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Different enzymatic antioxidative pathways operate within the sheep caruncular and intercaruncular endometrium throughout the estrous cycle and early pregnancy
Theriogenology 99 (2017) 111e118
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Different enzymatic antioxidative pathways operate within the sheep caruncular and intercaruncular endometrium throughout the estrous cycle and early pregnancy K.H. Al-Gubory a, *, P. Faure b, C. Garrel b UMR BDR, INRA, ENVA, Universit e Paris Saclay, 78350, Jouy en Josas, France Unit e de Biochimie Hormonale - Nutritionnelle, Centre Hospitalier Universitaire de Grenoble, D epartement de Biologie - Toxicologie - Pharmacologie, 38043 Grenoble cedex 9, France a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 June 2016 Received in revised form 3 May 2017 Accepted 22 May 2017 Available online 1 June 2017
There has been a growing interest in the role played by antioxidant enzymes in the regulation of endometrial function in mammals. However, little is known about enzymatic antioxidative pathways involved in conditioning the cyclic and early pregnant endometrium for conceptus attachment and implantation in domestic ruminants. We aimed to investigate changes in activities of superoxide dismutase 1 and 2 (SOD1, SOD2), glutathione peroxidase (GPX), glutathione reductase (GR) and catalase (CAT) in sheep caruncles (CAR) and intercaruncles (ICAR) endometrial tissues of cyclic and early pregnant ewes. Irrespective of day of cycle or pregnancy, CAR demonstrated higher activities of SOD1 and SOD2 than in ICAR. On day 12 of the estrous cycle, ICAR demonstrated higher activity of GPX and GR than in CAR tissues. On days 12 and 16 the estrous cycle, ICAR demonstrated higher activity of CAT than in CAR. CAR demonstrated higher activity of GPX on day 18 than on days 4, 8, 12 and 16 of the estrous cycle. CAR demonstrated higher activity of CAT on day 18 than on days 4, 8, 12 and 16 of the estrous cycle. ICAR demonstrated higher activity of CAT on day 18 than on days 4, 8, and 16 of the estrous cycle. The activity of CAT in ICAR increased from days 4 and 8 to day 12 of the estrous cycle. The activity of SOD2 in CAR increased from day 16 to day 18 of pregnancy. On day 12 of pregnancy, CAR demonstrated higher activity of GPX than in ICAR. On day 16 of pregnancy, ICAR demonstrated higher activity of GPX than in CAR. The activity of GPX in ICAR increased from day 12 to day 16 of pregnancy. The activity of GPX in CAR increased from day 16 to day 18 of pregnancy. The activity of GR in CAR and ICAR increased from days 12 and 16 to day 18 of pregnancy. On days 16 and 18 of pregnancy, ICAR demonstrated higher activity of CAT than in CAR. The activity of CAT in CAR decreased from day 12 to days 16 and 18 of pregnancy. The activity of CAT in ICAR decreased from day 12 to day 16 of pregnancy and then increased from day 16 to day 18 of pregnancy. In conclusion, different antioxidant mechanisms operate within CAR and ICAR endometrium throughout the estrous cycle and during early pregnancy. This might be related to the different but important roles of CAR and ICAR endometrial tissues for the establishment of pregnancy. © 2017 Published by Elsevier Inc.
Keywords: Sheep Caruncular and intercaruncular endometrium Antioxidant enzymes Estrous cycle Early pregnancy
1. Introduction The control of reactive oxygen species (ROS) by several antioxidant enzymes plays important roles in folliculogenesis, oocyte maturation, endometrial function, implantation, embryogenesis,
* Corresponding author. Institut National de la Recherche Agronomique (INRA), partement de Physiologie Animale et Syste mes d’Elevage, UMR 1198 Biologie du De veloppement et de la Reproduction, 78352 Jouy-en-Josas cedex, France. De E-mail addresses: [email protected], [email protected] (K.H. AlGubory). http://dx.doi.org/10.1016/j.theriogenology.2017.05.017 0093-691X/© 2017 Published by Elsevier Inc.
and prenatal development [1,2]. Establishment of pregnancy is a dynamic process associated with significant physiological changes in the enzymatic antioxidative pathways, which are important for sheep reproductive organ functions, such as the corpus luteum [3e5] and endometrium [6,7]. The endometrium provides nutrients, including glucose, amino acids, glutathione (GSH), calcium, and potassium for the unattached conceptus (embryo and associated extraembryonic membranes), as well as a vital biological surface for attachment of the extraembryonic membranes [8]. Worthy of note is that defective uterine environment is associated with inappropriate secretion of ovarian steroids and/or antioxidant
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enzyme activities, which contributes to early pregnancy failure [9]. There has been a growing interest in the role played by antioxidant enzymes in the regulation of rodent [10], guinea pig [11], human [12e15] and sheep [6,7,16] endometrial function aimed at favoring conceptus attachment and successful establishment of pregnancy. However, little is known about activities of antioxidant enzymes in ruminant endometrium throughout the estrous cycle and during early pregnancy. The sheep is an appropriate small ruminant model to explore endometrial antioxidant machinery and its regulation [6,7,17]. The sheep uterus (see Fig. 1 A for supplementary information), like that of goat and cattle, has developed specialized endometrial tissues [18], namely caruncle (CAR) and intercaruncle (ICAR) areas (see Fig. 1 B for supplementary information). CAR and ICAR are histoarchitecturally different playing important roles in the establishment of pregnancy [19]. CAR areas are glandless dense stromal protuberances and lack glands (see Fig. 1C & E for supplementary information). They represent specialized sites of initial attachment of the extraembryonic membrane. ICAR areas contain branched glands (see Fig. 1 D & F for supplementary information) that produce histotroph nutrition to support early conceptus development and survival [20]. Both CAR and ICAR are covered by a simple luminal epithelium (see Fig. 1E and F for supplementary information). Therefore, we aimed to investigate changes in activities of the key antioxidant enzymes, superoxide dismutase 1 and 2 (SOD1, SOD2), glutathione peroxidase (GPX), glutathione reductase (GR) and catalase (CAT), in sheep CAR and ICAR endometrial tissues collected on days 4, 8, 12, 16 and 18 of the estrous cycle (experiment 1) and on days 12, 16 and 18 of pregnancy, corresponding to conceptus pre-attachment, attachment and early post-attachment periods, respectively (experiment 2).
(Paris, France). The minimum detectable concentration of P4 was 0.1 ng/ml and the intra-assay coefficient of variation was less than 10%. 2.3. Tissue collection All the ewes were slaughtered at a local abattoir in accordance with protocols approved by the local institutional animal use committee at the Institut National de la Recherche Agronomique (INRA, Jouy-en-Josas, France). In experiment 1, ewes were randomly allocated for slaughter on days 4 (n ¼ 4 ewes), 8 (n ¼ 4 ewes), 12 (n ¼ 4 ewes), 16 (n ¼ 4 ewes) and 18 (n ¼ 4 ewes) of the estrous cycle. In experiment 2, ewes were randomly allocated for slaughter on days 12 (n ¼ 4 ewes), 16 (n ¼ 4 ewes) and 18 (n ¼ 4 ewes) of pregnancy corresponding to conceptus pre-attachment, attachment and early post-implantation period, respectively. The stages of pregnancy were confirmed by the presence of one or two conceptus in uterine flushing [20]. The reproductive tract of each ewe was collected within 10 min of death, placed on crushed ice and transported to the laboratory. All subsequent manipulation of the tissue was performed at 4 C. The uterine horns were opened and all CAR and ICAR were separately dissected from the entire two uterine horns of each ewe, snap-frozen in liquid nitrogen and then stored at 80 C until processed for activities of the superoxide ( O 2 ) scavenging antioxidant enzymes, SOD1 and SOD2, and the hydrogen peroxide (H2O2) scavenging antioxidant enzymes, GPX, GR and CAT. For morphological analysis, endometrial tissues were fixed in freshly prepared 4% paraformaldehyde in phosphatebuffered saline (PBS, pH 7.4) and then processed for routine histology or histochemistry. 2.4. Conventional histology
2. Materials and methods 2.1. Animals and management The French Ministry of Agriculture approved all procedures relating to care and use of animals according to the French regulation for animal experimentation (authorization no 78e34). This alpes-du-Sud breed (18 study used multiparous ewes of the Pre months of age, n ¼ 35 ewes). All the ewes were treated for 14 days with intravaginal sponges containing 40 mg fluorogestone acetate (Intervet, Angers, France) to synchronize oestrous. Ewes assigned to the pregnant group were mated twice at the time of the synchronized oestrus with fertile rams of the same breed at an interval of 12 h. Throughout the experiment, the ewes were housed under conditions of natural day-length and temperature, fed straw and 2 Kg of hay per day par animal, and had free access to mineral licks and water.
