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Expression of superoxide dismutases in the bovine oviduct during the estrous cycle
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Theriogenology 70 (2008) 836–842 www.theriojournal.com
Expression of superoxide dismutases in the bovine oviduct during the estrous cycle M. Roy a,b, D. Gauvreau a,b, J.-F. Bilodeau a,b,c,* a
Unite´ de Recherche en Ontoge´nie et Reproduction, Centre de Recherche du Centre Hospitalier de l’Universite´ Laval, Que´bec, Canada G1V 4G2 b Centre de Recherche en Biologie de la Reproduction (CRBR), Universite´ Laval, Que´bec, Canada GIV 4G2 c De´partement d’Obste´trique et Gyne´cologie, Faculte´ de Me´decine, Universite´ Laval, Que´bec, Canada G1K 7P4 Received 28 September 2007; received in revised form 30 January 2008; accepted 9 May 2008
Abstract The superoxide dismutases (SODs) are first-line enzymatic antioxidants that dismute superoxide anion (O2 ) to produce hydrogen peroxide (H2O2). The primary objective was to characterize, by western blot analysis, the expression of two SODs, the cytosolic (Cu,ZnSOD or SOD1) and the mitochondrial (MnSOD or SOD2) forms in three sections of the oviduct, i.e. isthmus (I), ishtmic–ampullary junction (IA), and ampulla (A), during the estrous cycle. The Cu,ZnSOD and MnSOD proteins were mostly expressed in the ampulla (I < IA < A; P < 0.0093). Expression of Cu,ZnSOD was lowest during metestrus (P < 0.0041) and it was mostly expressed in the oviduct contralateral to the CL (P = 0.0019). The expression of MnSOD was consistent throughout the cycle. Based on immunohistochemistry, the SODs were present in all cell types of the oviduct, with variations among oviductal sections. We inferred that the expression of Cu,ZnSOD was influenced by the hormonal milieu. Furthermore, variations among oviduct sections in protein expression profile of SODs suggested they have an important role in preserving and capacitating sperm. # 2008 Elsevier Inc. All rights reserved. Keywords: Oviduct; Superoxide dismutase; Reactive oxygen species; Estrous cycle; Cow
1. Introduction In cattle and other species, the oviducts play a critical role in early reproductive processes, including gamete maturation, fertilization, and early embryo development [1–3]. These processes require an appropriate balance between reactive oxygen species (ROS) and antioxidants, as shown by many in vivo and in vitro studies
* Corresponding author at: De´partement d’Obste´trique et Gyne´cologie, Faculte´ de Me´decine, Universite´ Laval, Que´bec, Canada. Tel.: +1 418 525 4444x46153; fax: +1 418 654 2765. E-mail address: [email protected] (J.F. Bilodeau). 0093-691X/$ – see front matter # 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2008.05.042
[4–6]. Reactive oxygen species (ROS) exist in several forms (O2 , H2O2, OH, NO, ONOO ) and can be either beneficial or detrimental to reproductive events [4,6,7]. Indeed, low levels of ROS can be beneficial in promoting the binding of sperm to the zona pellucida [8–10]. In cattle, the superoxide anion (O2 ) and hydrogen peroxide (H2O2) are essential for sperm capacitation and the acrosome reaction, respectively, in vitro [6,11]. However, high hydrogen peroxide (H2O2) concentrations reduced bull sperm motility in vitro [6] and may impair fertilization and embryo development [12,13]. Furthermore, interaction of O2 and H2O2 can generate the hydroxyl radical (OH), one of the most powerful ROS, which can damage lipids, proteins and nucleic acids [14]. The H2O2 can also form OH via the
M. Roy et al. / Theriogenology 70 (2008) 836–842
Fenton reaction in the presence of transition metals (e.g. iron) [14]. The O2 can also react with nitric oxide (NO) to form peroxynitrite (ONOO ), a powerful oxidant, which reduces the bioavailability of NO [15]. Production of ROS is controlled by various antioxidants. Nonenzymatic antioxidants include glutathione (GSH), a-tocopherol, b-carotene, ascorbate, and vitamin C [15,16]. The main antioxidant enzymes include superoxide dismutases (SODs) which neutralize superoxide anion (O2 ), catalases which scavenge hydrogen peroxide (H2O2), and glutathione peroxidases (GPx) which detoxify H2O2 and lipid hydroperoxides (ROOH) [15]. The SODs exist in three forms: the cytosolic (Cu,ZnSOD or SOD1), the mitochondrial (MnSOD or SOD2) and the extracellular form (ECSOD or SOD3) [17]. The SODs are the first line of defense against ROS in dismuting two superoxide anions (O2 ) into one hydrogen peroxide (H2O2) [18], which can be metabolized by either catalases or GPx [15]. Thus, SODs are important defense enzymes against O2 , preventing the formation of OH and ONOO . That Cu,ZnSOD female knock-out mice had reduced fertility implicates SODs in reproduction in the female [19]. Although the balance between antioxidants and ROS is critically important for reproductive success, few in vivo studies have characterized antioxidants in the oviduct, especially SODs in cattle. In mammalian oviducts, Cu,ZnSOD and MnSOD were best characterized in the mouse, rat, rabbit, and human [20–22]. We previously reported that antioxidant enzymes such as glutathione peroxidases (GPx) and prooxidant enzymes such as nitric oxide synthases (NOS) that produce NO were spatially and temporally regulated in the bovine oviduct during the estrous cycle [23,24]. However, the specific protein regulation of superoxide dismutases (SODs) remains to be determined in the cow. The primary objective of the present study was to determine the expression of Cu,ZnSOD and MnSOD proteins during the estrous cycle in three sections of each oviduct (both ipsilateral and contralateral to the CL). The secondary objective was to determine cellular localization of these SODs. 2. Materials and methods
