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Antioxidant enzymatic defence systems in sheep corpus luteum throughout pregnancy

UMR Biologie du Développement et de la Reproduction, Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas cedex, France and ¹ Laboratoire de Biochimie Médicale, CHU Necker, Institut National de la Santé et de la Recherche Médicale, 75015 Paris, France

Correspondence should be addressed to K H Al-Gubory; Email: algubory{at}jouy.inra.fr

	Abstract

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The activities of copper, zinc-superoxide dismutase (SOD1), manganese SOD (SOD2), glutathione peroxidase (GPX), glutathione reductase (GSSG-R) and glutathione S-transferase (GST) were studied in sheep corpora lutea (CL) obtained on days 15, 40, 60, 80 and 128 of pregnancy. Maintained enzymatic activity of SOD1, SOD2, GPX, GSSG-R and GST were found in the sheep CL throughout pregnancy. Enzymatic activity of SOD1, GPX and GST increased significantly from day 15 to day 40 of pregnancy, and thereafter remained constant until day 128. SOD2 and GSSG-R activities were not different between any days of pregnancy examined. Apoptotic luteal cells identified by the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick-end labelling were very rarely observed, and their incidence (less than 0.5%) was not different between days of pregnancy. These results showed that the activities of antioxidant enzymes in the sheep CL are subject to major changes during early pregnancy, suggesting that the CL of early pregnancy may be rescued from luteolysis through increasing activities of key antioxidant enzymes and inhibition of apoptosis. Maintained levels of antioxidant enzymes in the CL throughout pregnancy may be linked to reactive oxygen species continuously generated in the steroidogenically active luteal cells, and may be involved in the maintenance of luteal steroidogenic activity and cellular integrity.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Superoxide dismutases (SODs) and glutathione (GSH)-dependent enzymes are the key antioxidants that protect cells against the toxic and damaging effects of reactive oxygen species (ROS). The copper-zinc SOD (Cu,Zn-SOD or SOD1) which is located in the cytosol (McCord & Fridovich 1969) and manganese SOD (Mn-SOD or SOD2) which is located in the mitochondria (Weisiger & Fridovich 1973) constitute the first enzymatic step that plays a vital cellular protective role by catalyzing the conversion of superoxide radical (O₂^–) into hydrogen peroxide (H₂O₂). Glutathione peroxidase (GPX) (Mills 1957), the principal peroxidase in mammals, present in significant amounts in the cytoplasm, and catalase, found primarily within peroxisomes (Chance et al. 1979) both catalyze the conversion of H₂O₂ to H₂O. GPX detoxifies H₂O₂ to H₂O through the oxidation of reduced GSH (Chance et al. 1979). In addition, GPX catalyzes the degradation of lipid peroxides (LPO) and can metabolize lipid hydroperoxides to less reactive hydroxy fatty acids (Chance et al. 1979). Glutathione reductase (GSSG-R) is an important component of the cellular antioxidant defence mechanism. This enzyme catalyzes the reduction of the oxidized form of glutathione (GSSG) to GSH with NADPH as the reducing agent (Chance et al. 1979). Therefore GSSG-R is essential for the glutathione redox cycle that maintains adequate levels of reduced GSH. The ability of GPX to reduce H₂O₂ or other hydroperoxides is therefore dependent on the activity of GSSG-R. Glutathione S-transferases (GST) are a polyfunctional family of cell enzymes that play an important role in detoxifying reactive metabolites by catalyzing their conjugation with reduced GSH. They are involved in the intracellular transport of compounds and their delivery to sites for subsequent transformation and/or excretion (Jakoby 1978, Kaplowitz 1980).

