 65, 1634–1639. Okuda, K., Korzekwa, A., Shibaya, M., Murakami, S., Nishimura, R., Tsubouchi, M., Woclawek-Potocka, I., Skarzynski, D.J., 2004. Progesterone is a suppressor of apoptosis in bovine luteal cells. Biol. Reprod. 71, 2065–2071. Olson, L.M., Jones-Burton, C.M., Jablonka-Shariff, A., 1996. Nitric oxide decreases estradiol synthesis of rat luteinized ovarian cells: possible role for nitric oxide in functional luteal regression. Endocrinology 137, 3531–3539. Pate, J.L., Landis Keyes, P., 2001. Immune cells in the corpus luteum: friends or foes? Reproduction 122, 665–676. Reynolds, L.P., Grazul-Bilska, A.T., Redmer, D.A., 2000. Angiogenesis in the corpus luteum. Endocrine 12, 1–9. Riley, J.C.M., Behrman, H.R., 1991. In vivo generation of hydrogen peroxide in the rat corpus luteum during luteolysis. Endocrinology 128, 1749–1753. Robinson, R.S., Woad, K.J., Hammond, A.J., Laird, M., Hunter, M.G., Mann, G.E., 2009. Angiogenesis and vascular function in the ovary. Reproduction 138, 869–881. Rueda, B.R., Tilly, K.I., Hansen, T.R., Hoyer, P.B., Tilly, J.L., 1995. Expression of superoxide dismutase, catalase and glutathione peroxidase in the bovine corpus luteum: evidence supporting a role for oxidative stress in luteolysis. Endocrine 3, 227–232. Rueda, B.R., Hendry, I.R., Hendry, W.J., Stormshak, F., Slayden, O.D., Davis, J.S., 2000. Decreased progesterone levels and progesterone receptor antagonist promote apoptotic cell death in bovine luteal cells. Biol Reprod. 62, 269–276. Ryan, K.J., 1969. The foeto-placental unit. In: Pe ´cicle, A., Finzi, C. (Eds.), Theoretical Basis for Endocrine Control of Gestation——A Comparative Approach. Excerpta Medica Foundation, Amsterdam, pp. 120–131. Sawada, M., Carlson, J.C., 1989. Superoxide radical production in plasma membrane samples from regressing rat corpora lutea. Can. J. Physiol. Pharmacol. 67, 465–471. Sawada, M., Carlson, J.C., 1991. Rapid plasma membrane changes in superoxide radical formation, fluidity, and phospholipase A2 activity in the corpus luteum of the rat during induction of luteolysis. Endocrinology 128, 2992–2998. Sawada, M., Carlson, J.C., 1996. Intracellular regulation of progesterone secretion by the superoxide radical in the rat corpus luteum. Endocrinology 137, 1580–1584. Semenza, G.L., 1999. Regulation of mammalian O2 homeostasis by hypoxia- inducible factor 1. Annu. Rev. Cell Dev. Biol. 15, 551–578. Shikone, T., Yamoto, M., Kokawa, K., Yamashita, K., Nishimori, K., Nakano, R., 1996. Apoptosis of human corpora lutea during cyclic luteal regression and early pregnancy. J. Clin. Endocrinol. Metab. 81, 2376–2380. Shutt, D.A., Shearman, R.P., Lyneham, R.C., Clarke, A.H., McMahon, G.R., Goh, P., 1975. Radioimmunoassay of progesterone, 17-hydroxyprogesterone, estradiol-17b and prostaglandin F in human corpus luteum. Steroids 26, 299–310. Skarzynski, D.J., Jaroszewski, J.J., Bah, M.M., Deptula, K.M., Barszczewska, B., Gawronska, B., Hansel, W., 2003. Administration of a nitric oxide synthase inhibitor counteracts prostaglandin F2-induced luteolysis in cattle. Biol. Reprod. 68, 1674–1681. Skarzynski, D.J., Okuda, K., 2000. Different actions of noradrenaline and nitric oxide on the output of prostaglandins and progesterone in cultured bovine luteal cells. Prostaglandins Other Lipid Mediat. 60, 35–47. Smith, W.L., Garavito, R.M., DeWitt, D.L., 1996. Prostaglandin endoperoxide H synthases (cyclooxygenases)-1 and -2. J. Biol. Chem. 271, 33157–33160.