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tion of the ovaries, the tracts were divided according to stage of the cycle (3): metestrus (Days 0–3), diestrus (Days 10–15), and proestrus (Days 18–21). The locations of the current and previous CL were determined; only tracts with these CL on opposite ovaries were used. Furthermore, oviducts ipsilateral and contralateral to the current CL were studied separately. Oviducts were dissected (to remove blood vessels, ligaments, and other tissues) and cut in three sections of 2 cm: the isthmus was adjacent to the uterotubal junction; the isthmic–ampullary junction was the midpoint of the oviduct; and the ampulla was adjacent to the fimbria. Each section were frozen in liquid nitrogen and subsequently kept at 86 8C until analyzed. For each stage of the estrous cycle, five pairs of oviducts (ipsilateral and contralateral to the CL) were used (three sections per oviduct). 2.2. Western blot analysis A sample (0.5 cm) from each section of the oviduct was mixed in ice-cold PBS (Invitrogen, Carlsbad, CA, USA) containing a protease inhibitor cocktail (1 mM EDTA, 0.5 mg/mL Leupeptin, 1.4 mg/mL Pepstatin A, 70 mg/mL PMSF; Boehringer Mannheim, Laval, QC, Canada) and homogenized with an Ultra Turrax T25 (IKA Works, Inc., Wilmington, NC, USA). Homogenates were mixed in Laemmli sample buffer (5% (v:v) 2-mercaptoethanol) and boiled for 10 min [23,24]. The quantity of proteins was determined using a BCA protein assay, according to the manufacturer’s instructions (Pierce, Rockford, IL, USA). An aliquot (30 mg) of proteins from each oviductal section were loaded on a 15% polyacrylamide-SDS gel. After electrophoresis, the proteins were transferred to a nitrocellulose membrane (0.2 mm; Bio-Rad Laboratories, Montre´al, QC, Canada). The membranes were incubated for 1 h at room temperature with 5% dry milk in TBS-T, then incubated for 2 h at room temperature with primary rabbit polyclonal antibodies against bovine SOD1 (dilution 1:10,000) [23] and SOD2 (dilution 1:5000) (GeneTex, San Antonio, TX, USA). The signal was revealed using ECL Trade Mark (GE Healthcare Bio-Sciences, Baie d’Urfe´ QC, Canada) and a densitometry was done with an Alpha Imager 2000 (Alpha Innotech, San Leandro, CA, USA).
2.1. Oviducts 2.3. Immunohistochemistry Genital tracts were recovered from Holstein cows/ heifers at an abattoir and transported on ice to the laboratory within 4 h after death. Tracts with visible anomalies were eliminated. Based on visual examina-
Immunohistochemistry was performed as previously described [24]. Briefly, immediately after dissection, a portion of each section of the oviduct (isthmus, isthmic–
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ampullary junction and ampulla) was fixed overnight in 4% paraformaldehyde. Optimum cutting temperature medium (OCT; Canemco, St. Laurent, QC, Canada) was used to embed the sections before freezing in liquid nitrogen. Samples were cryosectioned (eight sections/ block, each section 8 mm thick) with a cryotome (Shandon, Pittsburgh, PA, USA), and spread on a microscope slide. Vectastain Elite ABC kit (Vector Laboratories Inc., Burlingame, CA, USA) was used, in accordance with the manufacturer’s instructions, to perform immunohistochemistry analysis. All incubations were done at room temperature unless otherwise stated. The slides were incubated for 30 min in 3% H2O2 (v:v) with methanol to inactivate endogenous peroxidases, incubated for 1 h with 10% goat serum (Sigma–Aldrich, Oakville, ON, Canada) in PBS to block non-specific binding, and then incubated overnight at 4 8C with primary antibody. The primary antibodies were rabbit polyclonal antibody against bovine SOD2 (dilution 1:1000; GeneTex) and SOD1 (dilution 1:1000; Stressgen, Victoria, BC, Canada). A negative control was also done with non-specific IgGs. After washing in PBS, the slides were incubated using a biotinylated goat anti-rabbit IgG (Vector Laboratories) for 1 h. Finally, the slides were incubated 30 min with the ABC elite reagent and with 3-amino-9-ethylcarbazole until the immunostaining was revealed. Mayer’s hematoxylin (Sigma–Aldrich) was used as a counterstain, An Axioskop 2 Plus microscope (Zeiss, Toronto, ON, Canada) coupled to a digital camera was used to record color images. The images from microscopy were analyzed subjectively for relative protein expression. 2.4. Statistical analysis Densitometry values obtained from the western blot analysis were normalized to b-actin. To study the
effects of the factors: stage of the estrous cycle, oviduct side and oviduct section on the gene expression, a threeway analysis of variance with repeated measures was used. To account for possible correlations between observations made on the same cow, we considered the factors: oviduct side and section, as repeated measures. The MIXED procedure of SAS software (SAS Institute Inc. 2005. SAS OnlineDoc1 9.1.3. Cary, NC, USA) was used with a repeated statement and the covariance structure that minimizes the Akaike criterion. The method of Kenward–Roger was used to calculate the degrees of freedom. The normality and the homogeneity of the variance assumptions were met. Pairwise comparisons were then made using protected Fisher LSD (least significant difference). For all analyses, P < 0.05 was considered significant. 3. Results Western blot analyses were performed to determine the protein expression of Cu,ZnSOD and MnSOD; one replicate is shown (Fig. 1). Both Cu,ZnSOD and MnSOD were detected in all three oviductal segments. The expression of Cu,ZnSOD varied significantly throughout the estrous cycle, along the oviduct and in the ispilateral and contralateral oviduct (Table 1), whereas MnSOD expression varied only in the three sections of the oviduct (Table 2). There were no interactions between the three factors studied (stage of the estrous cycle, side relative to the CL and section of the oviducts) for both Cu,ZnSOD and MnSOD (Tables 1 and 2). The expression for both Cu,ZnSOD and MnSOD differed significantly along the oviduct (Figs. 2A and 3). The Cu,ZnSOD levels were higher in the ampulla than in the isthmic–ampullary junction by 27% (P = 0.0001) and the isthmus by 56% (P < 0.0001), respectively. The
Fig. 1. Cu,ZnSOD, MnSOD protein levels in the isthmus (I), isthmic–ampullary junction (I–A) and ampulla (A) regions of the bovine oviduct (ipsilateral and contralateral to the CL) of cattle at metestrus, diestrus, and proestrus; (C): positive control. The loading control was b-actin. These results are a single replicate (tissues derived from one reproductive tract) and were representative of all five replicates.