The ROS, such as O₂^–0, H₂O₂, LPO and hydroxyl radicals (⁰OH) and their generation in biological systems are known to damage cellular components such as lipids, proteins and nucleic acids (Halliwell & Gutteridge 1989a, b) and ultimately lead to cell death. They are increased in the rat corpus luteum (CL) during luteolysis (Sawada & Carlson 1989, Riley and Behrman 1991) and have been shown to affect progesterone production in rat luteal cells by inhibiting gonadotrophic action (Gatzuli et al. 1991, Musicki et al. 1994) and cholesterol translocation to the mitochondria (Behrman & Aten 1991). Evidence has also accumulated to suggest that locally produced antioxidant enzymes within the CL are directly involved in the maintenance of luteal steroidogenic activity and possibly in the rescue of CL from luteolysis when pregnancy occurs in various mammalian species. This includes (1) the changes in SOD1 and manganese SOD2 activities in the rat CL in a manner similar to the change in serum progesterone concentrations throughout pregnancy (Sugino et al. 1993a) and pseudopregnancy (Shimamura et al. 1995), (2) the high level of expression of the extracellular SOD (EC-SOD or SOD3), SOD2 and catalase in the bovine CL during early pregnancy (Rueda et al. 1995), (3) the high expression and activity of SOD1 in human CL during early pregnancy (Sugino et al. 2000), (4) the ability of SOD1 and catalase to stimulate the secretion of progesterone in vivo by rat CL of late pregnancy (Sugino et al. 1993b) and (5) the isolation and identification of sheep SOD1 from the CL of pregnancy (AlGubory et al. 2003). The inhibition of nuclear DNA cleavage, characteristic of physiological cell death (apoptosis), in cultured isolated rabbit CL with SOD, catalase or a putative stimulator of endogenous GPX (Dharmarajan et al. 1999), the induction of apoptotic DNA fragmentation in bovine luteal cells by down-regulation of GPX (Nakamura et al. 2001) and the induction of apoptosis in cultured bovine luteal cells by simultaneous treatment with H₂O₂ and with a specific inhibitor of GPX (Nakamura & Sakamoto 2001) suggest a role for ROS in luteal cell apoptosis.

Despite these investigations, which have mostly been carried out in rat CL, SODs and the GSH-dependent system of antioxidant defences that control in vivo ROS levels in the CL, on the one hand, and the degree of in situ luteal cell death, on the other hand, have not been studied throughout pregnancy in mammalian CL. The present study was therefore designed to determine the activities of key intracellular antioxidant enzymes, namely SOD1, SOD2, GPX, GSSG-R and GST, and the degree of in situ apoptosis in the CL collected from ewes at early pregnancy (day 15) and during the first (day 40), second (days 60 and 80) and third (day 128) trimester of gestation.

	Materials and Methods

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Animals and tissue collection
All procedures relating to the care and use of animals were approved by the French Ministry of Agriculture according to the French regulations for animal experimentation (authorization no. 78–34). The study involved 22 pregnant ewes of the Préalpes-du-Sud breed. All the ewes were treated for 14 days with intravaginal sponges containing 40 mg fluorogestone acetate (Intervet, Angers, France) to synchronize oestrus. Each ewe received, immediately after removal of the sponges, an intramuscular injection of 400 IU equine chorionic gonadotrophin (Intervet) and they were mated at the time of the synchronized oestrus with fertile rams. The ewes were killed at a local abattoir in accordance with protocols approved by the local institutional animal use committee. After the slaughter of the ewes, the reproductive tracts were collected and immediately transported to the laboratory. The CL were dissected from the surrounding ovarian tissue and weighed. For the determination of apoptosis, a piece of each CL was fixed overnight in freshly prepared 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4, washed in PBS, dehydrated through a series of increasing concentrations of ethanol (70–100%), cleared in xylene and embedded in paraffin wax. The rest of the CL was snap-frozen in liquid nitrogen and then stored at –80 °C until processed for anti-oxidant enzyme activities. The CL were obtained during early pregnancy (day 15, n = 4 ewes), the first (day 40, n = 5 ewes), second (day 60, n = 5 ewes, day 80, n = 5 ewes) and third (day 128, n = 4 ewes) trimester of gestation.