Endometrial tissue was washed in PBS, dehydrated through a series of increasing concentrations of ethanol, cleared with butanol:ethanol (V:V), butanol, embedded in paraffin wax, and sectioned at 7 mm. Sections were deparaffinised in toluene, hydrated through decreasing concentrations of ethanol, washed in distilled water and stained with hematoxylin and eosin. 2.5. Histochemistry Endometrium sections (7 mm) were treated for permeabilization and coloration in 1.25 mg/ml FITC-Phalloidin (Sigma, St. Quentin Fallavier, France) and 0.05% saponin in PBS for 40 min. After washing in PBS, the sections were then incubated 10 min with 1 mg/ ml DAPI (Sigma) in PBS for nucleus localization. After washing in PBS, the sections were mounted in Vectashield mounting medium (Vector Laboratories, Peterborough, UK). Confocal microscopy observations were performed with a Zeiss confocal microscope.
2.2. Blood sampling and progesterone assay 2.6. Antioxidant enzyme activity assays In the present study daily plasma progesterone (P4) was determined during one entire estrous cycle of 3 ewes. In addition, plasma P4 was determined on days 4, 8, 12, 16 and 18 of the estrous cycle (experiment 1), and on days 12, 16 and 18 of pregnancy (experiment 2). Blood samples were taken from the jugular veins into evacuated heparinized tubes. After centrifugation (3000 g, 4 C) for 30 min, plasma was collected and stored at 20 C until assayed for P4 concentrations. The concentrations of P4 were determined by radioimmunoassay (RIA) in unextracted plasma as described [21] and validated for sheep jugular venous plasma with slight modifications [22]. Tritiated P4 (1,2,6, 7-3H-P4, sp act 88 Ci/ mmol) was obtained from Amersham (Bucks, UK), and a specific anti-progesterone antibody was obtained from the Pasteur Institute
The CAR or ICAR were homogenized in cold phosphate buffer (50 mM, pH 7.4) and then the homogenates were centrifuged at 15000 g for 30 min, 4 C. The resulting supernatant was used for determination of protein concentration [23]. A standard SOD assay [24] that had been validated for different sheep reproductive tissues [3,6,17,25] was used. Total SOD activity was measured using the pyrogallol assay based on the competition between pyrogallol oxidation by O 2 and O2 dismutation by SOD. Enzymatic activity of manganese-SOD (SOD2) was determined by assaying for SOD activity in the presence of sodium cyanide, which selectively inhibits copper/zinc-SOD (SOD1) but not SOD2 [26]. SOD1 activity was calculated by subtracting SOD2 activity from total SOD activity. The
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rate of auto-oxidation is taken from the increase in the absorbance at 420 nm. CAT activity was determined as described previously [27]. Activity was assayed by determining the rate of decomposition of H2O2 by CAT in potassium phosphate buffer (pH 7). The reaction rate was related to the amount of CAT present in the mixture. The rate of H2O2 decomposition by CAT was followed at 240 nm. GPX activity was measured using the glutathione reductase-NADPH method. Activity was determined by a coupled assay system [27] in which oxidation of GSH was coupled to NADPH oxidation catalyzed by GR. The rate of GSH oxidized by tertiary butyl hydroperoxide was evaluated by the decrease of NADPH in the presence of EDTA, excess GSH and GR. The rate of decrease in concentration of NADPH was recorded at 340 nm. GR activity was assayed by the standard method of NADPH oxidation. In this assay, GSSG is reduced to GSH by GR, which oxidizes NADPH to NADPþ. NADPH consumption was determined at 340 nm. 2.7. Statistical analysis Data was subjected to ANOVA and the Newman-Keuls multiple comparison test (PRISM Graph Pad version 4, Graph Pad Software, San Diego, CA). Data were presented as the mean ± SEM. The acceptable level of significance was set at P < 0.05. 3. Results 3.1. Progesterone concentrations throughout the estrous cycle The mean daily P4 concentrations in plasma of jugular blood collected throughout the estrous cycle are shown in Fig. 2 (see
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supplementary information). P4 concentration increased gradually from the mean basal value of 0.5 ± 0.3 ng/ml on day 0 (C0) to 2.4 ± 0.7 ng/ml and 4.3 ± 1.0 on day 4 (C4) and day 8 (C8) of the estrous cycle, respectively. The peak value of P4 concentration of 4.9 ± 1.0 ng/ml occurs on day 12 (C12) of the estrous cycle when the CL is fully mature. P4 concentration declined gradually by day 13 to reach a nadir of less than 1 ng/ml on days 16 (C16) and 18 (C18) when the CL was fully regressed. 3.2. Progesterone concentrations during the estrous cycle and early pregnancy The mean P4 concentrations in plasma of jugular blood collected on days 4 (C4), 8 (C8), 12 (C12), 16 (C16) and 18 (C18) of the estrous cycle and on days 12 (P12), 16 (P16) and 18 (P18) of pregnancy are shown in Fig. 1. Plasma concentrations of P4 on day 16 of the estrous cycle were lower than those on days 8 and 12 of the estrous cycle and on days 12, 16 and 18 of pregnancy. Plasma concentrations of P4 on day 18 of the estrous cycle were lower than those on days 4, 8, 12 and 16 of the estrous cycle and on days 12, 16 and 18 of pregnancy. 3.3. Enzymatic activities in CAR and ICAR during the estrous cycle Activities of TSOD, SOD1 and SOD2 in CAR and ICAR collected on days 4, 8, 12, 16 and 18 of the estrous cycle are shown in Fig. 2. Irrespective of day of cycle, CAR demonstrated higher activities of TSOD, SOD1 and SOD2 than in ICAR. The activities of TSOD, SOD1 and SOD2 in CAR or ICAR were not different between any stages of the estrous cycle examined. Activities of GPX, GR and CAT in CAR
Fig. 1. Concentrations of progesterone in plasma of jugular blood collected on days 4 (C4), 8 (C8), 12 (C12), 16 (C16) and 18 (C18) of the estrous cycle and on days 12 (P12), 16 (P16) and 18 (P18) of pregnancy. Values are shown as mean ± SEM for the number of ewes used (n ¼ 4 ewes per group). The acceptable level of significance was set at P < 0.05.