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Table 1 Analysis of variance for Cu,ZnSOD protein expression in the bovine oviduct (summary of fixed-effects statistics) Effect
DFN
DFD
F-value
P-value
Stage Side Stage side Section Stage section Side section Stage side section
2 1 2 2 4 2 4
21.9 11.1 11.1 20 20 34.6 34.6
10.43 16.29 0.09 31.12 0.19 1.56 0.46
0.0007 0.0019 0.9135 <0.0001 0.9401 0.2246 0.7667
Stage: stage of estrous cycle (metestrus, diestrus, proestrus); Side: contralateral or ipsilateral oviduct (relative to the CL); Section: section of the oviduct (isthmus, isthmic–ampullary junction, ampulla); DFN: degrees of freedom in numerator; DFD: degrees of freedom in denominator.
expression for Cu,ZnSOD was also higher in the isthmic–ampullary junction than the isthmus (P = 0.0049; Fig. 2A). Similarly, MnSOD expression was higher in the ampulla than in the isthmic–ampullary junction (+23%; P = 0.0093) and the isthmus (+64%; P < 0.0001), while being higher in the isthmic– ampullary junction than in the isthmus (P = 0.0064; Fig. 3). The expression for Cu,ZnSOD also varied throughout the estrous cycle (Fig. 2B). Indeed the Cu,ZnSOD expression was lower in metestrus than in diestrus ( 20%; P = 0.0041) and proestrus ( 26%; P = 0.0002). However, there was no difference between diestrus and proestrus stages (P = 0.2388). Furthermore, the expression for Cu,ZnSOD was higher in the contralateral than in the ipsilateral oviduct (+18%; P = 0.0019) Fig. 2C). In the absence of relative staining (immunohistochemistry) differences between cell types for the SODs during the estrous cycle, we showed only one stage Table 2 Analysis of variance for MnSOD protein expression in the bovine oviduct (summary of fixed-effects statistics) Effect
DFN
DFD
F-value
P-value
Stage Side Stage side Section Stage section Side section Stage side section
2 1 2 2 4 2 4
17.3 18.1 18.1 29.4 29.4 28.3 28.3
0.04 3.21 2.24 16.38 0.51 0.21 0.20
0.9644 0.0899 0.1351 <0.0001 0.7286 0.8101 0.9362
Stage: stage of estrous cycle (metestrus, diestrus, proestrus); Side: contralateral or ipsilateral oviduct (relative to the CL); Section: section of the oviduct (isthmus, isthmic–ampullary junction, ampulla); DFN: degrees of freedom in numerator; DFD: degrees of freedom in denominator.
Fig. 2. Mean (S.E.M.) Cu,ZnSOD protein expression (densitometry of western blots) in bovine oviducts. The expression of Cu,ZnSOD in the isthmus, isthmic–ampullary junction and ampulla (A). Cu,ZnSOD expression during the estrous cycle (metestrus, diestrus and proestrus; (B) and Cu,ZnSOD expression between ipsilateral and contralateral oviducts (C). a–cColumns without a common superscript differed (P < 0.05).
(Fig. 4). In the isthmus, isthmic–ampullary junction and ampulla, Cu,ZnSOD was found in each cell type of the oviduct, but the staining intensities varied among cell types of each section (Fig. 4). Indeed, in the isthmus Cu,ZnSOD was mostly detected in the lamina propria, whereas in the isthmic–ampullary junction, the staining was homogenous. Interestingly, in the ampulla, Cu,ZnSOD was mostly observed in the epithelial cells. The MnSOD was also observed in all cell types in each section of the oviduct. Specifically, in the isthmus and isthmic–ampullary junction, the staining intensity was stronger in the epithelial cells and in the lamina propria than in the smooth muscle cells. In the ampulla section, MnSOD was mostly observed in the epithelial cells, as seen with Cu,ZnSOD (Fig. 4).
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Fig. 3. Mean (S.E.M.) MnSOD protein expression (densitometry of western blots) in the isthmus, isthmic–ampullary junction and ampulla of the bovine oviduct. a–cColumns without a common superscript differed (P < 0.05).
4. Discussion In the present study, Cu,ZnSOD and MnSOD proteins were least expressed in the isthmus; this was consistent with our previous findings that total SOD activity was lower in the oviductal fluid of the isthmus than in the ampulla [23]. Based on in vitro studies, O2 was important in capacitation of cryopreserved bovine sperm [6,11]. In cattle, the isthmus is considered a sperm reservoir and the site of capacitation [1]. Perhaps
low levels of Cu,ZnSOD and MnSOD are needed to ensure that there is sufficient superoxide to allow capacitation to occur. The SODs dismute O2 to produce H2O2, which is metabolized by the glutathione peroxidase family (GPx) or by catalase [23,25,26]. That H2O2 decreases motility of bovine sperm in vitro [6] probably accounts for high catalase and total GPx activities that detoxify H2O2 in oviductal fluid, thereby counterbalancing the activity of SODs [23]. Moreover, we detected the mRNA of the extracellular GPx (eGPx or GPx-3) mainly in the isthmus, a known sperm reservoir [23]. It was noteworthy that the protein expression of Cu,ZnSOD in the present study seemed similar to the profile of expression of eNOS in our previous report [24]. Indeed, the highest expression for both proteins was in the ampulla. In addition, both of these proteins were located in the epithelium, in close proximity to gametes. This was noteworthy, as O2 can react with NO, produced by NOS, to generate peroxynitrite (ONOO ), a strong oxidant [15]. Thus, Cu,ZnSOD could protect against the formation of toxic ONOO , which was already detected in vivo as nitrotyrosine residues along the oviduct [24].
Fig. 4. Immunolocalization of Cu,ZnSOD and MnSOD in various sections of the bovine oviduct. Three animals were analyzed at each stage of the estrous cycle. Since no difference in relative staining was observed throughout the estrous cycle, only the three sections are shown. The red color represents the positive results. The counterstain was Mayer’s hematoxylin. Final magnification 200 and 400.
