Just Accepted by Free Radical Research
Abortion-prone mating influences placental antioxidant status and adversely affects placental and foetal development Kaïs H. Al-Gubory, Krawiec Angele, Sandra Grange, Patrice Faure and Catherine Garrel Doi: 10.3109/10715762.2014.967690
Free Radic Res Downloaded from informahealthcare.com by University of Newcastle on 10/06/14 For personal use only.
Abstract Oxidative stress is associated with decreased female fertility and adversely affects prenatal development. Mammalian cells have developed a network of enzymatic and non-enzymatic antioxidant defence systems to prevent oxidative stress. Little attention has been paid to the antioxidative pathways in placentas of normal and disturbed pregnancies, leaving a gap in our knowledge about the role of antioxidants in the control of foeto-placental development. The challenges in studying early human pregnancy can partly be overcome by designing animal models of abnormal pregnancy. We aimed to determine whether the antioxidant status of placentas from the CBA/J x DBA/2 abortion-prone pregnant mice differed from that of normal pregnant mice. The foetal/placental weight ratio was lower in abortion-prone matings compared with non abortion-prone matings. The increased placental lipid peroxidation, the end products of lipid peroxidation, with concomitants alterations in placental antioxidants, namely copper-zinc containing superoxide dismutase (SOD1), manganese containing (SOD2), glutathione peroxidases (GPX), glutathione reductase (GR) and catalase (CAT) activities may be involved in placental and foetal growth restriction. We show that placental oxidative stress is linked with poor prenatal development and pregnancy losses in CBA/J x DBA/2 mice matings. This animal model may be useful in the evaluation of nutritional antioxidant therapies of oxidative stress and associated prenatal developmental disorders.
© 2014 Informa UK, Ltd. This provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. DISCLAIMER: The ideas and opinions expressed in the journal’s Just Accepted articles do not necessarily reflect those of Informa Healthcare (the Publisher), the Editors or the journal. The Publisher does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of the material contained in these articles. The reader is advised to check the appropriate medical literature and the product information currently provided by the manufacturer of each drug to be administered to verify the dosages, the method and duration of administration, and contraindications. It is the responsibility of the treating physician or other health care professional, relying on his or her independent experience and knowledge of the patient, to determine drug dosages and the best treatment for the patient. Just Accepted articles have undergone full scientific review but none of the additional editorial preparation, such as copyediting, typesetting, and proofreading, as have articles published in the traditional manner. There may, therefore, be errors in Just Accepted articles that will be corrected in the final print and final online version of the article. Any use of the Just Accepted articles is subject to the express understanding that the papers have not yet gone through the full quality control process prior to publication.
1
Abortion-prone mating influences placental antioxidant status and adversely affects placental and foetal development
Kaïs H. Al-Gubory1, Krawiec Angele2, Sandra Grange2, Patrice Faure2 and Catherine
PT ED
Garrel2 1
INRA, UMR1198 Biologie du Développement et Reproduction, Département de
Physiologie Animale et Systèmes d’Elevage, 78350 Jouy-en-Josas, France, 2Unité de
Grenoble, Département de Biologie - Toxicologie - pharmacologie, 38043 Grenoble cedex 9, France
Short title: Placental antioxidant status of abortion-prone mating *
CE
Correspondence should be addressed to Kaïs H. Al-Gubory, Institut National de la Recherche Agronomique (INRA), Département de Physiologie Animale et Systèmes d’Elevage, UMR 1198 Biologie du Développement et de la Reproduction, 78352 Jouy-en-Josas cedex, France. Tel: 33 1 34652362, Fax: 33 1 34652364, Email: [email protected] Abstract
ST
AC
Oxidative stress is associated with decreased female fertility and adversely affects prenatal development. Mammalian cells have developed a network of enzymatic and non-enzymatic antioxidant defence systems to prevent oxidative stress. Little attention has been paid to the antioxidative pathways in placentas of normal and disturbed pregnancies, leaving a gap in our knowledge about the role of antioxidants in the control of foeto-placental development. The challenges in studying early human pregnancy can partly be overcome by designing animal models of abnormal pregnancy. We aimed to determine whether the antioxidant status of placentas from the CBA/J x DBA/2 abortion-prone pregnant mice differed from that of normal pregnant mice. The foetal/placental weight ratio was lower in abortion-prone matings compared with non abortion-prone matings. The increased placental lipid peroxidation, the end products of lipid peroxidation, with concomitants alterations in placental antioxidants, namely copper-zinc containing superoxide dismutase (SOD1), manganese containing (SOD2), glutathione peroxidases (GPX), glutathione reductase (GR) and catalase (CAT) activities may be involved in placental and foetal growth restriction. We show that placental oxidative stress is linked with poor prenatal development and pregnancy losses in CBA/J x DBA/2 mice matings. This animal model may be useful in the evaluation of nutritional antioxidant therapies of oxidative stress and associated prenatal developmental disorders.
JU
Free Radic Res Downloaded from informahealthcare.com by University of Newcastle on 10/06/14 For personal use only.
Biochimie Hormonale et Nutritionnelle, Centre Hospitalier Universitaire de
2
Key words: Abortion-prone mating, Oxidative stress, Antioxidants, Placenta, Foeto-placental development
1. Introduction The incidence of early pregnancy failure is high in humans, estimated to be 30% prior to conceptus implantation and 30% before 6 weeks of pregnancy [1].
PT ED
Uncontrolled reactive oxygen species (ROS) production by antioxidants can result in oxidative damage to cellular macromolecules, including lipids, proteins and
DNA lesions [2] that adversely affect prenatal developmental outcome [3,4]. ROSinduced oxidative stress has been reported to be involved in a number of pregnancy disorders such as abortion, intra-uterine growth restriction (IUGR) of foetus and
CE
prenatal mortality [5,6].