560 Stadler, J., Trockfeld, J., Schmalix, W.A., Brill, T., Siewert, J.R., Greim, H., Doehmer, J., 1994. Inhibition of cytochromes P4501A by nitric oxide. Proc. Natl. Acad. Sci. USA 91, 3559–3563. Stocco, C., Telleria, C., Gibori, G., 2007. The molecular control of corpus luteum formation, function, and regression. Endocr. Rev. 28, 117–149. Sugino, N., 2006. Roles of reactive oxygen species in the corpus luteum. Anim. Sci. J. 77, 556–565. Sugino, N., Okuda, K., 2007. Species-related differences in the mechanism of apoptosis during structural luteolysis. J. Reprod. Dev. 53, 977–986. Sugino, N., Nakamura, Y., Takeda, O., Ishimatsu, M., Kato, H., 1993a. Changes in activities of superoxide dismutase and lipid peroxide in corpus luteum during pregnancy in rats. J. Reprod. Fert. 97, 347–351. Sugino, N., Nakamura, Y., Okuno, N., Ishimatu, M., Teyama, T., Kato, H., 1993b. Effects of ovarian ischemia–reperfusion on luteal function in pregnant rats. Biol. Reprod. 49, 354–358. Sugino, N., Shimamura, K., Tamura, H., Ono, M., Nakamura, Y., Ogino, K., Kato, H., 1996. Progesterone inhibits superoxide radical production by mononuclear phagocytes in pseudopregnant rats. Endocrinology 137, 749–754. Sugino, N., Telleria, C.M., Gibori, G., 1998a. Differential regulation of copper–zinc superoxide dismutase and manganese superoxide dismutase in the rat corpus luteum: induction of manganese superoxide dismutase messenger ribonucleic acid by inflammatory cytokines. Biol. Reprod. 59, 208–215. Sugino, N., Hirosawa-Takamori, M., Zhong, L., Telleria, C.M., Shiota, K., Gibori, G., 1998b. Hormonal regulation of copper–zinc superoxide dismutase and manganese superoxide dismutase messenger ribonucleic acid in the rat corpus luteum: induction by prolactin and placental lactogens. Biol. Reprod. 59, 599–605. Sugino, N., Takiguchi, S., Kashida, S., Takayama, H., Yamagata, Y., Nakamura, Y., Kato, H., 1999. Suppression of intracellular superoxide dismutase activity by antisense oligonucleotides causes inhibition of progesterone production by rat luteal cells. Biol. Reprod. 61, 1133–1138. Sugino, N., Takiguchi, S., Kashida, S., Karube, A., Nakamura, Y., Kato, H., 2000a. Superoxide dismutase expression in the human corpus luteum during the menstrual cycle and in early pregnancy. Mol. Hum. Reprod. 6, 19–25. Sugino, N., Suzuki, T., Kashida, S., Karube, A., Takiguchi, S., Kato, H., 2000b. Expression of Bcl-2 and Bax in the human corpus luteum during the menstrual cycle and in early pregnancy: regulation by human chorionic gonadotropin. J. Clin. Endocrinol. Metab. 85, 4379–4386. Takiguchi, S., Sugino, N., Esato, K., Karube-Harada, A., Sakata, A., Nakamura, Y., Ishikawa, H., Kato, H., 2004. Differential regulation of apoptosis in the corpus luteum of pregnancy and newly formed corpus luteum after parturition in rats. Biol. Reprod. 70, 313–318. Tam, K.K., Russell, D.L., Peet, D.J., Bracken, C.P., Rodgers, R.J., Thompson, J.G., Kind, K.L., 2010. Hormonally regulated follicle

KH Al-Gubory et al. differentiation and luteinization in the mouse is associated with hypoxia inducible factor activity. Mol. Cell. Endocrinol. 327, 47–55. Tanaka, M., Miyazaki, T., Tanigaki, S., Kasai, K., Minegishi, K., Miyakoshi, K., Ishimoto, H., Yoshimura, Y., 2000. Participation of reactive oxygen species in PGF2alpha-induced apoptosis in rat luteal cells. J. Reprod. Fert. 120, 239–245. Taniguchi, K., Matsuoka, A., Kizuka, F., Lee, L., Tamura, I., Maekawa, R., Asada, H., Taketani, T., Tamura, H., Sugino, N., 2010. Prostaglandin F2a (PGF2a) stimulates PTGS2 expression and PGF2a synthesis through NFKB activation via reactive oxygen species in the corpus luteum of pseudopregnant rats. Reproduction 140, 885–892. Telleria, C.M., Goyeneche, A.A., Cavicchia, J.C., Stati, A.O., Deis, R.P., 2001. Apoptosis induced by antigestagen RU486 in rat corpus luteum of pregnancy. Endocrine 15, 147–155. Valko, M., Leibfritz, D., Moncol, J., Cronin, M.T., Mazur, M., Telser, J., 2007. Free radicals and antioxidants in normal physiological functions and human disease. Int. J. Biochem. Cell Biol. 39, 44–84. van den Driesche, S., Myers, M., Gay, E., Thong, K.J., Duncan, W.C., 2008. HCG up-regulates hypoxia inducible factor-1 alpha in luteinized granulosa cells: implications for the hormonal regulation of vascular endothelial growth factor A in the human corpus luteum. Mol. Hum. Reprod. 14, 455–464. Van Voorhis, B.J., Dunn, M.S., Snyder, G.D., Weiner, C.P., 1994. Nitric oxide: an autocrine regulator of human granulosa–luteal cell steroidogenesis. Endocrinology 135, 1799–1806. Vega, M., Johnson, M.C., Diaz, H.A., Stocco, C., Palomino, A., Devoto, L., 1998. Regulation of human luteal steroidogenesis in vitro by nitric oxide. Endocrine 8, 185–191. Weisiger, R.A., Fridovich, I., 1973. Mitochondrial superoxide dismutase. Site of synthesis and intramitochondrial localisation. J. Biol. Chem. 248, 4791–4793. Wink, D.A., Osawa, Y., Darbyshire, J.F., Jones, C.R., Eshenaur, S.C., Nims, R.W., 1993. Inhibition of cytochromes P450 by nitric oxide and a nitric oxide-releasing agent. Arch. Biochem. Biophys. 300, 115–123. Wu, X.M., Sawada, M., Carlson, J.C., 1992. Stimulation of phospholipase A2 by xanthine oxidase in the rat corpus luteum. Biol. Reprod. 47, 1053–1058. Zangar, R.C., Davydov, D.R., Verma, S., 2004. Mechanisms that regulate production of reactive oxygen species by cytochrome P450. Toxicol. Appl. Pharmacol. 199, 316–331. Zorov, D.B., Juhaszova, M., Sollott, S.J., 2006. Mitochondrial ROS-induced ROS release: an update and review. Biochim. Biophys. Acta 1757, 509–517. Declaration: The authors report no financial or commercial conflicts of interest. Received 1 March 2012; refereed 2 May 2012; accepted 21 August 2012.