In situ detection of apoptosis by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling (TUNEL) method
Luteal apoptotic cells were identified with the in situ cell death detection fluorescein kit (Roche Diagnostics, Mannheim, Germany). This assay detects nuclear DNA fragmentation in apoptotic cells by the TdT-mediated TUNEL method and thus provides useful information about the fate of an individual cell in a given cell population, particularly on conventional histological sections without disruption of the tissue morphology. Briefly, 6 µm thick tissue sections were prepared, mounted on silane-coated slides (3-amino-propyltriethoxysilane-coated slides; Sigma-Aldrich), rehydrated through a series of decreasing concentrations of ethanol (100–70%) to water. They were then treated with 100 µg/ml Proteinase K (Sigma-Aldrich Chimie, Sarl, Saint Quentin Fallavier, France) in 10 mM Tris, pH 8.0, for 30 min at 37 °C and washed with PBS, pH 7.4. The slides were submerged in 0.1 M sodium citrate for 30 min at 70 °C and then washed with PBS. Sections were incubated 60 min at 37 °C with the TUNEL mixture, washed with PBS, counterstained with an aqueous solution of propidium iodide (DNA-PREP stain; Beckman Coulter Co., Miami, FL, USA) for 60 min at room temperature for detecting the nuclei of the cells in red. Tissue sections were immediately analyzed under a fluorescent microscope. Negative control sections were processed identically except that the labelling enzyme (TdT) was omitted. Four different optical fields (magnification x 250) were selected in a random manner for each CL tissue section and used to calculate the percentage of cells with apoptotic nuclei. Adjacent sections to those used for TUNEL were processed for routine histology and stained with haematoxylin and eosin for morphological analysis.

Antioxidant enzyme activity assays
Unfrozen luteal tissues corresponding to each stage of pregnancy were homogenized separately in 50 mM phosphate buffer, pH 7.4 and centrifuged at 15 000 g for 1 h at 4 °C. The resulting supernatant was used for determination of protein concentration and measurement of enzymatic activities. Protein concentrations were determined by the method of Lowry et al.(1951). Enzyme activities of SOD1, SOD2, GPX, GSSG-R and GST in the supernatant of each CL were determined in triplicate on an automatic Cobas-Bio centrifugal analyzer (Hoffman-LaRoche, Basel, Switzerland) as described in detail elsewhere (Wheeler et al. 1990, Ceballos-Picot et al. 1992, Guégan et al. 1999).

Blood sampling and progesterone assay
Blood samples were taken from the jugular vein into evacuated heparinized tubes. After centrifugation (3000 g, 4 °C) for 30 min, plasma was stored at –20 °C until assayed. The concentrations of progesterone were determined by radioimmunoassay in unextracted plasma as described (Schanbacher 1979), except that charcoal–dextran solution was used instead of polyethylene glycol for the separation of bound and free radioactivity. Tritiated progesterone ([1,2,6,7-³H]progesterone, specific activity. 88 Ci/mmol) was obtained from Amersham International (Amersham, Bucks, UK) and a specific anti-progesterone antibody was obtained from the Institut Pasteur (Paris, France). All plasma samples were run in duplicate in a single assay. The minimum detectable concentration of progesterone was 0.1 ng/ml and the intra-assay coefficient of variation was less than 10%.

Statistical analysis
Data were analyzed by ANOVA and the new Duncan’s multiple range test. Differences were considered to be significant if P < 0.05. The number of positively stained apoptotic cells was counted in four random fields/section per CL and results are expressed as the percentage of apoptotic cells of the total number of cells counted at each reproductive stage.

	Results

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CL mass and plasma progesterone level
The mean ± S.E.M. weight of CL (mg) recovered at day 15 of pregnancy (692 ± 59) was not different from that recovered at days 40 (611 ± 38), 60 (691 ± 98), 80 (675 ± 53) and 128 (589 ± 51) of pregnancy. As the sheep placenta begins to secrete large amounts of progesterone after day 60 of pregnancy (Ricketts & Flint 1980), concentrations of progesterone were determined only on days 15, 40 and 60 of pregnancy. The mean ± S.E.M. plasma concentration of progesterone (ng/ml) at day 15 of pregnancy (5.00 ± 0.40) was not different from those at days 40 (5.13 ± 0.47) and 60 (6.19 ± 0.72).