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In the present study, neither the CL nor the dominant follicle affected MnSOD protein expression during the estrous cycle. However, there was a significant difference in Cu,ZnSOD expression between the ipsilateral and contralateral oviduct and throughout the estrous cycle in contrast to what we have found earlier for Cu,ZnSOD mRNA expression [23]. Previous studies have shown that the CL or the dominant follicle had an influence on the expression of many genes [24,25,27]. Indeed, more than 27 genes were more expressed in the ispsilateral than in the contralateral (to the CL) oviduct in bovine epithelial cells [27], whereas the expression of some antioxidant genes like catalase were unaffected [23]. However, the latter studies did not include the analysis of SODs protein expression. In the present study, Cu,ZnSOD and MnSOD were present in all cell types in each section of the oviduct; only staining intensities varied among cell types among the three sections of the oviduct. Of note, the oviduct is composed of epithelial cells, lamina propria and smooth muscle cells [28]. There is little information regarding the presence of SODs in the oviducts. The MnSOD protein was in the epithelium of human fallopian tubes and Cu,ZnSOD protein was detected in the epithelial cells of the isthmus, but not in the ampulla of the oviduct, in contrast to what we have reported in bovine [21]. In the rat, MnSOD mRNA was not detected in the epithelial cells of the oviduct [22]. In summary, both Cu,ZnSOD and MnSOD were specifically expressed along the oviduct, and there were differences among stages of the cycle in the expression of Cu,ZnSOD, apparently due to direct or indirect actions of hormones. In general, levels of SODs increased from the isthmus to the ampulla, and specifically towards the end of the estrous cycle in diestrus and proestrus for Cu,ZnSOD. The proximity of dominant follicle appeared to enhance the expression of Cu,ZnSOD. The regulation of the SODs can directly affect O2 levels and hence, its role on sperm capacitation and hyperactivation. Moreover, due to the interaction of O2 with other ROS (e.g. NO and H2O2), the SODs apparently have important roles in the regulation of physiological and deleterious effects of ROS in reproductive events. Acknowledgements This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (NSERC grant #238570-02 to J.-F.B.). J.-F. B. was the recipient of a Canadian Institutes of Health Research
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(CIHR), Institute of Aging, New Investigators award. M.R. was the recipient of a scholarship from Fonds de Recherche sur la Nature et les Technologies (FQRNT). The authors thank Ms. Vale´rie Jomphe (Laval University, Statistical service) for assistance with statistical analysis. References [1] Lefebvre R, Chenoweth PJ, Drost M, LeClear CT, MacCubbin M, Dutton JT, et al. Characterization of the oviductal sperm reservoir in cattle. Biol Reprod 1995;53:1066–74. [2] Talbot P, Geiske C, Knoll M. Oocyte pickup by the mammalian oviduct. Mol Biol Cell 1999;10:5–8. [3] Parrish JJ, Susko-Parrish JL, Handrow RR, Sims MM, First NL. Capacitation of bovine spermatozoa by oviduct fluid. Biol Reprod 1989;40:1020–5. [4] Blondin P, Coenen K, Sirard MA. The impact of reactive oxygen species on bovine sperm fertilizing ability and oocyte maturation. J Androl 1997;18:454–60. [5] O’Flaherty C, Breininger E, Beorlegui N, Beconi MT. Acrosome reaction in bovine spermatozoa: role of reactive oxygen species and lactate dehydrogenase C4. Biochim Biophys Acta 2005; 1726:96–101. [6] O’Flaherty CM, Beorlegui NB, Beconi MT. Reactive oxygen species requirements for bovine sperm capacitation and acrosome reaction. Theriogenology 1999;52:289–301. [7] Riley JC, Behrman HR. Oxygen radicals and reactive oxygen species in reproduction. Proc Soc Exp Biol Med 1991;198: 781–91. [8] Sengoku K, Tamate K, Yoshida T, Takaoka Y, Miyamoto T, Ishikawa M. Effects of low concentrations of nitric oxide on the zona pellucida binding ability of human spermatozoa. Fertil Steril 1998;69:522–7. [9] Aitken RJ, Clarkson JS, Fishel S. Generation of reactive oxygen species, lipid peroxidation, and human sperm function. Biol Reprod 1989;41:183–97. [10] de Lamirande E, Jiang H, Zini A, Kodama H, Gagnon C. Reactive oxygen species and sperm physiology. Rev Reprod 1997;2:48–54. [11] O’Flaherty C, Beorlegui N, Beconi MT. Participation of superoxide anion in the capacitation of cryopreserved bovine sperm. Int J Androl 2003;26:109–14. [12] Ozawa M, Nagai T, Fahrudin M, Karja NW, Kaneko H, Noguchi J, et al. Addition of glutathione or thioredoxin to culture medium reduces intracellular redox status of porcine IVM/IVF embryos, resulting in improved development to the blastocyst stage. Mol Reprod Dev 2006;73:998–1007. [13] Velez-Pardo C, Morales AT, Del Rio MJ, Olivera-Angel M. Endogenously generated hydrogen peroxide induces apoptosis via mitochondrial damage independent of NF-kappaB and p53 activation in bovine embryos. Theriogenology 2007;67: 1285–96. [14] Halliwell B, Gutteridge JM. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem J 1984;219:1–14. [15] Chaudiere J, Ferrari-Iliou R. Intracellular antioxidants: from chemical to biochemical mechanisms. Food Chem Toxicol 1999;37:949–62. [16] Agarwal A, Gupta S, Sharma RK. Role of oxidative stress in female reproduction. Reprod Biol Endocrinol 2005;3:28.
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[17] Zelko IN, Mariani TJ, Folz RJ. Superoxide dismutase multigene family: a comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression. Free Radic Biol Med 2002;33:337–49. [18] Fridovich I. Superoxide dismutases. Annu Rev Biochem 1975; 44:147–59. [19] Ho YS, Gargano M, Cao J, Bronson RT, Heimler I, Hutz RJ. Reduced fertility in female mice lacking copper-zinc superoxide dismutase. J Biol Chem 1998;273:7765–9. [20] El Mouatassim S, Guerin P, Menezo Y. Mammalian oviduct and protection against free oxygen radicals: expression of genes encoding antioxidant enzymes in human and mouse. Eur J Obstet Gynecol Reprod Biol 2000;89:1–6. [21] Tamate K, Sengoku K, Ishikawa M. The role of superoxide dismutase in the human ovary and fallopian tube. J Obstet Gynaecol 1995;21:401–9. [22] Nomura T, Sasaki J, Mori H, Sato EF, Watanabe S, Kanda S, et al. Expression of manganese superoxide dismutase mRNA in reproductive organs during the ovulatory process and the estrous cycle of the rat. Histochem Cell Biol 1996;105:1–6.