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Fig. 2. Activities of total superoxide dismutase (SOD), SOD1 and SOD2 in caruncle (CAR) and intercaruncle (ICAR) endometrium tissues derived from the entire two uterine horns of each ewe on days 4 (C4), 8 (C8), 12 (C12), 16 (C16) and 18 (C18) of the estrous cycle. Values are means ± SEM for the number of ewes used (n ¼ 4 ewes per group). The acceptable level of significance was set at P < 0.05.
and ICAR collected on days 4, 8, 12, 16 and 18 of the estrous cycle are shown in Fig. 3. On day 12 of the estrous cycle, ICAR demonstrated higher activity of GPX and GR than in CAR tissues. On days 12 and 16 the estrous cycle, ICAR demonstrated higher activity of CAT than in CAR. While GPX activity in ICAR did not alter throughout the estrous cycle, CAR demonstrated higher activity of GPX on day 18 than on days 4, 8, 12 and 16 of the estrous cycle. The activity of GR in CAR and ICAR was not different between any stages of the estrous cycle examined. CAR demonstrated higher activity of CAT on day 18 than on days 4, 8, 12 and 16 of the estrous cycle. ICAR demonstrated higher activity of CAT on day 18 than on days 4, 8, and 16 of the estrous cycle. The activity of CAT in ICAR increased from days 4 and 8 to day 12 of the estrous cycle. 3.4. Enzymatic activities in CAR and ICAR during early pregnancy Activities of TSOD, SOD1 and SOD2 in CAR and ICAR collected on days 12, 16 and 18 of pregnancy are shown in Fig. 4. Irrespective of the day of pregnancy, CAR demonstrated higher activity of TSOD, SOD1 and SOD2 than that in ICAR. The activities of TSOD and SOD1 in CAR or ICAR were not different between any stages of the
pregnancy examined. The activity of SOD2 in CAR or ICAR was not different between days 12 and 16 of pregnancy. The activity of SOD2 in CAR increased from day 16 to day 18 of pregnancy. Activities of GPX, GR and CAT in CAR and ICAR collected on days 12, 16 and 18 of pregnancy are shown in Fig. 5. On day 12 of pregnancy, CAR demonstrated higher activity of GPX than in ICAR. On day 16 of pregnancy, ICAR demonstrated higher activity of GPX than in CAR. The activity of GPX in ICAR increased from day 12 to day 16 of pregnancy. The activity of GPX in CAR increased from day 16 to day 18 of pregnancy. Irrespective of the day of pregnancy, the activity of GR was not different between CAR and ICAR. The activity of GR in CAR and ICAR increased from days 12 and 16 to day 18 of pregnancy. On days 16 and 18 of pregnancy, ICAR demonstrated higher activity of CAT than in CAR. The activity of CAT in CAR decreased from day 12 to days 16 and 18 of pregnancy. The activity of CAT in ICAR decreased from day 12 to day 16 of pregnancy and then increased from day 16 to day18 of pregnancy. 4. Discussion The present study showed that antioxidant enzyme activities in
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Fig. 3. Activities of glutathione peroxidase (GPX), glutathione reductase (GR) and catalase (CAT) in caruncle (CAR) and intercaruncle (ICAR) endometrium tissues derived from the entire two uterine horns of each ewe on days 4 (C4), 8 (C8), 12 (C12), 16 (C16) and 18 (C18) of the estrous cycle. Values are means ± SEM for the number of ewes used (n ¼ 4 ewes per group). The acceptable level of significance was set at P < 0.05.
CAR and ICAR areas of sheep endometrium undergo specific changes during the estrous cycle and early pregnancy that might have regulatory roles in uterine physiology, which could represent one of the underlying biochemical mechanisms conditioning endometrial receptivity. The SOD family is a ubiquitously distrib uted group of metalloenzymes. By catalyzing the conversion of O 2 into H2O2, SOD1 and SOD2 are believed to play a major role in the first line of defense against cellular oxidative damage and its subsequent effects on tissues of biological systems. SOD1 is a dimeric protein, essentially located in the cytoplasm [28], whereas manganese-containing SOD2 is a homotetrameric protein, located in the mitochondria [29]. There is ample evidence that SOD1 and SOD2 play major roles in the process of implantation and pregnancy outcome. Defects in conceptus implantation or premature death of the fetuses have been observed in mutant mice lacking SOD1 [30]. Neonatal lethality [31] or postnatal development restriction [32] has been reported in mutant mice lacking SOD2. In the spontaneous abortion, SOD1 activity in the human endometrium was lower than that in normal pregnancy, suggesting that SOD1 may contribute to the maintenance of pregnancy by pre venting the accumulation of O 2 [33].
Although CAR and ICAR have efficient O 2 scavenging antioxidative capacities throughout the estrous cycle, SOD1 and SOD2 activities were higher in CAR than in ICAR irrespective of the day of the cycle examined (present study). These differences were not negated during the peri-attachment period of pregnancy. Initial attachment of the elongated conceptus is restricted to the CAR epithelium and interdigitation between microvilli of CAR epithelial cells and conceptus trophectoderm cells induces extensive endometrial structural modifications and an increase in vascular remodeling [34]. Therefore, CAR areas, privileged sites for conceptus-uterine communication where early placentation occurs in ruminants, may need appropriate and strong SOD1 and SOD2 activities most likely to contribute to attachment, development and survival of the genetically foreign semiallogenic conceptus. In ruminants, including sheep [35] and cattle [36], the uterine endometrium undergoes structural and functional changes in response to progesterone secreted by the CL during the estrous cycle. Among the antioxidant enzymes studied, only the activity of GPX in CAR, and the activity of CAT in CAR and ICAR increases at day 18 of the estrous cycle when plasma P4 level is very low. Although one can speculate that P4 may play a role in the regulation of GPX
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Fig. 4. Activities of total superoxide dismutase (SOD), SOD1 and SOD2 in caruncle (CAR) and intercaruncle (ICAR) endometrium tissues derived from the entire two uterine horns of each ewe on days 12 (P12), 16 (P16) and 18 (P18) of pregnancy. Values are means ± SEM for the number of ewes used (n ¼ 4 ewes per group). The acceptable level of significance was set at P < 0.05.
and CAT bioactivity, specific in vivo and/or in vitro studies seem necessary to shed light on this point. Cell-to-cell communications between the extraembryonic membranes and the luminal epithelium of the uterine endometrium is among the cellular processes essential for successful conceptus implantation, early post-implantation development and survival [37]. Our results showed that SOD2 activity in CAR endometrium increase from the time of attachment (day 16) to early post-attachment (day 18) period of pregnancy. There is evidence to suggest that conceptus-derived factors regulate protein expression and endometrial enzyme activities of SOD2 during early pregnancy. This is demonstrated by the following observations: 1) SOD2 activity in human endometrium decreases at the late secretory phase of the menstrual cycle and increases early in pregnancy [12] and 2) sheep CAR endometrial tissues demonstrate increased activity [6] and protein expression [7] of SOD2 from day 16 to day 20 of
Fig. 5. Activities of glutathione peroxidase (GPX), glutathione reductase (GR) and catalase (CAT) in caruncle (CAR) and intercaruncle (ICAR) endometrium tissues derived from the entire two uterine horns of each ewe on days 12 (P12), 16 (P16) and 18 (P18) of pregnancy. Values are means ± SEM for the number of ewes used (n ¼ 4 ewes per group). The acceptable level of significance was set at P < 0.05.
pregnancy. Our present data support and reinforce the idea that conceptus-derived factors up-regulate SOD2 activity in the endo metrium. By controlling the production of mitochondrial O 2 , SOD2 may play an important role in conceptus-endometrium communication, which is necessary for successful establishment of pregnancy. There may be an alternative mechanism for the increase of SOD2 activity in endometrium during the peri-attachment period. The endometrium of early pregnancy is a cytokine-rich environment because a variety of immune cells increase at the conceptus attachment site of the endometrium, which are important source of cytokines [38]. Cytokines induce O 2 production in the mitochondria [39]. Of note, SOD2 is immediately induced by cytokines and scavenges O 2 in the mitochondria of human endometrial cells, indicating the protective roles of SOD2 against cytokine-mediated oxidative stress [40]. Interestingly, blockage of SOD2 expression
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causes decreased SOD2 activity, elevated ROS production and apoptosis in rabbit endothelial cells [41]. Therefore, SOD2 probably work to protect the sheep endometrium against oxidative stress for successful conceptus attachment and development. In the presence of transition metals, H2O2 can be converted into the extremely reactive hydroxyl radical ( OH). Therefore, the control of H2O2 generated by SODs is an important component of de fense against OH-induced oxidative insult [42]. The GPX, located within the mitochondrial matrix and the cytoplasm [43] and CAT, found primarily within peroxisomes [44] are responsible for the conversion of H2O2 to water. GR maintains adequate levels of reduced glutathione necessary for the catalytic activity of GPX [45]. It is important to note that the differences of GPX, CAT and GR activities observed between the peri-implantation periods (present study) depend on the endometrium tissues examined. GPX activity in ICAR endometrium increases from the day 12 to day 16 of pregnancy whereas in CAR endometrium it increases from the day 16 to day 18 of pregnancy. CAT activity decreases from the day 12 to day 16 of pregnancy in both CAR and ICAR endometrium. Then after, CAT activity differed only in ICAR endometrium, being higher at day 18 than that at day 16 of pregnancy. GR activity increases from the days 12 and 16 to day 18 of pregnancy in both CAR and ICAR endometrium. The increase GPX activity in CAR, CAT activity in ICAR and GR activity in both CAR and ICAR during the transition from the implantation to the post-implantation periods of pregnancy observed in the present study provide additional support for the finding that conceptus-derived signals play key roles in the upregulation of multiple functional proteins, including antioxidants enzymes, in sheep endometrium during early pregnancy [46]. The different mechanisms operating within sheep CAR and ICAR to regulate GPX, CAT and GR activities during early pregnancy are yet to be clarified. 5. Conclusions Different cellular antioxidant mechanisms operate within the sheep CAR and ICAR endometrial tissues throughout the estrous cycle and early pregnancy that might be related to the different functions of these highly differentiated endometrial areas. Our present data support and further reinforce the idea that the early developing conceptus plays important roles in the regulation of antioxidant enzyme activities in both CAR and ICAR. The networks of ROS scavenging enzymes and their regulation during conceptus pre-attachment, attachment and early post-attachment periods may play an important role in the establishment of pregnancy in sheep. Declaration of interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. Funding This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector. Author contributions KHA conceived and designed the study, performed tissue collection, acquisition and statistical analysis of data and wrote the manuscript. CG and PF provided reagents and materials and took responsibility for the integrity and the accuracy of the biochemical analysis. All authors have approved the final article.