Activities of SODs and GSH-dependent enzymes
Changes in activities of SODs and GSH-dependent enzymes in the CL of pregnancy are shown in Fig. 1. The activities of SOD1, GPX and GST significantly increased from day 15 to day 40 and thereafter remained relatively constant until day 128. The activities of SOD2 and GSSG-R were not different between any days of pregnancy examined.

View larger version (18K): [in this window] [in a new window]   Figure 1 Changes in the activities of SOD1, SOD2, GPX, GSSG-R and GST in the sheep CL collected from ewes at early pregnancy (day 15, n = 4 ewes) and during the first (day 40, n = 5 ewes), second (day 60, n = 5 ewes and day 80, n = 5 ewes) and third (day 128, n = 4 ewes) trimester of pregnancy. Values are means ± S.E.M. for the number of animals used per stage. *P < 0.05, **P < 0.01, ***P < 0.001 compared with values at day 15.

View larger version (148K): [in this window] [in a new window]   Figure 2 Histological features and in situ identification of apoptotic cells by fluorescence labelling of nicked DNA of the luteal tissues at day 15 (upper panels), day 40 (middle panels) and day 60 (lower panels) of pregnancy (x 400). Sections (6 µm) were stained with haematoxylin and eosin (left panels) or subjected to TUNEL analysis (right panels). In the haematoxylin and eosin photographs, large luteal cells (L) can be distinguished from small luteal cells (S) by size and nuclear morphology. In the TUNEL photographs, non-fragmented nuclei are stained red (propidium iodide) whereas apoptotic nuclei with fragmented DNA are stained red and yellow (arrowheads). The general morphology and the intensity of TUNEL-positive cells at days 80 and 128 (not shown) are similar to those at days 15, 40 and 60 of pregnancy.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

The SODs constitute the first and most important line of antioxidant enzyme defence systems against O₂^–0 radicals. Our results showed that the magnitude of enzymatic activity of SOD1 and SOD2 in sheep CL was relatively similar throughout pregnancy. However, the changes in enzymatic activity of SOD1 and SOD2 during early pregnancy show a different pattern. The activity of SOD1 was significantly increased from day 15 to day 40 of pregnancy, whereas that of SOD2 remained relatively stable, indicating that these enzymes are controlled differently in the CL during early pregnancy. Although the sheep CL is necessary to maintain pregnancy at least during the first 60 days (Al-Gubory et al. 1999), it remains functional for nearly the whole period (duration about 150 days) (Linzell & Heap 1968, Moore et al. 1972). The presence of high levels of luteal tissue progesterone until day 142 of pregnancy in sheep indicates that luteal cells have a sustained and high steroidogenic capacity until late pregnancy (OShea & McCoy 1988). The first and rate-limiting step in the synthesis of progesterone in all steroidogenic organs, including the CL (Niswender 2002, Christenson & Devoto 2003), is the transfer of cholesterol from the outer mitochondrial membrane to the inner membrane where it is converted into pregnenolone by the enzyme cytochrome P450 side chain cleavage. It is well known that ROS are produced during enzyme reaction, particularly by the cytochrome P-450 family (Cross & Jones 1991) and by the respiratory system of mitochondria (Cadenas & Davies 2000), and thus they are considered as a by-product of steroid synthesis in the CL. As the steroidogenic capacity of the ovine CL shows no changes throughout pregnancy (OShea & McCoy 1988), the maintained enzymatic activity of SOD2 in sheep CL during pregnancy (present study) may therefore be related to the steady-state concentrations of ROS produced during steroidogenesis in functionally active luteal cells.