[23] Lapointe J, Bilodeau JF. Antioxidant defenses are modulated in the cow oviduct during the estrous cycle. Biol Reprod 2003;68: 1157–64. [24] Lapointe J, Roy M, St-Pierre I, Kimmins S, Gauvreau D, MacLaren LA, et al. Hormonal and spatial regulation of nitric oxide synthases (NOS) (neuronal NOS, inducible NOS, and endothelial NOS) in the oviducts. Endocrinology 2006;147:5600–10. [25] Lapointe J, Kimmins S, Maclaren LA, Bilodeau JF. Estrogen selectively up-regulates the phospholipid hydroperoxide glutathione peroxidase in the oviducts. Endocrinology 2005;146: 2583–92. [26] Mates JM, Sanchez-Jimenez F. Antioxidant enzymes and their implications in pathophysiologic processes. Front Biosci 1999; 4:D339–45. [27] Bauersachs S, Blum H, Mallok S, Wenigerkind H, Rief S, Prelle K, et al. Regulation of ipsilateral and contralateral bovine oviduct epithelial cell function in the postovulation period: a transcriptomics approach. Biol Reprod 2003;68:1170–7. [28] Ellington JE. The bovine oviduct and its role in reproduction: a review of the literature. Cornell Vet 1991;81:313–28.
Theriogenology 70 (2008) 836–842 www.theriojournal.com
Expression of superoxide dismutases in the bovine oviduct during the estrous cycle M. Roy a,b, D. Gauvreau a,b, J.-F. Bilodeau a,b,c,* a
Unite´ de Recherche en Ontoge´nie et Reproduction, Centre de Recherche du Centre Hospitalier de l’Universite´ Laval, Que´bec, Canada G1V 4G2 b Centre de Recherche en Biologie de la Reproduction (CRBR), Universite´ Laval, Que´bec, Canada GIV 4G2 c De´partement d’Obste´trique et Gyne´cologie, Faculte´ de Me´decine, Universite´ Laval, Que´bec, Canada G1K 7P4 Received 28 September 2007; received in revised form 30 January 2008; accepted 9 May 2008
Abstract The superoxide dismutases (SODs) are first-line enzymatic antioxidants that dismute superoxide anion (O2 ) to produce hydrogen peroxide (H2O2). The primary objective was to characterize, by western blot analysis, the expression of two SODs, the cytosolic (Cu,ZnSOD or SOD1) and the mitochondrial (MnSOD or SOD2) forms in three sections of the oviduct, i.e. isthmus (I), ishtmic–ampullary junction (IA), and ampulla (A), during the estrous cycle. The Cu,ZnSOD and MnSOD proteins were mostly expressed in the ampulla (I < IA < A; P < 0.0093). Expression of Cu,ZnSOD was lowest during metestrus (P < 0.0041) and it was mostly expressed in the oviduct contralateral to the CL (P = 0.0019). The expression of MnSOD was consistent throughout the cycle. Based on immunohistochemistry, the SODs were present in all cell types of the oviduct, with variations among oviductal sections. We inferred that the expression of Cu,ZnSOD was influenced by the hormonal milieu. Furthermore, variations among oviduct sections in protein expression profile of SODs suggested they have an important role in preserving and capacitating sperm. # 2008 Elsevier Inc. All rights reserved. Keywords: Oviduct; Superoxide dismutase; Reactive oxygen species; Estrous cycle; Cow
1. Introduction In cattle and other species, the oviducts play a critical role in early reproductive processes, including gamete maturation, fertilization, and early embryo development [1–3]. These processes require an appropriate balance between reactive oxygen species (ROS) and antioxidants, as shown by many in vivo and in vitro studies
* Corresponding author at: De´partement d’Obste´trique et Gyne´cologie, Faculte´ de Me´decine, Universite´ Laval, Que´bec, Canada. Tel.: +1 418 525 4444x46153; fax: +1 418 654 2765. E-mail address: [email protected] (J.F. Bilodeau). 0093-691X/$ – see front matter # 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2008.05.042
[4–6]. Reactive oxygen species (ROS) exist in several forms (O2 , H2O2, OH, NO, ONOO ) and can be either beneficial or detrimental to reproductive events [4,6,7]. Indeed, low levels of ROS can be beneficial in promoting the binding of sperm to the zona pellucida [8–10]. In cattle, the superoxide anion (O2 ) and hydrogen peroxide (H2O2) are essential for sperm capacitation and the acrosome reaction, respectively, in vitro [6,11]. However, high hydrogen peroxide (H2O2) concentrations reduced bull sperm motility in vitro [6] and may impair fertilization and embryo development [12,13]. Furthermore, interaction of O2 and H2O2 can generate the hydroxyl radical (OH), one of the most powerful ROS, which can damage lipids, proteins and nucleic acids [14]. The H2O2 can also form OH via the
M. Roy et al. / Theriogenology 70 (2008) 836–842
Fenton reaction in the presence of transition metals (e.g. iron) [14]. The O2 can also react with nitric oxide (NO) to form peroxynitrite (ONOO ), a powerful oxidant, which reduces the bioavailability of NO [15]. Production of ROS is controlled by various antioxidants. Nonenzymatic antioxidants include glutathione (GSH), a-tocopherol, b-carotene, ascorbate, and vitamin C [15,16]. The main antioxidant enzymes include superoxide dismutases (SODs) which neutralize superoxide anion (O2 ), catalases which scavenge hydrogen peroxide (H2O2), and glutathione peroxidases (GPx) which detoxify H2O2 and lipid hydroperoxides (ROOH) [15]. The SODs exist in three forms: the cytosolic (Cu,ZnSOD or SOD1), the mitochondrial (MnSOD or SOD2) and the extracellular form (ECSOD or SOD3) [17]. The SODs are the first line of defense against ROS in dismuting two superoxide anions (O2 ) into one hydrogen peroxide (H2O2) [18], which can be metabolized by either catalases or GPx [15]. Thus, SODs are important defense enzymes against O2 , preventing the formation of OH and ONOO . That Cu,ZnSOD female knock-out mice had reduced fertility implicates SODs in reproduction in the female [19]. Although the balance between antioxidants and ROS is critically important for reproductive success, few in vivo studies have characterized antioxidants in the oviduct, especially SODs in cattle. In mammalian oviducts, Cu,ZnSOD and MnSOD were best characterized in the mouse, rat, rabbit, and human [20–22]. We previously reported that antioxidant enzymes such as glutathione peroxidases (GPx) and prooxidant enzymes such as nitric oxide synthases (NOS) that produce NO were spatially and temporally regulated in the bovine oviduct during the estrous cycle [23,24]. However, the specific protein regulation of superoxide dismutases (SODs) remains to be determined in the cow. The primary objective of the present study was to determine the expression of Cu,ZnSOD and MnSOD proteins during the estrous cycle in three sections of each oviduct (both ipsilateral and contralateral to the CL). The secondary objective was to determine cellular localization of these SODs. 2. Materials and methods