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Acknowledgements We thank Mrs Angele Krawiec, Sandra Grange and Laurence Puillet-Anselme (CHU Grenoble, France) for their expert technical assistance and the staff of the sheep sheds of Jouy-en-Josas (INRA, France) for outstanding technical help and animal management. The authors also thank Dr. Laurent Galio for Histochemistry of endometrium sections. The authors are grateful to Mrs Clare Gaffney for critical reading of the article. The authors would also like to thank the anonymous reviewers for their close examination of this article and their useful comments. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.theriogenology.2017.05.017. References [1] Al-Gubory KH, Fowler PA, Garrel C. The roles of cellular reactive oxygen species, oxidative stress and antioxidants in pregnancy outcomes. Int J Biochem Cell Biol 2010;42:1634e50. [2] Agarwal A, Aponte-Mellado A, Premkumar BJ, Shaman A, Gupta S. The effects of oxidative stress on female reproduction: a review. Reprod Biol Endocrinol 2012;10:49. [3] Al-Gubory KH, Bolifraud P, Germain G, Nicole A, Ceballos-Picot I. Antioxidant enzymatic defence systems in sheep corpus luteum throughout pregnancy. Reproduction 2004;128:767e74. [4] Arianmanesh M, McIntosh RH, Lea RG, Fowler PA, Al-Gubory KH. Ovine corpus luteum proteins, with functions including oxidative stress and lipid metabolism, show complex alterations during implantation. J Endocrinol 2011;210:47e58. [5] Al-Gubory KH, Garrel C, Faure P, Sugino N. Roles of antioxidant enzymes in corpus luteum rescue from reactive oxygen species-induced oxidative stress. Reprod Biomed Online 2012;25:551e60. [6] Al-Gubory KH, Garrel C. Antioxidative signalling pathways regulate the level of reactive oxygen species at the endometrial-extraembryonic membranes interface during early pregnancy. Int J Biochem Cell Biol 2012;44:1511e8. [7] Al-Gubory KH, Arianmanesh M, Garrel C, Bhattacharya S, Cash P, Fowler PA. Proteomic analysis of the sheep caruncular and intercaruncular endometrium reveals changes in functional proteins crucial for the establishment of pregnancy. Reproduction 2014;147:599e614. [8] Gao H, Wu G, Spencer TE, Johnson GA, Li X, Bazer FW. Select nutrients in the ovine uterine lumen. I. Amino acids, glucose, and ions in uterine lumenal flushings of cyclic and pregnant ewes. Biol Reprod 2009;80:86e93. [9] Ramos RS, Oliveira ML, Izaguirry AP, Vargas LM, Soares MB, Mesquita FS, Santos FW, Binelli M. The periovulatory endocrine milieu affects the uterine redox environment in beef cows. Reprod Biol Endocrinol 2015; May 10;13:39. http://dx.doi.org/10.1186/s12958-015-0036-x. [10] Laloraya M, Kumar GP, Laloraya MM. Changes in the superoxide radical and superoxide dismutase levels in the uterus of Rattus norvegicus during the estrous cycle and a possible role for superoxide radical in uterine oedema and cell proliferation at proestrus. Biochem Cell Biol 1991;69:313e6. [11] Makker A, Bansode FW, Srivastava VM, Singh MM. Antioxidant defense system during endometrial receptivity in the Guinea pig: effect of ormeloxifene, a selective estrogen receptor modulator. J Endocrinol 2006;188:121e34. [12] Sugino N, Shimamura K, Takiguchi S, Tamura H, Ono M, Nakata M, Nakamura Y, Ogino K, Uda T, Kato H. Changes in activity of superoxide dismutase in the human endometrium throughout the menstrual cycle and in early pregnancy. Hum Reprod 1996;11:1073e8. [13] Sugino N, Karube-Harada A, Sakata A, Takiguchi S, Kato H. Different mechanisms for the induction of copper-zinc superoxide dismutase and manganese superoxide dismutase by progesterone in human endometrial stromal cells. Hum Reprod 2002;17:1709e14. [14] Sugino N, Karube-Harada A, Kashida S, Takiguchi S, Kato H. Differential regulation of copper-zinc superoxide dismutase and manganese superoxide dismutase by progesterone withdrawal in human endometrial stromal cells. Mol Hum Reprod 2002;8:68e74. [15] Sugino N. The role of oxygen radical-mediated signaling pathways in endometrial function. Placenta 2007;28(Suppl A):S133e6. [16] Al-Gubory KH, Bolifraud P, Garrel C. Regulation of key antioxidant enzymatic systems in the sheep endometrium by ovarian steroids. Endocrinology 2008;149:4428e34. [17] Al-Gubory KH, Bolifraud P, Garrel C. Regulation of key antioxidant enzymatic systems in the sheep endometrium by ovarian steroids. Endocrinology 2008;149:4428e34. [18] Bartol FF, Wiley AA, Coleman DA, Wolfe DF, Riddell MG. Ovine uterine morphogenesis: effects of age and progestin administration and withdrawal on neonatal endometrial development and DNA synthesis. J Anim Sci
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1996;93:9782e7. [33] Sugino N, Nakata M, Kashida S, Karube A, Takiguchi S, Kato H. Decreased superoxide dismutase expression and increased concentrations of lipid peroxide and prostaglandin F(2alpha) in the deciduas of failed pregnancy. Mol Hum Reprod 2000;6:642e7. [34] Igwebuike UM. A review of uterine structural modifications that influence conceptus implantation and development in sheep and goats. Ani Reprod Sci 2009;112:1e7. [35] Gray CA, Bazer FW, Spencer TE. Effects of neonatal progestin exposure on female reproductive tract structure and function in the adult ewe. Biol Reprod 2001;64:797e804. [36] Wang CK, Robinson RS, Flint AP, Mann GE. Quantitative analysis of changes in endometrial gland morphology during the bovine oestrous cycle and their association with progesterone levels. Reproduction 2007;134:365e71. [37] Spencer TE, Burghardt RC, Johnson GA, Bazer FW. Conceptus signals for establishment and maintenance of pregnancy. Anim Reprod Sci 2004;82e83: 537e50. [38] Tamura I, Taketani T, Lee L, Kizuka F, Taniguchi K, Maekawa R, Asada H, Tamura H, Sugino N. Differential effects of progesterone on COX-2 and MnSOD expressions are associated with histone acetylation status of the promoter region in human endometrial stromal cells. J Clin Endocrinol Metab 2011;96:E1073e82. [39] Yang D, Elner SG, Bian ZM, Till GO, Petty HR, Elner VM. Pro-inflammatory cytokines increase reactive oxygen species through mitochondria and NADPH oxidase in cultured RPE cells. Exp Eye Res 2007;85:462e72. [40] Karube-Harada A, Sugino N, Kashida S, Takiguchi S, Takayama H, Yamagata Y, Nakamura Y, Kato H. Induction of manganese superoxide dismutase by tumor necrosis factor-alpha in human endometrial stromal cells. Mol Hum Reprod 2001;7:1065e72. [41] Liu C, Ogando D, Bonanno JA. SOD2 contributes to anti-oxidative capacity in rabbit corneal endothelial cells. Mol Vis 2001;17:2473e81. [42] Chen SX, Schopfer P. Hydroxyl-radical production in physiological reactions. A novel function of peroxidase. Eur J Biochem 1999;260:726e35. [43] Michiels C, Raes M, Toussaint O, Remacle J. Importance of Se-glutathione peroxidase, catalase, and Cu/Zn-SOD for cell survival against oxidative stress. Free Radic Biol Med 1994;17:235e48. [44] Chance B, Sies H, Boveris A. Hydroperoxide metabolism in mammalian organs. Physiol Rev 1979;59:527e605. [45] Hayes JD, McLellan LI. Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defence against oxidative stress. Free Radic Res 1999;31:273e300. [46] Arianmanesh M, Fowler PA, Al-Gubory KH. The sheep conceptus modulates proteome profiles in caruncular endometrium during early pregnancy. Anim Reprod Sci 2016;175:48e56.