In the defence against ROS and oxidative stress, GSH-dependent antioxidant enzymes, like GPX, GSSG-R and GST, play an important role (Hayes & McLellan 1999). Investigations into these enzymes and their role in reproduction have been carried out mostly in humans (Knapen 2000). However, studies on key GSH-dependent antioxidant enzymes in ruminant reproductive organs are relatively absent (Sesh et al. 2001), particularly in the CL throughout pregnancy. In the present study, the changes in the activity of these enzymes in the sheep CL of different gestational stages were investigated. Unlike O₂^–0 radicals, H₂O₂ is relatively stable and has a higher oxidant potential. In the presence of iron, H₂O₂ and O₂^–0 can interact in a Haber–Weiss reaction to generate ⁰OH (Kehrer 2000) which is thought to be an extremely powerful initiator of lipid peroxidation (Halliwell & Gutteridge 1989a). Therefore, GPX activity has a major antioxidant role within cells by removing H₂O₂ before it reacts with metal catalyst to form extremely toxic ROS. GPX is also of utmost importance for cell protection as it catalyzes the degradation of LPO and can metabolize lipid hydroperoxides to less reactive hydroxy fatty acids (Chance et al. 1979). In rat luteal cells, H₂O₂ has been shown to inhibit steroidogenesis by blocking luteinizing hormone (LH)-stimulated cAMP (Behrman & Preston 1989) and cholesterol transport into mitochondria (Behrman & Aten 1991). In rat luteal cells, the antisteroidogenic effect of LPO appears to be due to stimulation of phospholipase-A₂, which is a rate-limiting step in prostaglandin synthesis (Kodamen et al. 1994). In the present study, the high enzymatic activity of GPX, and consequently the increased ability of luteal cells to scavenge H₂O₂ and LPO, would contribute to the maintenance of luteal cell steroidogenesis and integrity. Possibly, luteal cells induce enhancement of GPX to counteract a steady-state concentration of H₂O₂ due to high enzymatic activity of SODs. This is one of the described antioxidative cellular strategy representing an adaptive cellular mechanism (Ceballos et al. 1988). GSSG-R catalyzes the reduction of GSSG to GSH with NADPH as the reducing agent (Chance et al. 1979). Therefore this enzyme is essential for the glutathione redox cycle that maintains adequate levels of reduced GSH. The significant and maintained activity of GSSG-R in sheep CL throughout pregnancy reported in the present study may continuously supply GSH which, besides being a cofactor essential for both GPX and GST activity, is also one of the most efficient non-enzymatic antioxidants.

The activity of GST was found to be present in relatively high levels in the sheep CL during pregnancy (present study). Among the known biological functions of GST, besides its detoxification action, are intracellular transport and metabolism of steroid hormones (Listowsky et al. 1988). Furthermore, several investigations have suggested that ovarian GST is involved in steroidogenesis in various mammalian species. These include (1) the high localization of GST to the active steroid-producing cells of human ovaries throughout the menstrual cycle (Rahilly et al. 1991), (2) the increase in GST activity in porcine ovarian follicles during growth and the high activity of GST in the CL (Keira et al. 1994), (3) the close relationship between changes in GST activity induced by follicle-stimulating hormone and LH in porcine cultured granulosa cells and progesterone production (Keira et al. 1994), (4) the high GST activity in rat ovary observed at the oestrous stage (Singh & Pandey 1996) and (5) the high expression of GST in steroidogenically active bovine follicular and luteal cells (Rabahi et al. 1999). Although the significance of high activity of GST in the CL throughout pregnancy (present study) is at present unknown, it may suggest a role in the regulation of CL steroidogenesis.

The present results further demonstrated that the SOD1, GPX and GST activities in the CL markedly increased from day 15 to day 40 of pregnancy and remained elevated thereafter during all the stages of pregnancy examined (i.e. days 60, 80 and 128). Structural integrity of both large and small luteal cells in the CL of pregnancy was well preserved and relatively conserved irrespective of CL age, up to day 128 of pregnancy. Apoptotic luteal cells identified by fluorescence labelling of nicked DNA were very rarely observed and their incidence was not different between days of pregnancy. In the present study, plasma progesterone concentrations were not measured at days 80 and 128 of pregnancy because the sheep placenta begins to secrete large amounts of progesterone after day 60 (Ricketts & Flint 1980) and thus peripheral progesterone levels would not reflect the functional property of luteal cells. If levels of progesterone in the ovarian vein are a measure of secretory activity of the CL, the sheep CL is therefore actively secreting progesterone at a constant rate until day 130 of pregnancy (Moore et al. 1972). It can be suggested, therefore, that the reported increases in enzymatic activities of SODs, GPX and GST after day 15 of pregnancy, which remained at that level during the last two-thirds of pregnancy, and consequently the increased and maintained ability of luteal cells to scavenge O₂^–0 radicals, H₂O₂ and LPO, would contribute to the maintenance of luteal cell steroidogenesis, integrity and CL life span throughout the whole period of pregnancy in sheep.