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tion of the ovaries, the tracts were divided according to stage of the cycle (3): metestrus (Days 0–3), diestrus (Days 10–15), and proestrus (Days 18–21). The locations of the current and previous CL were determined; only tracts with these CL on opposite ovaries were used. Furthermore, oviducts ipsilateral and contralateral to the current CL were studied separately. Oviducts were dissected (to remove blood vessels, ligaments, and other tissues) and cut in three sections of 2 cm: the isthmus was adjacent to the uterotubal junction; the isthmic–ampullary junction was the midpoint of the oviduct; and the ampulla was adjacent to the fimbria. Each section were frozen in liquid nitrogen and subsequently kept at 86 8C until analyzed. For each stage of the estrous cycle, five pairs of oviducts (ipsilateral and contralateral to the CL) were used (three sections per oviduct). 2.2. Western blot analysis A sample (0.5 cm) from each section of the oviduct was mixed in ice-cold PBS (Invitrogen, Carlsbad, CA, USA) containing a protease inhibitor cocktail (1 mM EDTA, 0.5 mg/mL Leupeptin, 1.4 mg/mL Pepstatin A, 70 mg/mL PMSF; Boehringer Mannheim, Laval, QC, Canada) and homogenized with an Ultra Turrax T25 (IKA Works, Inc., Wilmington, NC, USA). Homogenates were mixed in Laemmli sample buffer (5% (v:v) 2-mercaptoethanol) and boiled for 10 min [23,24]. The quantity of proteins was determined using a BCA protein assay, according to the manufacturer’s instructions (Pierce, Rockford, IL, USA). An aliquot (30 mg) of proteins from each oviductal section were loaded on a 15% polyacrylamide-SDS gel. After electrophoresis, the proteins were transferred to a nitrocellulose membrane (0.2 mm; Bio-Rad Laboratories, Montre´al, QC, Canada). The membranes were incubated for 1 h at room temperature with 5% dry milk in TBS-T, then incubated for 2 h at room temperature with primary rabbit polyclonal antibodies against bovine SOD1 (dilution 1:10,000) [23] and SOD2 (dilution 1:5000) (GeneTex, San Antonio, TX, USA). The signal was revealed using ECL Trade Mark (GE Healthcare Bio-Sciences, Baie d’Urfe´ QC, Canada) and a densitometry was done with an Alpha Imager 2000 (Alpha Innotech, San Leandro, CA, USA).
2.1. Oviducts 2.3. Immunohistochemistry Genital tracts were recovered from Holstein cows/ heifers at an abattoir and transported on ice to the laboratory within 4 h after death. Tracts with visible anomalies were eliminated. Based on visual examina-
Immunohistochemistry was performed as previously described [24]. Briefly, immediately after dissection, a portion of each section of the oviduct (isthmus, isthmic–
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ampullary junction and ampulla) was fixed overnight in 4% paraformaldehyde. Optimum cutting temperature medium (OCT; Canemco, St. Laurent, QC, Canada) was used to embed the sections before freezing in liquid nitrogen. Samples were cryosectioned (eight sections/ block, each section 8 mm thick) with a cryotome (Shandon, Pittsburgh, PA, USA), and spread on a microscope slide. Vectastain Elite ABC kit (Vector Laboratories Inc., Burlingame, CA, USA) was used, in accordance with the manufacturer’s instructions, to perform immunohistochemistry analysis. All incubations were done at room temperature unless otherwise stated. The slides were incubated for 30 min in 3% H2O2 (v:v) with methanol to inactivate endogenous peroxidases, incubated for 1 h with 10% goat serum (Sigma–Aldrich, Oakville, ON, Canada) in PBS to block non-specific binding, and then incubated overnight at 4 8C with primary antibody. The primary antibodies were rabbit polyclonal antibody against bovine SOD2 (dilution 1:1000; GeneTex) and SOD1 (dilution 1:1000; Stressgen, Victoria, BC, Canada). A negative control was also done with non-specific IgGs. After washing in PBS, the slides were incubated using a biotinylated goat anti-rabbit IgG (Vector Laboratories) for 1 h. Finally, the slides were incubated 30 min with the ABC elite reagent and with 3-amino-9-ethylcarbazole until the immunostaining was revealed. Mayer’s hematoxylin (Sigma–Aldrich) was used as a counterstain, An Axioskop 2 Plus microscope (Zeiss, Toronto, ON, Canada) coupled to a digital camera was used to record color images. The images from microscopy were analyzed subjectively for relative protein expression. 2.4. Statistical analysis Densitometry values obtained from the western blot analysis were normalized to b-actin. To study the
effects of the factors: stage of the estrous cycle, oviduct side and oviduct section on the gene expression, a threeway analysis of variance with repeated measures was used. To account for possible correlations between observations made on the same cow, we considered the factors: oviduct side and section, as repeated measures. The MIXED procedure of SAS software (SAS Institute Inc. 2005. SAS OnlineDoc1 9.1.3. Cary, NC, USA) was used with a repeated statement and the covariance structure that minimizes the Akaike criterion. The method of Kenward–Roger was used to calculate the degrees of freedom. The normality and the homogeneity of the variance assumptions were met. Pairwise comparisons were then made using protected Fisher LSD (least significant difference). For all analyses, P < 0.05 was considered significant. 3. Results Western blot analyses were performed to determine the protein expression of Cu,ZnSOD and MnSOD; one replicate is shown (Fig. 1). Both Cu,ZnSOD and MnSOD were detected in all three oviductal segments. The expression of Cu,ZnSOD varied significantly throughout the estrous cycle, along the oviduct and in the ispilateral and contralateral oviduct (Table 1), whereas MnSOD expression varied only in the three sections of the oviduct (Table 2). There were no interactions between the three factors studied (stage of the estrous cycle, side relative to the CL and section of the oviducts) for both Cu,ZnSOD and MnSOD (Tables 1 and 2). The expression for both Cu,ZnSOD and MnSOD differed significantly along the oviduct (Figs. 2A and 3). The Cu,ZnSOD levels were higher in the ampulla than in the isthmic–ampullary junction by 27% (P = 0.0001) and the isthmus by 56% (P < 0.0001), respectively. The
Fig. 1. Cu,ZnSOD, MnSOD protein levels in the isthmus (I), isthmic–ampullary junction (I–A) and ampulla (A) regions of the bovine oviduct (ipsilateral and contralateral to the CL) of cattle at metestrus, diestrus, and proestrus; (C): positive control. The loading control was b-actin. These results are a single replicate (tissues derived from one reproductive tract) and were representative of all five replicates.
