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Catalase and Glutathione Peroxidase Expression in Bovine Corpus Luteum During the Estrous

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Catalase and Glutathione Peroxidase Expression in Bovine Corpus Luteum During the Estrous Anim. Reprod., v.11, n.2, p.74-84, Apr./Jun. 2014 Catalase and glutathione peroxidase expression in bovine corpus luteum during the estrous cycle and their modulation by prostaglandin F2α and H2O2 H.V. Vu, T.J. Acosta1 Laboratory of Reproductive Physiology, Graduate School of Environmental and Life Science, Okayama University, Okayama, Japan. Abstract (Skarzynski et al., 2009). If pregnancy does not occur, the CL regresses and loses its capacity to produce P4 Antioxidant enzymes seem to play roles in (Agarwal et al., 2012). Regression of the CL (luteolysis) controlling the luteal function and the luteolytic action is crucial to reset the ovarian cycle, so that the animal of prostaglandin F2α (PGF). The aim of this study was can return to estrus and have another opportunity to to clarify the roles of catalase (CAT) and glutathione become pregnant (Gibson, 2008). peroxidase (GPx) in the luteolytic action of PGF in both Prostaglandin F2α (PGF) is a luteolytic factor corpus luteum (CL) and cultured luteal cells. Corpora in mammals. In the cow, both endogenous PGF lutea were collected at the early (days 2-3), developing synthesized by the uterus at the late-luteal stage (days 5-6), mid (days 8-12), late (days 15-17) and (McCracken et al., 1999) and exogenous PGF given regressed (days 19-21) luteal stages (n = 5 CL/stage) during the mid-luteal stage (Schallenberger et al., 1984) and at 0, 2 and 24 h after luteolytic PGF administration cause irreversible luteal regression that is characterized (0 h) on day 10 (n = 5 cows/time point). Catalase by a rapid decrease in P4 production (functional protein and the activities of CAT and GPx increased luteolysis) followed by a decrease in the size of the CL from the early to mid-luteal stage, then all decreased (structural luteolysis; Juengel et al., 1993; Acosta et al., (P < 0.05), reaching their lowest levels at the regressed 2002). In addition, in the cow (Pate, 1988) and ewe luteal stage. The levels of GPx1 protein were lower in (Rexroad and Guthrie, 1979; Lee et al., 2012) the CL the regressed luteal stage than in other stages (P < 0.05). also synthesizes PGF. Nevertheless, the mechanisms Immunohistochemical examination also revealed the regulating the luteolytic action of PGF remain unclear. expression of CAT and GPx1 protein in the bovine CL Reactive oxygen species (ROS), the byproducts tissue. Injection of a luteolytic dose of PGF increased of normal aerobic metabolism, are highly cytotoxic, and luteal GPx1 protein and GPx activities at 2 h but thus act as apoptotic factors (Garrel et al., 2007). suppressed them at 24 h. Catalase protein and CAT Reactive oxygen species include superoxide radicals, activity did not change at 2 h but CAT activity hydrogen peroxide and hydroxyl radicals (Kato et al., decreased (P < 0.05) at 24 h. Prostaglandin F2α (1 µM) 1997). Antioxidant enzymes control the ROS and H2O2 (10 µM) decreased CAT and GPx1 protein concentration. The balance between ROS generation expression and activity at 24 h in cultured luteal cells and ROS elimination by antioxidant enzymes helps isolated from mid-luteal stage CL (n = 3 CL per each maintain cellular function. An increase in intracellular experiment). Interestingly, CAT protein and activity did ROS levels by an increase in ROS production or a not change while GPx1 protein and activity increased at decrease in antioxidant enzyme levels can lead to cell 2 h in luteal cells treated with PGF and H2O2 (P < 0.05). death (Garrel et al., 2007) by apoptosis (Juengel et al., The down-regulation of CAT and GPx, and their 1993; Buttke and Sandstrom, 1994). Reactive oxygen activities during structural luteolysis might enhance the species have been implicated in the regulation of luteal accumulation of reactive oxygen species, which would function, including luteo-protective (Kawaguchi et al., result in both increasing luteal PGF production and cell 2013) and luteolytic roles (Riley and Behrman, 1991b; death to complete CL regression in cattle. Carlson et al., 1993; Tanaka et al., 2000; Hayashi et al., 2003; Al-Gubory et al., 2010; Taniguchi et al., 2010). Keywords: bovine corpus luteum, catalase, glutathione Reactive oxygen species generation is induced by PGF peroxidase, luteolysis, prostaglandin F2α. in the ovine (Hayashi et al., 2003), rat (Tanaka et al., 2000) and bovine CL (Vu et al., 2013). Intraluteal ROS Introduction increases during luteal regression (Riley and Behrman, 1991b; Shimamura et al., 1995) and it has the capacity The corpus luteum (CL) forms in the ovary to inhibit P4 production (Sawada and Carlson, 1991; after ovulation and produces progesterone (P4), the Sugino et al., 1993). In addition, we and others found hormone responsible for the maintenance of pregnancy that PGF production is induced by ROS in bovine luteal ________________________________ 1Corresponding author: [email protected] Phone: +81(86)251-8349; Fax: +81(86)251-8349 