In conclusion, these results show that the activities of key antioxidant enzymes in the sheep CL are subject to major changes during early pregnancy, suggesting that the CL of early pregnancy may be rescued from luteolysis through increasing activities of major antioxidant enzymes and inhibition of apoptosis. The high levels of antioxidant enzymes in the CL throughout pregnancy may be linked to ROS continuously generated in the steroidogenically healthy and active luteal cells, and may be involved in the maintenance of luteal steroidogenic activity and cellular integrity.

	Acknowledgements

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
The authors would like to thank the staff of the sheep sheds of Brouëssy and Jouy-en-Josas for outstanding technical help and animal management, and C Poirier for assistance with photomicrograph preparation.

	Footnotes

 
Received 23 June 2004
First decision 30 July 2004
Accepted 10 September 2004

	References

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 

Al-Gubory KH, Solari A & Mirman B 1999 Effects of lutectomy on the maintenance of pregnancy, circulating progesterone concentrations and lambing performance in sheep. Reproduction, Fertility and Development 11 317–322.[CrossRef][Medline]

Al-Gubory KH, Huet JC, Pernollet JC, Martal J & Locatelli A 2003 Corpus luteum derived copper, zinc dismutase serves as a luteinizing hormone-release inhibiting factor in sheep. Molecular and Cellular Endocrinology 199 1–9.[CrossRef][ISI][Medline]

Behrman HR & Preston SL 1989 Luteolytic actions of peroxide in rat ovarian cells. Endocrinology 124 2895–2900.[Abstract]

Behrman HR & Aten RF 1991 Evidence that hydrogen peroxide blocks hormone-sensitive cholesterol transport into mitochondria of rat luteal cells. Endocrinology 128 2958–2966.[Abstract]

Cadenas E & Davies KJ 2000 Mitochondrial free radical generation, oxidative stress, and aging. Free Radical Biology and Medicine 29 222–230.[CrossRef][ISI][Medline]

Ceballos I, Delabar JM, Nicole A, Lynch RE, Hallewell RA, Kamoun P & Sinet PM 1988 Expression of transfected human Cu, Zn-superoxide dismutase gene in mouse L cells and NS20Y neuroblastoma cells induces enhancement of glutathione peroxidase activity. Biochimica et Biophysica Acta 949 58–64.[Medline]

Ceballos-Picot I, Nicole A, Clément M, Bourre JM & Sinet PM 1992 Age-related changes in antioxidant enzymes and lipid peroxidation in brains of control and transgenic mice overexpressing copper-zinc superoxide dismutase. Mutation Research 275 281–293.[CrossRef][ISI][Medline]

Chance B, Sies H & Boveris A 1979 Hydroperoxide metabolism in mammalian organs. Physiological Reviews 59 527–605.[Free Full Text]

Christenson LK & Devoto L 2003 Cholesterol transport and steroido-genesis by the corpus luteum. Reproductive Biology and Endocrinology 1 1–9.