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Different enzymatic antioxidative pathways operate within the sheep caruncular and intercaruncular endometrium throughout the estrous cycle and early pregnancy K.H. Al-Gubory a, *, P. Faure b, C. Garrel b UMR BDR, INRA, ENVA, Universit e Paris Saclay, 78350, Jouy en Josas, France Unit e de Biochimie Hormonale - Nutritionnelle, Centre Hospitalier Universitaire de Grenoble, D epartement de Biologie - Toxicologie - Pharmacologie, 38043 Grenoble cedex 9, France a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 June 2016 Received in revised form 3 May 2017 Accepted 22 May 2017 Available online 1 June 2017
There has been a growing interest in the role played by antioxidant enzymes in the regulation of endometrial function in mammals. However, little is known about enzymatic antioxidative pathways involved in conditioning the cyclic and early pregnant endometrium for conceptus attachment and implantation in domestic ruminants. We aimed to investigate changes in activities of superoxide dismutase 1 and 2 (SOD1, SOD2), glutathione peroxidase (GPX), glutathione reductase (GR) and catalase (CAT) in sheep caruncles (CAR) and intercaruncles (ICAR) endometrial tissues of cyclic and early pregnant ewes. Irrespective of day of cycle or pregnancy, CAR demonstrated higher activities of SOD1 and SOD2 than in ICAR. On day 12 of the estrous cycle, ICAR demonstrated higher activity of GPX and GR than in CAR tissues. On days 12 and 16 the estrous cycle, ICAR demonstrated higher activity of CAT than in CAR. CAR demonstrated higher activity of GPX on day 18 than on days 4, 8, 12 and 16 of the estrous cycle. CAR demonstrated higher activity of CAT on day 18 than on days 4, 8, 12 and 16 of the estrous cycle. ICAR demonstrated higher activity of CAT on day 18 than on days 4, 8, and 16 of the estrous cycle. The activity of CAT in ICAR increased from days 4 and 8 to day 12 of the estrous cycle. The activity of SOD2 in CAR increased from day 16 to day 18 of pregnancy. On day 12 of pregnancy, CAR demonstrated higher activity of GPX than in ICAR. On day 16 of pregnancy, ICAR demonstrated higher activity of GPX than in CAR. The activity of GPX in ICAR increased from day 12 to day 16 of pregnancy. The activity of GPX in CAR increased from day 16 to day 18 of pregnancy. The activity of GR in CAR and ICAR increased from days 12 and 16 to day 18 of pregnancy. On days 16 and 18 of pregnancy, ICAR demonstrated higher activity of CAT than in CAR. The activity of CAT in CAR decreased from day 12 to days 16 and 18 of pregnancy. The activity of CAT in ICAR decreased from day 12 to day 16 of pregnancy and then increased from day 16 to day 18 of pregnancy. In conclusion, different antioxidant mechanisms operate within CAR and ICAR endometrium throughout the estrous cycle and during early pregnancy. This might be related to the different but important roles of CAR and ICAR endometrial tissues for the establishment of pregnancy. © 2017 Published by Elsevier Inc.
Keywords: Sheep Caruncular and intercaruncular endometrium Antioxidant enzymes Estrous cycle Early pregnancy
1. Introduction The control of reactive oxygen species (ROS) by several antioxidant enzymes plays important roles in folliculogenesis, oocyte maturation, endometrial function, implantation, embryogenesis,
* Corresponding author. Institut National de la Recherche Agronomique (INRA), partement de Physiologie Animale et Syste mes d’Elevage, UMR 1198 Biologie du De veloppement et de la Reproduction, 78352 Jouy-en-Josas cedex, France. De E-mail addresses: [email protected], [email protected] (K.H. AlGubory). http://dx.doi.org/10.1016/j.theriogenology.2017.05.017 0093-691X/© 2017 Published by Elsevier Inc.
and prenatal development [1,2]. Establishment of pregnancy is a dynamic process associated with significant physiological changes in the enzymatic antioxidative pathways, which are important for sheep reproductive organ functions, such as the corpus luteum [3e5] and endometrium [6,7]. The endometrium provides nutrients, including glucose, amino acids, glutathione (GSH), calcium, and potassium for the unattached conceptus (embryo and associated extraembryonic membranes), as well as a vital biological surface for attachment of the extraembryonic membranes [8]. Worthy of note is that defective uterine environment is associated with inappropriate secretion of ovarian steroids and/or antioxidant
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enzyme activities, which contributes to early pregnancy failure [9]. There has been a growing interest in the role played by antioxidant enzymes in the regulation of rodent [10], guinea pig [11], human [12e15] and sheep [6,7,16] endometrial function aimed at favoring conceptus attachment and successful establishment of pregnancy. However, little is known about activities of antioxidant enzymes in ruminant endometrium throughout the estrous cycle and during early pregnancy. The sheep is an appropriate small ruminant model to explore endometrial antioxidant machinery and its regulation [6,7,17]. The sheep uterus (see Fig. 1 A for supplementary information), like that of goat and cattle, has developed specialized endometrial tissues [18], namely caruncle (CAR) and intercaruncle (ICAR) areas (see Fig. 1 B for supplementary information). CAR and ICAR are histoarchitecturally different playing important roles in the establishment of pregnancy [19]. CAR areas are glandless dense stromal protuberances and lack glands (see Fig. 1C & E for supplementary information). They represent specialized sites of initial attachment of the extraembryonic membrane. ICAR areas contain branched glands (see Fig. 1 D & F for supplementary information) that produce histotroph nutrition to support early conceptus development and survival [20]. Both CAR and ICAR are covered by a simple luminal epithelium (see Fig. 1E and F for supplementary information). Therefore, we aimed to investigate changes in activities of the key antioxidant enzymes, superoxide dismutase 1 and 2 (SOD1, SOD2), glutathione peroxidase (GPX), glutathione reductase (GR) and catalase (CAT), in sheep CAR and ICAR endometrial tissues collected on days 4, 8, 12, 16 and 18 of the estrous cycle (experiment 1) and on days 12, 16 and 18 of pregnancy, corresponding to conceptus pre-attachment, attachment and early post-attachment periods, respectively (experiment 2).