Received: January 9, 2014 Accepted: April 1, 2014 Vu and Acosta. Modulation of CAT and GPx in bovine corpus luteum. cells and that ovarian ROS production is rapidly Materials and Methods inducible by PGF in vitro (Margolin and Behrman, 1992; Vu et al., 2013) and in vivo (Acosta et al., 2009). Collection of bovine corpus luteum tissues throughout However, the effects of ROS on the CL may differ the luteal stages depending on time or dose (Vega et al., 1995). Treatment of human mid luteal cells with a low Uteri and ovaries with CL were collected from concentration of hydrogen peroxide (H2O2; 0.01 μM) Holstein cows at a local slaughterhouse within 10-20 min significantly stimulated P4 secretion while treatment after exsanguinations and transported to the laboratory with a higher concentration (100 μM) markedly (Laboratory of Reproductive Physiology, Graduate inhibited human chorionic gonadotropin (hCG)- School of Environmental and Life Science, Okayama stimulated P4 and estradiol secretion (Vega et al., 1995). University, Okayama 700-8530, Japan) within 1-1.5 h on Since the local ROS concentrations are ice. Only ovaries containing CL from apparently normal controlled by antioxidant enzymes, it is possible that reproductive tracts based on uterine characteristics (size, these enzymes are involved in regulating the luteolytic color, tonus, consistency and mucus) were used in the action of PGF (Minegishi et al., 2002). Antioxidant present study. Luteal stages were classified as early (days enzymes include superoxide dismutases (SODs), 2-3 after ovulation), developing (days 5-7), mid (days 8- catalase (CAT) and glutathione peroxidases (GPx). 12), late (days 15-17) and regressed (days 19-21) luteal Superoxide dismutases are located in the nucleus and stages by macroscopic observation of the ovary and cytoplasm (type 1), in the mitochondria (type 2) and in corpus luteum as described previously (Okuda et al., the extracellular matrix (type 3). Catalase is usually 1988; Miyamoto et al., 2000; Vu et al., 2012). The CL located in a cellular organelle called the peroxisome tissues were immediately used for cell isolation and cell (Mueller et al., 2002). Glutathione peroxidases include culture (CL tissue at mid luteal stage, n = 3 CL per each several isozymes that differ according to their cellular experiment), fixed for immunohistochemical trial (CL location and substrate specificity (Muller et al., 2007). tissue at mid luteal stage, n = 5 CL), or dissected from Glutathione peroxidase type 1 (GPx1), the most the ovaries and stored at -80°C (n = 5 CL per each abundant type, scavenge H2O2 in the cytoplasm (Muller luteal stage) until protein and enzyme activity et al., 2007). All of these antioxidant enzymes are found analyses. in nearly all living organisms exposed to oxygen (Chelikani et al., 2004). Superoxide dismutases convert Collection of bovine corpus luteum tissues during PGF- oxygen radicals into H2O2. H2O2 is then converted to induced luteolysis water and oxygen by CAT (Chelikani et al., 2004) and GPx (Al-Gubory et al., 2008). Therefore, an increase of The collection procedures were approved by SOD without elevation of CAT or GPx may enhance the the local institutional animal care and use committee accumulation of H2O2. The down-regulation of of Polish Academy of Sciences in Olsztyn, Poland glutathione peroxidase causes bovine luteal cell (Agreement No. 5/2007, 6/2007 and 88/2007). Healthy apoptosis during structural luteolysis (Nakamura et al., normally cycling Polish Holstein Black and White 2001). In bovine CL, the expression of CAT and SOD cows were used for collection of CL. Estrus was are low during luteal regression, which suggests that synchronized in the cows by two injections of a PGF oxidative stress has a role in inducing luteolysis (Rueda analogue (Dinoprost, Dinolytic; Pharmacia & Upjohn, et al., 1995). Our previous study (Vu et al., 2012) found Belgium) with an 11-day interval according to the that the SOD expression and activity increased during manufacturer’s direction. Ovulation was determined by functional luteolysis. The increase of SOD could induce a veterinarian via transrectal ultrasonographic different effects in luteal cells depending on the levels examination. Then, corpora lutea were collected with of CAT and GPx: 1) SOD protects the luteal cells when the Colpotomy technique using a Hauptner’s effeninator it increases concomitantly with CAT and/or GPx; 2) (Hauptner and Herberholz, Solingen, Germany) on day SOD enhances cell death when it increases without 10 post ovulation, i.e. just before administration of a elevation of CAT or GPx to scavenge H2O2 (Vu et al., luteolytic dose of a PGF analogue (Dinoprost, 2012). Thus, CAT or GPx seem to control luteal Dinolytic; Pharmacia; 0 h), and at 2 and 24 h post- function by regulating the intraluteal actions of PGF. treatment (n = 5 cows per time point). Corpus luteum The aim of this study was to clarify the role of CAT and tissues were dissected from the ovaries and then GPx on the luteolytic action of PGF in both CL and immediately stored at -80°C until protein and enzyme cultured luteal cells.
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