Cross AR & Jones OT 1991 Enzymic mechanisms of superoxide production. Biochimica et Biophysica Acta 1057 281–298.[Medline]

Dharmarajan AM, Hisheh S, Singh B, Parkinson S, Tilly KI & Tilly JL 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.[Abstract/Free Full Text]

Gatzuli E, Aten RF & Behrman HR 1991 Inhibition of gondatropic action and progesterone synthesis by xanthine oxidase in rat luteal cells. Endocrinology 128 2253–2258.[Abstract]

Guégan C, Ceballos-Picot I, Chevalier E, Nicole A, Onténiente B & Sola B 1999 Reduction of ischemic damage in NGF-transgenic mice: correlation with enhancement of antioxidant enzyme activities. Neurobiology of Disease 6 180–189.[CrossRef][ISI][Medline]

Halliwell B & Gutteridge JMC 1989a The chemistry of oxygen radicals and other derived species. In Free Radicals in Biology and Medicine, pp 22–85. Eds B Halliwell & JMC Gutteridge. Oxford: Clarendon Press.

Halliwell B & Gutteridge JMC 1989b Protection against oxidants in biological systems: the superoxide theory of oxygen toxicity. In Free Radicals in Biology and Medicine, pp 86–123. Eds B Halli-well & JMC Gutteridge. Oxford: Clarendon Press.

Hayes JD & McLellan LI 1999 Glutathione and glutathione-dependent enzymes represent a coordinately regulated defence against oxidative stress. Free Radical Research 31 273–300.[ISI][Medline]

Jakoby WB 1978 The glutathione S-transferases: a group of multifunctional detoxification proteins. Advances in Enzymology and Related Areas of Molecular Biology 46 383–414.[Medline]

Kaplowitz N 1980 Physiological significance of glutathione S-transferase. American Journal of Physiology 239 G439–G444.[ISI][Medline]

Kehrer JP 2000 The Haber–Weiss reaction and mechanisms of toxicity. Toxicology 149 43–50.[CrossRef][ISI][Medline]

Keira M, Nishihira J, Ishibashi T, Tanaka T & Fujimoto S 1994 Identification of a molecular species in porcine ovarian luteal glutathione S-transferase and its hormonal regulation by pituitary gonadotropins. Archives of Biochemistry and Biophysics 308 126–132.[CrossRef][ISI][Medline]

Knapen MF 2000 The glutathione/glutathione-related enzyme system in reproduction. European Journal of Obstetrics, Gynecology and Reproductive Biology 91 127–129.[CrossRef][ISI][Medline]

Kodaman PH, Aten RF & Behrman HR 1994 Lipid hydroperoxides evoke antigonadotropic and antosteroidogenic activity in rat luteal cells. Endocrinology 135 2723–2730.[Abstract]

Linzell JL & Heap RB 1968 A comparison of progesterone metabolism in the pregnant sheep and goat: source of production and an estimation of uptake by some target organs. Journal of Endocrinology 41 433–438.[ISI][Medline]

Listowsky I, Abramovitz M, Homma H & Niitsu Y 1988 Intracellular binding and transport of hormones and xenobiotics by glutathione S-transferases. Drug Metabolism Reviews 19 305–318.[ISI][Medline]

Lowry OH, Rosebrough NJ, Farr AL & Randall RF 1951 Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193 265–275.[Free Full Text]

McCord JM & Fridovich I 1969 Superoxide dismutase. An enzymatic function for erythrocuprein. Journal of Biological Chemistry 244 6049–6055.[Abstract/Free Full Text]

Mills GC 1957 Hemoglobin catabolism. I Glutathione peroxidase, an erythrocyte enzyme which protects hemoglobin from oxidative breakdown. Journal of Biological Chemistry 229 189–197.[Free Full Text]

Moore NW, Barrett S & Brown JB 1972 Progesterone concentration in maternal and foetal blood plasma of ewes. Journal of Endocrinology 53 187–194.[ISI][Medline]

Musicki B, Aten RE & Behrman HR 1994 Inhibition of protein synthesis and hormone-sensitive steroidogenesis in response to hydrogen peroxide in rat luteal cells. Endocrinology 134 588–595.[Abstract]

Nakamura T & Sakamoto K 2001 Reactive oxygen species up-regulates cyclooxygenase-2, p53 and Bax mRNA expression in bovine luteal cells. Biochemical and Biophysical Research Communications 284 203–210.[CrossRef][ISI][Medline]