(Paris, France). The minimum detectable concentration of P4 was 0.1 ng/ml and the intra-assay coefficient of variation was less than 10%. 2.3. Tissue collection All the ewes were slaughtered at a local abattoir in accordance with protocols approved by the local institutional animal use committee at the Institut National de la Recherche Agronomique (INRA, Jouy-en-Josas, France). In experiment 1, ewes were randomly allocated for slaughter on days 4 (n ¼ 4 ewes), 8 (n ¼ 4 ewes), 12 (n ¼ 4 ewes), 16 (n ¼ 4 ewes) and 18 (n ¼ 4 ewes) of the estrous cycle. In experiment 2, ewes were randomly allocated for slaughter on days 12 (n ¼ 4 ewes), 16 (n ¼ 4 ewes) and 18 (n ¼ 4 ewes) of pregnancy corresponding to conceptus pre-attachment, attachment and early post-implantation period, respectively. The stages of pregnancy were confirmed by the presence of one or two conceptus in uterine flushing [20]. The reproductive tract of each ewe was collected within 10 min of death, placed on crushed ice and transported to the laboratory. All subsequent manipulation of the tissue was performed at 4 C. The uterine horns were opened and all CAR and ICAR were separately dissected from the entire two uterine horns of each ewe, snap-frozen in liquid nitrogen and then stored at 80 C until processed for activities of the superoxide ( O 2 ) scavenging antioxidant enzymes, SOD1 and SOD2, and the hydrogen peroxide (H2O2) scavenging antioxidant enzymes, GPX, GR and CAT. For morphological analysis, endometrial tissues were fixed in freshly prepared 4% paraformaldehyde in phosphatebuffered saline (PBS, pH 7.4) and then processed for routine histology or histochemistry. 2.4. Conventional histology
2. Materials and methods 2.1. Animals and management The French Ministry of Agriculture approved all procedures relating to care and use of animals according to the French regulation for animal experimentation (authorization no 78e34). This alpes-du-Sud breed (18 study used multiparous ewes of the Pre months of age, n ¼ 35 ewes). All the ewes were treated for 14 days with intravaginal sponges containing 40 mg fluorogestone acetate (Intervet, Angers, France) to synchronize oestrous. Ewes assigned to the pregnant group were mated twice at the time of the synchronized oestrus with fertile rams of the same breed at an interval of 12 h. Throughout the experiment, the ewes were housed under conditions of natural day-length and temperature, fed straw and 2 Kg of hay per day par animal, and had free access to mineral licks and water.
Endometrial tissue was washed in PBS, dehydrated through a series of increasing concentrations of ethanol, cleared with butanol:ethanol (V:V), butanol, embedded in paraffin wax, and sectioned at 7 mm. Sections were deparaffinised in toluene, hydrated through decreasing concentrations of ethanol, washed in distilled water and stained with hematoxylin and eosin. 2.5. Histochemistry Endometrium sections (7 mm) were treated for permeabilization and coloration in 1.25 mg/ml FITC-Phalloidin (Sigma, St. Quentin Fallavier, France) and 0.05% saponin in PBS for 40 min. After washing in PBS, the sections were then incubated 10 min with 1 mg/ ml DAPI (Sigma) in PBS for nucleus localization. After washing in PBS, the sections were mounted in Vectashield mounting medium (Vector Laboratories, Peterborough, UK). Confocal microscopy observations were performed with a Zeiss confocal microscope.
2.2. Blood sampling and progesterone assay 2.6. Antioxidant enzyme activity assays In the present study daily plasma progesterone (P4) was determined during one entire estrous cycle of 3 ewes. In addition, plasma P4 was determined on days 4, 8, 12, 16 and 18 of the estrous cycle (experiment 1), and on days 12, 16 and 18 of pregnancy (experiment 2). Blood samples were taken from the jugular veins into evacuated heparinized tubes. After centrifugation (3000 g, 4 C) for 30 min, plasma was collected and stored at 20 C until assayed for P4 concentrations. The concentrations of P4 were determined by radioimmunoassay (RIA) in unextracted plasma as described [21] and validated for sheep jugular venous plasma with slight modifications [22]. Tritiated P4 (1,2,6, 7-3H-P4, sp act 88 Ci/ mmol) was obtained from Amersham (Bucks, UK), and a specific anti-progesterone antibody was obtained from the Pasteur Institute
The CAR or ICAR were homogenized in cold phosphate buffer (50 mM, pH 7.4) and then the homogenates were centrifuged at 15000 g for 30 min, 4 C. The resulting supernatant was used for determination of protein concentration [23]. A standard SOD assay [24] that had been validated for different sheep reproductive tissues [3,6,17,25] was used. Total SOD activity was measured using the pyrogallol assay based on the competition between pyrogallol oxidation by O 2 and O2 dismutation by SOD. Enzymatic activity of manganese-SOD (SOD2) was determined by assaying for SOD activity in the presence of sodium cyanide, which selectively inhibits copper/zinc-SOD (SOD1) but not SOD2 [26]. SOD1 activity was calculated by subtracting SOD2 activity from total SOD activity. The
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rate of auto-oxidation is taken from the increase in the absorbance at 420 nm. CAT activity was determined as described previously [27]. Activity was assayed by determining the rate of decomposition of H2O2 by CAT in potassium phosphate buffer (pH 7). The reaction rate was related to the amount of CAT present in the mixture. The rate of H2O2 decomposition by CAT was followed at 240 nm. GPX activity was measured using the glutathione reductase-NADPH method. Activity was determined by a coupled assay system [27] in which oxidation of GSH was coupled to NADPH oxidation catalyzed by GR. The rate of GSH oxidized by tertiary butyl hydroperoxide was evaluated by the decrease of NADPH in the presence of EDTA, excess GSH and GR. The rate of decrease in concentration of NADPH was recorded at 340 nm. GR activity was assayed by the standard method of NADPH oxidation. In this assay, GSSG is reduced to GSH by GR, which oxidizes NADPH to NADPþ. NADPH consumption was determined at 340 nm. 2.7. Statistical analysis Data was subjected to ANOVA and the Newman-Keuls multiple comparison test (PRISM Graph Pad version 4, Graph Pad Software, San Diego, CA). Data were presented as the mean ± SEM. The acceptable level of significance was set at P < 0.05. 3. Results 3.1. Progesterone concentrations throughout the estrous cycle The mean daily P4 concentrations in plasma of jugular blood collected throughout the estrous cycle are shown in Fig. 2 (see
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supplementary information). P4 concentration increased gradually from the mean basal value of 0.5 ± 0.3 ng/ml on day 0 (C0) to 2.4 ± 0.7 ng/ml and 4.3 ± 1.0 on day 4 (C4) and day 8 (C8) of the estrous cycle, respectively. The peak value of P4 concentration of 4.9 ± 1.0 ng/ml occurs on day 12 (C12) of the estrous cycle when the CL is fully mature. P4 concentration declined gradually by day 13 to reach a nadir of less than 1 ng/ml on days 16 (C16) and 18 (C18) when the CL was fully regressed. 3.2. Progesterone concentrations during the estrous cycle and early pregnancy The mean P4 concentrations in plasma of jugular blood collected on days 4 (C4), 8 (C8), 12 (C12), 16 (C16) and 18 (C18) of the estrous cycle and on days 12 (P12), 16 (P16) and 18 (P18) of pregnancy are shown in Fig. 1. Plasma concentrations of P4 on day 16 of the estrous cycle were lower than those on days 8 and 12 of the estrous cycle and on days 12, 16 and 18 of pregnancy. Plasma concentrations of P4 on day 18 of the estrous cycle were lower than those on days 4, 8, 12 and 16 of the estrous cycle and on days 12, 16 and 18 of pregnancy. 3.3. Enzymatic activities in CAR and ICAR during the estrous cycle Activities of TSOD, SOD1 and SOD2 in CAR and ICAR collected on days 4, 8, 12, 16 and 18 of the estrous cycle are shown in Fig. 2. Irrespective of day of cycle, CAR demonstrated higher activities of TSOD, SOD1 and SOD2 than in ICAR. The activities of TSOD, SOD1 and SOD2 in CAR or ICAR were not different between any stages of the estrous cycle examined. Activities of GPX, GR and CAT in CAR
Fig. 1. Concentrations of progesterone in plasma of jugular blood collected on days 4 (C4), 8 (C8), 12 (C12), 16 (C16) and 18 (C18) of the estrous cycle and on days 12 (P12), 16 (P16) and 18 (P18) of pregnancy. Values are shown as mean ± SEM for the number of ewes used (n ¼ 4 ewes per group). The acceptable level of significance was set at P < 0.05.