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Table 1 Analysis of variance for Cu,ZnSOD protein expression in the bovine oviduct (summary of fixed-effects statistics) Effect
DFN
DFD
F-value
P-value
Stage Side Stage side Section Stage section Side section Stage side section
2 1 2 2 4 2 4
21.9 11.1 11.1 20 20 34.6 34.6
10.43 16.29 0.09 31.12 0.19 1.56 0.46
0.0007 0.0019 0.9135 <0.0001 0.9401 0.2246 0.7667
Stage: stage of estrous cycle (metestrus, diestrus, proestrus); Side: contralateral or ipsilateral oviduct (relative to the CL); Section: section of the oviduct (isthmus, isthmic–ampullary junction, ampulla); DFN: degrees of freedom in numerator; DFD: degrees of freedom in denominator.
expression for Cu,ZnSOD was also higher in the isthmic–ampullary junction than the isthmus (P = 0.0049; Fig. 2A). Similarly, MnSOD expression was higher in the ampulla than in the isthmic–ampullary junction (+23%; P = 0.0093) and the isthmus (+64%; P < 0.0001), while being higher in the isthmic– ampullary junction than in the isthmus (P = 0.0064; Fig. 3). The expression for Cu,ZnSOD also varied throughout the estrous cycle (Fig. 2B). Indeed the Cu,ZnSOD expression was lower in metestrus than in diestrus ( 20%; P = 0.0041) and proestrus ( 26%; P = 0.0002). However, there was no difference between diestrus and proestrus stages (P = 0.2388). Furthermore, the expression for Cu,ZnSOD was higher in the contralateral than in the ipsilateral oviduct (+18%; P = 0.0019) Fig. 2C). In the absence of relative staining (immunohistochemistry) differences between cell types for the SODs during the estrous cycle, we showed only one stage Table 2 Analysis of variance for MnSOD protein expression in the bovine oviduct (summary of fixed-effects statistics) Effect
DFN
DFD
F-value
P-value
Stage Side Stage side Section Stage section Side section Stage side section
2 1 2 2 4 2 4
17.3 18.1 18.1 29.4 29.4 28.3 28.3
0.04 3.21 2.24 16.38 0.51 0.21 0.20
0.9644 0.0899 0.1351 <0.0001 0.7286 0.8101 0.9362
Stage: stage of estrous cycle (metestrus, diestrus, proestrus); Side: contralateral or ipsilateral oviduct (relative to the CL); Section: section of the oviduct (isthmus, isthmic–ampullary junction, ampulla); DFN: degrees of freedom in numerator; DFD: degrees of freedom in denominator.
Fig. 2. Mean (S.E.M.) Cu,ZnSOD protein expression (densitometry of western blots) in bovine oviducts. The expression of Cu,ZnSOD in the isthmus, isthmic–ampullary junction and ampulla (A). Cu,ZnSOD expression during the estrous cycle (metestrus, diestrus and proestrus; (B) and Cu,ZnSOD expression between ipsilateral and contralateral oviducts (C). a–cColumns without a common superscript differed (P < 0.05).
(Fig. 4). In the isthmus, isthmic–ampullary junction and ampulla, Cu,ZnSOD was found in each cell type of the oviduct, but the staining intensities varied among cell types of each section (Fig. 4). Indeed, in the isthmus Cu,ZnSOD was mostly detected in the lamina propria, whereas in the isthmic–ampullary junction, the staining was homogenous. Interestingly, in the ampulla, Cu,ZnSOD was mostly observed in the epithelial cells. The MnSOD was also observed in all cell types in each section of the oviduct. Specifically, in the isthmus and isthmic–ampullary junction, the staining intensity was stronger in the epithelial cells and in the lamina propria than in the smooth muscle cells. In the ampulla section, MnSOD was mostly observed in the epithelial cells, as seen with Cu,ZnSOD (Fig. 4).
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Fig. 3. Mean (S.E.M.) MnSOD protein expression (densitometry of western blots) in the isthmus, isthmic–ampullary junction and ampulla of the bovine oviduct. a–cColumns without a common superscript differed (P < 0.05).
4. Discussion In the present study, Cu,ZnSOD and MnSOD proteins were least expressed in the isthmus; this was consistent with our previous findings that total SOD activity was lower in the oviductal fluid of the isthmus than in the ampulla [23]. Based on in vitro studies, O2 was important in capacitation of cryopreserved bovine sperm [6,11]. In cattle, the isthmus is considered a sperm reservoir and the site of capacitation [1]. Perhaps
low levels of Cu,ZnSOD and MnSOD are needed to ensure that there is sufficient superoxide to allow capacitation to occur. The SODs dismute O2 to produce H2O2, which is metabolized by the glutathione peroxidase family (GPx) or by catalase [23,25,26]. That H2O2 decreases motility of bovine sperm in vitro [6] probably accounts for high catalase and total GPx activities that detoxify H2O2 in oviductal fluid, thereby counterbalancing the activity of SODs [23]. Moreover, we detected the mRNA of the extracellular GPx (eGPx or GPx-3) mainly in the isthmus, a known sperm reservoir [23]. It was noteworthy that the protein expression of Cu,ZnSOD in the present study seemed similar to the profile of expression of eNOS in our previous report [24]. Indeed, the highest expression for both proteins was in the ampulla. In addition, both of these proteins were located in the epithelium, in close proximity to gametes. This was noteworthy, as O2 can react with NO, produced by NOS, to generate peroxynitrite (ONOO ), a strong oxidant [15]. Thus, Cu,ZnSOD could protect against the formation of toxic ONOO , which was already detected in vivo as nitrotyrosine residues along the oviduct [24].