Nakamura T, Ishigami T, Makino N & Sakamoto K 2001 The down-regulation of glutathione peroxidase causes bovine luteal cell apoptosis during structural luteolysis. Journal of Biochemistry 129 937–942.[Abstract/Free Full Text]

Niswender GD 2002 Molecular control of luteal secretion of progesterone. Reproduction 123 333–339.[Abstract]

O’Shea JD & McCoy K 1988 Weight, composition, mitosis, cell death and content of progesterone and DNA in the corpus luteum of pregnancy in the ewe. Journal of Reproduction and Fertility 83 107–117.[Abstract]

Rabahi F, Brûlé S, Sirois J, Beckers JF, Silversides DW & Lussier JG 1999 High expression of bovine glutathione S-transferase GSTA1, GSTA2 subunits is mainly associated with steroidogenically active cells and regulated by gonadotropins in bovine ovarian follicles. Endocrinology 140 3507–3517.[Abstract/Free Full Text]

Rahilly M, Carder PJ, Al Nafussi A & Harrison DJ 1991 Distribution of glutathione S-transferase isoenzymes in human ovary. Journal of Reproduction and Fertility 93 303–311.[Abstract]

Riley JCM & Behrman HR 1991 In vivo generation of hydrogen peroxide in the rat corpus luteum during luteolysis. Endocrinology 128 1749–1753.[Abstract]

Ricketts AP & Flint APF 1980 Onset of synthesis of progesterone by ovine placenta. Journal of Endocrinology 86 337–347.[Abstract]

Rueda BR, Tilly KI, Hansen TR, Hoyer PB & Tilly JL 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.[ISI]

Sawada M & Carlson JC 1989 Superoxide radical production in plasma membrane samples from regressing rat corpora lutea. Canadian Journal of Physiology and Pharmacology 67 465–471.[ISI][Medline]

Schanbacher BD 1979 Radioimmunoassay of ovine and bovine serum progesterone without extraction and chromatography. Endocrine Research Communications 6 265–277.[ISI][Medline]

Sesh PS, Singh D, Sharma MK & Pandey RS 2001 Activity of glutathione related enzymes and ovarian steroid hormones in different sizes of follicles from goat and sheep ovary of different reproductive stages. Indian Journal of Experimental Biology 39 1156–1159.[Medline]

Shimamura K, Sugino N, Yoshida Y, Nakamura Y, Ogino K & Kato H 1995 Changes in lipid peroxide and antioxidant enzymes activities in corpora lutea during pseudopregnancy in rats. Journal of Reproduction and Fertility 105 253–257.[Abstract]

Singh D & Pandey RS 1996 Glutathione-S-transferase in rat ovary: its changes during estrous cycle and increase in its activity by estradiol-17 beta. Indian Journal of Experimental Biology 34 1158–1160.[Medline]

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. Journal of Reproduction and Fertility 97 347–351.[Abstract]

Sugino N, Nakamura Y, Okuno N, Ishimatu M, Teyama T & Kato H 1993b Effects of ovarian ischemia-reperfusion on luteal function in pregnant rats. Journal of Reproduction and Fertility 49 354–358.[CrossRef]

Sugino N, Takiguchi S, Kashida S, Karube A, Nakamura Y & Kato H 2000 Superoxide dismutase expression in the human corpus luteum during the menstrual cycle and in early pregnancy. Molecular Human Reproduction 6 19–25.[Abstract/Free Full Text]

Weisiger RA & Fridovich I 1973 Mitochondrial superoxide dismutase. Site of synthesis and intramitochondrial localisation. Journal of Biological Chemistry 248 4793–4796.[Abstract/Free Full Text]

Wheeler CR, Salzman JA, Elsayed NM, Omaye ST & Korte DW Jr 1990 Autommated assays of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activity. Analytical Biochemistry 184 193–199.[CrossRef][ISI][Medline]

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This Article Abstract Full Text (PDF) An erratum has been published Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Al-Gubory, K. H Articles by Ceballos-Bicot, I. Search for Related Content PubMed PubMed Citation Articles by Al-Gubory, K. H Articles by Ceballos-Bicot, I.