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Fig. 2. Activities of total superoxide dismutase (SOD), SOD1 and SOD2 in caruncle (CAR) and intercaruncle (ICAR) endometrium tissues derived from the entire two uterine horns of each ewe on days 4 (C4), 8 (C8), 12 (C12), 16 (C16) and 18 (C18) of the estrous cycle. Values are means ± SEM for the number of ewes used (n ¼ 4 ewes per group). The acceptable level of significance was set at P < 0.05.
and ICAR collected on days 4, 8, 12, 16 and 18 of the estrous cycle are shown in Fig. 3. On day 12 of the estrous cycle, ICAR demonstrated higher activity of GPX and GR than in CAR tissues. On days 12 and 16 the estrous cycle, ICAR demonstrated higher activity of CAT than in CAR. While GPX activity in ICAR did not alter throughout the estrous cycle, CAR demonstrated higher activity of GPX on day 18 than on days 4, 8, 12 and 16 of the estrous cycle. The activity of GR in CAR and ICAR was not different between any stages of the estrous cycle examined. CAR demonstrated higher activity of CAT on day 18 than on days 4, 8, 12 and 16 of the estrous cycle. ICAR demonstrated higher activity of CAT on day 18 than on days 4, 8, and 16 of the estrous cycle. The activity of CAT in ICAR increased from days 4 and 8 to day 12 of the estrous cycle. 3.4. Enzymatic activities in CAR and ICAR during early pregnancy Activities of TSOD, SOD1 and SOD2 in CAR and ICAR collected on days 12, 16 and 18 of pregnancy are shown in Fig. 4. Irrespective of the day of pregnancy, CAR demonstrated higher activity of TSOD, SOD1 and SOD2 than that in ICAR. The activities of TSOD and SOD1 in CAR or ICAR were not different between any stages of the
pregnancy examined. The activity of SOD2 in CAR or ICAR was not different between days 12 and 16 of pregnancy. The activity of SOD2 in CAR increased from day 16 to day 18 of pregnancy. Activities of GPX, GR and CAT in CAR and ICAR collected on days 12, 16 and 18 of pregnancy are shown in Fig. 5. On day 12 of pregnancy, CAR demonstrated higher activity of GPX than in ICAR. On day 16 of pregnancy, ICAR demonstrated higher activity of GPX than in CAR. The activity of GPX in ICAR increased from day 12 to day 16 of pregnancy. The activity of GPX in CAR increased from day 16 to day 18 of pregnancy. Irrespective of the day of pregnancy, the activity of GR was not different between CAR and ICAR. The activity of GR in CAR and ICAR increased from days 12 and 16 to day 18 of pregnancy. On days 16 and 18 of pregnancy, ICAR demonstrated higher activity of CAT than in CAR. The activity of CAT in CAR decreased from day 12 to days 16 and 18 of pregnancy. The activity of CAT in ICAR decreased from day 12 to day 16 of pregnancy and then increased from day 16 to day18 of pregnancy. 4. Discussion The present study showed that antioxidant enzyme activities in
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Fig. 3. Activities of glutathione peroxidase (GPX), glutathione reductase (GR) and catalase (CAT) in caruncle (CAR) and intercaruncle (ICAR) endometrium tissues derived from the entire two uterine horns of each ewe on days 4 (C4), 8 (C8), 12 (C12), 16 (C16) and 18 (C18) of the estrous cycle. Values are means ± SEM for the number of ewes used (n ¼ 4 ewes per group). The acceptable level of significance was set at P < 0.05.
CAR and ICAR areas of sheep endometrium undergo specific changes during the estrous cycle and early pregnancy that might have regulatory roles in uterine physiology, which could represent one of the underlying biochemical mechanisms conditioning endometrial receptivity. The SOD family is a ubiquitously distrib uted group of metalloenzymes. By catalyzing the conversion of O 2 into H2O2, SOD1 and SOD2 are believed to play a major role in the first line of defense against cellular oxidative damage and its subsequent effects on tissues of biological systems. SOD1 is a dimeric protein, essentially located in the cytoplasm [28], whereas manganese-containing SOD2 is a homotetrameric protein, located in the mitochondria [29]. There is ample evidence that SOD1 and SOD2 play major roles in the process of implantation and pregnancy outcome. Defects in conceptus implantation or premature death of the fetuses have been observed in mutant mice lacking SOD1 [30]. Neonatal lethality [31] or postnatal development restriction [32] has been reported in mutant mice lacking SOD2. In the spontaneous abortion, SOD1 activity in the human endometrium was lower than that in normal pregnancy, suggesting that SOD1 may contribute to the maintenance of pregnancy by pre venting the accumulation of O 2 [33].
Although CAR and ICAR have efficient O 2 scavenging antioxidative capacities throughout the estrous cycle, SOD1 and SOD2 activities were higher in CAR than in ICAR irrespective of the day of the cycle examined (present study). These differences were not negated during the peri-attachment period of pregnancy. Initial attachment of the elongated conceptus is restricted to the CAR epithelium and interdigitation between microvilli of CAR epithelial cells and conceptus trophectoderm cells induces extensive endometrial structural modifications and an increase in vascular remodeling [34]. Therefore, CAR areas, privileged sites for conceptus-uterine communication where early placentation occurs in ruminants, may need appropriate and strong SOD1 and SOD2 activities most likely to contribute to attachment, development and survival of the genetically foreign semiallogenic conceptus. In ruminants, including sheep [35] and cattle [36], the uterine endometrium undergoes structural and functional changes in response to progesterone secreted by the CL during the estrous cycle. Among the antioxidant enzymes studied, only the activity of GPX in CAR, and the activity of CAT in CAR and ICAR increases at day 18 of the estrous cycle when plasma P4 level is very low. Although one can speculate that P4 may play a role in the regulation of GPX
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Fig. 4. Activities of total superoxide dismutase (SOD), SOD1 and SOD2 in caruncle (CAR) and intercaruncle (ICAR) endometrium tissues derived from the entire two uterine horns of each ewe on days 12 (P12), 16 (P16) and 18 (P18) of pregnancy. Values are means ± SEM for the number of ewes used (n ¼ 4 ewes per group). The acceptable level of significance was set at P < 0.05.
and CAT bioactivity, specific in vivo and/or in vitro studies seem necessary to shed light on this point. Cell-to-cell communications between the extraembryonic membranes and the luminal epithelium of the uterine endometrium is among the cellular processes essential for successful conceptus implantation, early post-implantation development and survival [37]. Our results showed that SOD2 activity in CAR endometrium increase from the time of attachment (day 16) to early post-attachment (day 18) period of pregnancy. There is evidence to suggest that conceptus-derived factors regulate protein expression and endometrial enzyme activities of SOD2 during early pregnancy. This is demonstrated by the following observations: 1) SOD2 activity in human endometrium decreases at the late secretory phase of the menstrual cycle and increases early in pregnancy [12] and 2) sheep CAR endometrial tissues demonstrate increased activity [6] and protein expression [7] of SOD2 from day 16 to day 20 of
Fig. 5. Activities of glutathione peroxidase (GPX), glutathione reductase (GR) and catalase (CAT) in caruncle (CAR) and intercaruncle (ICAR) endometrium tissues derived from the entire two uterine horns of each ewe on days 12 (P12), 16 (P16) and 18 (P18) of pregnancy. Values are means ± SEM for the number of ewes used (n ¼ 4 ewes per group). The acceptable level of significance was set at P < 0.05.