Fig. 4. Immunolocalization of Cu,ZnSOD and MnSOD in various sections of the bovine oviduct. Three animals were analyzed at each stage of the estrous cycle. Since no difference in relative staining was observed throughout the estrous cycle, only the three sections are shown. The red color represents the positive results. The counterstain was Mayer’s hematoxylin. Final magnification 200 and 400.
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In the present study, neither the CL nor the dominant follicle affected MnSOD protein expression during the estrous cycle. However, there was a significant difference in Cu,ZnSOD expression between the ipsilateral and contralateral oviduct and throughout the estrous cycle in contrast to what we have found earlier for Cu,ZnSOD mRNA expression [23]. Previous studies have shown that the CL or the dominant follicle had an influence on the expression of many genes [24,25,27]. Indeed, more than 27 genes were more expressed in the ispsilateral than in the contralateral (to the CL) oviduct in bovine epithelial cells [27], whereas the expression of some antioxidant genes like catalase were unaffected [23]. However, the latter studies did not include the analysis of SODs protein expression. In the present study, Cu,ZnSOD and MnSOD were present in all cell types in each section of the oviduct; only staining intensities varied among cell types among the three sections of the oviduct. Of note, the oviduct is composed of epithelial cells, lamina propria and smooth muscle cells [28]. There is little information regarding the presence of SODs in the oviducts. The MnSOD protein was in the epithelium of human fallopian tubes and Cu,ZnSOD protein was detected in the epithelial cells of the isthmus, but not in the ampulla of the oviduct, in contrast to what we have reported in bovine [21]. In the rat, MnSOD mRNA was not detected in the epithelial cells of the oviduct [22]. In summary, both Cu,ZnSOD and MnSOD were specifically expressed along the oviduct, and there were differences among stages of the cycle in the expression of Cu,ZnSOD, apparently due to direct or indirect actions of hormones. In general, levels of SODs increased from the isthmus to the ampulla, and specifically towards the end of the estrous cycle in diestrus and proestrus for Cu,ZnSOD. The proximity of dominant follicle appeared to enhance the expression of Cu,ZnSOD. The regulation of the SODs can directly affect O2 levels and hence, its role on sperm capacitation and hyperactivation. Moreover, due to the interaction of O2 with other ROS (e.g. NO and H2O2), the SODs apparently have important roles in the regulation of physiological and deleterious effects of ROS in reproductive events. Acknowledgements This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (NSERC grant #238570-02 to J.-F.B.). J.-F. B. was the recipient of a Canadian Institutes of Health Research
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(CIHR), Institute of Aging, New Investigators award. M.R. was the recipient of a scholarship from Fonds de Recherche sur la Nature et les Technologies (FQRNT). The authors thank Ms. Vale´rie Jomphe (Laval University, Statistical service) for assistance with statistical analysis. References [1] Lefebvre R, Chenoweth PJ, Drost M, LeClear CT, MacCubbin M, Dutton JT, et al. Characterization of the oviductal sperm reservoir in cattle. Biol Reprod 1995;53:1066–74. [2] Talbot P, Geiske C, Knoll M. Oocyte pickup by the mammalian oviduct. Mol Biol Cell 1999;10:5–8. [3] Parrish JJ, Susko-Parrish JL, Handrow RR, Sims MM, First NL. Capacitation of bovine spermatozoa by oviduct fluid. Biol Reprod 1989;40:1020–5. [4] Blondin P, Coenen K, Sirard MA. The impact of reactive oxygen species on bovine sperm fertilizing ability and oocyte maturation. J Androl 1997;18:454–60. [5] O’Flaherty C, Breininger E, Beorlegui N, Beconi MT. Acrosome reaction in bovine spermatozoa: role of reactive oxygen species and lactate dehydrogenase C4. Biochim Biophys Acta 2005; 1726:96–101. [6] O’Flaherty CM, Beorlegui NB, Beconi MT. Reactive oxygen species requirements for bovine sperm capacitation and acrosome reaction. Theriogenology 1999;52:289–301. [7] Riley JC, Behrman HR. Oxygen radicals and reactive oxygen species in reproduction. Proc Soc Exp Biol Med 1991;198: 781–91. [8] Sengoku K, Tamate K, Yoshida T, Takaoka Y, Miyamoto T, Ishikawa M. Effects of low concentrations of nitric oxide on the zona pellucida binding ability of human spermatozoa. Fertil Steril 1998;69:522–7. [9] Aitken RJ, Clarkson JS, Fishel S. Generation of reactive oxygen species, lipid peroxidation, and human sperm function. Biol Reprod 1989;41:183–97. [10] de Lamirande E, Jiang H, Zini A, Kodama H, Gagnon C. Reactive oxygen species and sperm physiology. Rev Reprod 1997;2:48–54. [11] O’Flaherty C, Beorlegui N, Beconi MT. Participation of superoxide anion in the capacitation of cryopreserved bovine sperm. Int J Androl 2003;26:109–14. [12] Ozawa M, Nagai T, Fahrudin M, Karja NW, Kaneko H, Noguchi J, et al. Addition of glutathione or thioredoxin to culture medium reduces intracellular redox status of porcine IVM/IVF embryos, resulting in improved development to the blastocyst stage. Mol Reprod Dev 2006;73:998–1007. [13] Velez-Pardo C, Morales AT, Del Rio MJ, Olivera-Angel M. Endogenously generated hydrogen peroxide induces apoptosis via mitochondrial damage independent of NF-kappaB and p53 activation in bovine embryos. Theriogenology 2007;67: 1285–96. [14] Halliwell B, Gutteridge JM. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem J 1984;219:1–14. [15] Chaudiere J, Ferrari-Iliou R. Intracellular antioxidants: from chemical to biochemical mechanisms. Food Chem Toxicol 1999;37:949–62. [16] Agarwal A, Gupta S, Sharma RK. Role of oxidative stress in female reproduction. Reprod Biol Endocrinol 2005;3:28.
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