pregnancy. Our present data support and reinforce the idea that conceptus-derived factors up-regulate SOD2 activity in the endo metrium. By controlling the production of mitochondrial O 2 , SOD2 may play an important role in conceptus-endometrium communication, which is necessary for successful establishment of pregnancy. There may be an alternative mechanism for the increase of SOD2 activity in endometrium during the peri-attachment period. The endometrium of early pregnancy is a cytokine-rich environment because a variety of immune cells increase at the conceptus attachment site of the endometrium, which are important source of cytokines [38]. Cytokines induce O 2 production in the mitochondria [39]. Of note, SOD2 is immediately induced by cytokines and scavenges O 2 in the mitochondria of human endometrial cells, indicating the protective roles of SOD2 against cytokine-mediated oxidative stress [40]. Interestingly, blockage of SOD2 expression
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causes decreased SOD2 activity, elevated ROS production and apoptosis in rabbit endothelial cells [41]. Therefore, SOD2 probably work to protect the sheep endometrium against oxidative stress for successful conceptus attachment and development. In the presence of transition metals, H2O2 can be converted into the extremely reactive hydroxyl radical ( OH). Therefore, the control of H2O2 generated by SODs is an important component of de fense against OH-induced oxidative insult [42]. The GPX, located within the mitochondrial matrix and the cytoplasm [43] and CAT, found primarily within peroxisomes [44] are responsible for the conversion of H2O2 to water. GR maintains adequate levels of reduced glutathione necessary for the catalytic activity of GPX [45]. It is important to note that the differences of GPX, CAT and GR activities observed between the peri-implantation periods (present study) depend on the endometrium tissues examined. GPX activity in ICAR endometrium increases from the day 12 to day 16 of pregnancy whereas in CAR endometrium it increases from the day 16 to day 18 of pregnancy. CAT activity decreases from the day 12 to day 16 of pregnancy in both CAR and ICAR endometrium. Then after, CAT activity differed only in ICAR endometrium, being higher at day 18 than that at day 16 of pregnancy. GR activity increases from the days 12 and 16 to day 18 of pregnancy in both CAR and ICAR endometrium. The increase GPX activity in CAR, CAT activity in ICAR and GR activity in both CAR and ICAR during the transition from the implantation to the post-implantation periods of pregnancy observed in the present study provide additional support for the finding that conceptus-derived signals play key roles in the upregulation of multiple functional proteins, including antioxidants enzymes, in sheep endometrium during early pregnancy [46]. The different mechanisms operating within sheep CAR and ICAR to regulate GPX, CAT and GR activities during early pregnancy are yet to be clarified. 5. Conclusions Different cellular antioxidant mechanisms operate within the sheep CAR and ICAR endometrial tissues throughout the estrous cycle and early pregnancy that might be related to the different functions of these highly differentiated endometrial areas. Our present data support and further reinforce the idea that the early developing conceptus plays important roles in the regulation of antioxidant enzyme activities in both CAR and ICAR. The networks of ROS scavenging enzymes and their regulation during conceptus pre-attachment, attachment and early post-attachment periods may play an important role in the establishment of pregnancy in sheep. Declaration of interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. Funding This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector. Author contributions KHA conceived and designed the study, performed tissue collection, acquisition and statistical analysis of data and wrote the manuscript. CG and PF provided reagents and materials and took responsibility for the integrity and the accuracy of the biochemical analysis. All authors have approved the final article.
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Acknowledgements We thank Mrs Angele Krawiec, Sandra Grange and Laurence Puillet-Anselme (CHU Grenoble, France) for their expert technical assistance and the staff of the sheep sheds of Jouy-en-Josas (INRA, France) for outstanding technical help and animal management. The authors also thank Dr. Laurent Galio for Histochemistry of endometrium sections. The authors are grateful to Mrs Clare Gaffney for critical reading of the article. The authors would also like to thank the anonymous reviewers for their close examination of this article and their useful comments. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.theriogenology.2017.05.017. References [1] Al-Gubory KH, Fowler PA, Garrel C. The roles of cellular reactive oxygen species, oxidative stress and antioxidants in pregnancy outcomes. Int J Biochem Cell Biol 2010;42:1634e50. [2] Agarwal A, Aponte-Mellado A, Premkumar BJ, Shaman A, Gupta S. The effects of oxidative stress on female reproduction: a review. Reprod Biol Endocrinol 2012;10:49. [3] Al-Gubory KH, Bolifraud P, Germain G, Nicole A, Ceballos-Picot I. Antioxidant enzymatic defence systems in sheep corpus luteum throughout pregnancy. Reproduction 2004;128:767e74. [4] Arianmanesh M, McIntosh RH, Lea RG, Fowler PA, Al-Gubory KH. Ovine corpus luteum proteins, with functions including oxidative stress and lipid metabolism, show complex alterations during implantation. J Endocrinol 2011;210:47e58. [5] Al-Gubory KH, Garrel C, Faure P, Sugino N. Roles of antioxidant enzymes in corpus luteum rescue from reactive oxygen species-induced oxidative stress. Reprod Biomed Online 2012;25:551e60. [6] Al-Gubory KH, Garrel C. Antioxidative signalling pathways regulate the level of reactive oxygen species at the endometrial-extraembryonic membranes interface during early pregnancy. Int J Biochem Cell Biol 2012;44:1511e8. [7] Al-Gubory KH, Arianmanesh M, Garrel C, Bhattacharya S, Cash P, Fowler PA. Proteomic analysis of the sheep caruncular and intercaruncular endometrium reveals changes in functional proteins crucial for the establishment of pregnancy. Reproduction 2014;147:599e614. [8] Gao H, Wu G, Spencer TE, Johnson GA, Li X, Bazer FW. Select nutrients in the ovine uterine lumen. I. Amino acids, glucose, and ions in uterine lumenal flushings of cyclic and pregnant ewes. Biol Reprod 2009;80:86e93. [9] Ramos RS, Oliveira ML, Izaguirry AP, Vargas LM, Soares MB, Mesquita FS, Santos FW, Binelli M. The periovulatory endocrine milieu affects the uterine redox environment in beef cows. Reprod Biol Endocrinol 2015; May 10;13:39. http://dx.doi.org/10.1186/s12958-015-0036-x. [10] Laloraya M, Kumar GP, Laloraya MM. Changes in the superoxide radical and superoxide dismutase levels in the uterus of Rattus norvegicus during the estrous cycle and a possible role for superoxide radical in uterine oedema and cell proliferation at proestrus. Biochem Cell Biol 1991;69:313e6. [11] Makker A, Bansode FW, Srivastava VM, Singh MM. Antioxidant defense system during endometrial receptivity in the Guinea pig: effect of ormeloxifene, a selective estrogen receptor modulator. J Endocrinol 2006;188:121e34. [12] Sugino N, Shimamura K, Takiguchi S, Tamura H, Ono M, Nakata M, Nakamura Y, Ogino K, Uda T, Kato H. Changes in activity of superoxide dismutase in the human endometrium throughout the menstrual cycle and in early pregnancy. Hum Reprod 1996;11:1073e8. [13] Sugino N, Karube-Harada A, Sakata A, Takiguchi S, Kato H. Different mechanisms for the induction of copper-zinc superoxide dismutase and manganese superoxide dismutase by progesterone in human endometrial stromal cells. Hum Reprod 2002;17:1709e14. [14] Sugino N, Karube-Harada A, Kashida S, Takiguchi S, Kato H. Differential regulation of copper-zinc superoxide dismutase and manganese superoxide dismutase by progesterone withdrawal in human endometrial stromal cells. Mol Hum Reprod 2002;8:68e74. [15] Sugino N. The role of oxygen radical-mediated signaling pathways in endometrial function. Placenta 2007;28(Suppl A):S133e6. [16] Al-Gubory KH, Bolifraud P, Garrel C. Regulation of key antioxidant enzymatic systems in the sheep endometrium by ovarian steroids. Endocrinology 2008;149:4428e34. [17] Al-Gubory KH, Bolifraud P, Garrel C. Regulation of key antioxidant enzymatic systems in the sheep endometrium by ovarian steroids. Endocrinology 2008;149:4428e34. [18] Bartol FF, Wiley AA, Coleman DA, Wolfe DF, Riddell MG. Ovine uterine morphogenesis: effects of age and progestin administration and withdrawal on neonatal endometrial development and DNA synthesis. J Anim Sci
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