Mammalian cells have developed a network of enzymatic and non-enzymatic antioxidant defence systems to control ROS production and prevent their spread and dissemination within and out of cellular organelles. The first enzymatic
AC
antioxidative protective pathway is the dismutation of superoxide anion (•O2−) into hydrogen peroxide (H2O2) by copper-zinc containing superoxide dismutase (SOD1), which is a dimeric protein essentially located in the cytoplasm [7] and manganese containing SOD (SOD2), which is a homotetrameric protein located in the
ST
mitochondria [8]. Glutathione peroxidases (GPXs), located within the mitochondrial matrix and the cytoplasm, and catalase (CAT), found within peroxisomes, both belong to the secondary antioxidative pathway that catalyzes the conversion of H2O2 to H2O. Hence, GPXs and CAT represent the second main cellular defence
JU
Free Radic Res Downloaded from informahealthcare.com by University of Newcastle on 10/06/14 For personal use only.
nucleic acids, and consequently leads to lipid peroxidation, protein degradation and
pathways against oxidative damage [9,10] by limiting the generation and
propagation of highly reactive and toxic H2O2-derived ROS, mainly hydroxyl radical (•OH). Glutathione reductase (GR) catalyzes the reduction of glutathione
disulphide (GSSG) to glutathione (GSH) with NADPH as the reducing agent [11] and is considered to be an essential antioxidant enzyme for the glutathione redox
3
cycle that ensures adequate levels of GSH necessary for the maintenance of cells in a reduced state [12]. The placenta provides an interface for exchange of nutrients and gases between the maternal and foetal blood circulation [13] that is prerequisite for proper foetal development and successful pregnancy. However, no attention has been paid to
PT ED
enzymatic and non-enzymatic antioxidants in the placenta of normal and disturbed pregnancies, leaving a gap in our knowledge about the importance of antioxidant
many challenges, including the ethical impossibility of obtaining human placental tissue samples during pregnancy. Therefore, appropriate animal models of normal and disturbed pregnancy might help answer this important question of biochemistry, developmental biology and reproductive medicine. In addition, understanding the
CE
roles of antioxidants in the control of placental and foetal development is of great interest because it may help in the design of nutritional antioxidant therapies for treatment of prenatal developmental dysfunction and complications [14,15] and in
AC
utero programmable adulthood metabolic and endocrine disorders [16-18]. Rodents are valuable models for assessing disruption of reproduction and fertility. In the present study, the ♀CBA/J x ♂DBA/2 mice mating, which is known for its high rate of foetal-placental resorption, and the ♀C57BL/6 x ♂DBA/2 mice mating were
ST
used as models of disturbed and normal pregnancy, respectively [19]. The non-T suppressor cells that arise in mouse decidua, the maternal part of the placenta, prevent the expression of transplantation immunity at the foeto-maternal interface and ensure the success of the foetal allograft [20]. The decidua of the ♀CBA/J x
JU
Free Radic Res Downloaded from informahealthcare.com by University of Newcastle on 10/06/14 For personal use only.
mechanisms in the control of placental and foetal development. Such studies face
♂DBA/2 mating is deficient in non-T suppressor cell activity before the onset of foetal death [21]. The antioxidant status of placentas in relation to foeto-placental development has been determined in these models of non abortion-prone and abortion-prone matings. The activities of the antioxidant enzymes, SOD1, SOD2, GPX, GR, CAT, the content of the non-enzymatic antioxidant, GSH and GSSG, and
4
the content of malondialdehyde (MDA), a
commonly
used
biomarker
of
peroxidative stress, were determined in placental tissues collected at day 14.5 of pregnancy. 2. Materials and Methods 2.1. Animals and experimental design
PT ED
Virgin females CBA/J and C57Bl/6, and males DBA/2 mice were obtained from Charles River Laboratories France (L’arbresle, France) at 8 weeks of age. They
before mating. They were breed overnight in a 2:1 female to male ratio. Mating was confirmed by detection of a vaginal plug and the morning of sighting was designed as day 0.5 of pregnancy. Throughout the experiment, animals were individually housed in plastic cages in an environmentally controlled room with constant
CE
temperature (22 °C) and a 12:12 h light-dark cycle. Diet and water were freely available. All procedures were approved by the institutional animal care and use committee according to the French regulation for animal experimentation
AC
(authorisation no° 78-34).
2.2. Tissue collection and extraction
The animals were euthanized by cervical dislocation at day 14.5 of pregnancy since the placenta is yet formed and after day 12 of pregnancy there is a clear difference
ST
between resorbing foetal-placental units and normal units [22]. For each mouse, the total number of implantation sites and number of foetal resorption was recorded. The rate of post-implantation foetal-placental resorption (%R) was determined by the following formula: %R = 100 x RF/(RF + VF) where RF, number of resorbed
JU
Free Radic Res Downloaded from informahealthcare.com by University of Newcastle on 10/06/14 For personal use only.
were kept for two weeks in our rodent experimental unit facility (Jouy-en-Josas)
foetuses and VF, number of viable foetuses [23]. The viable foetus with the placenta and yolk sac attached and intact is removed and then the placentas were separated from the foetus and extra-foetal membranes. Each foetus and its placenta obtained were weighed individually. Placentas were pooled separately for each litter prior to
biochemical analysis. The pooled placental tissue samples were snap frozen in liquid
5
nitrogen then stored at -80°C. Placentas were homogenized separately in cold phosphate buffer (50 mM, pH 7.4) and then the homogenates were centrifuged at 15,000 x g for 30 min at 4 °C. The resulting supernatant were stored at -80°C until processed for the activities of SOD1, SOD2, CAT, GPX, GR and the contents of GSH, GSSG and MDA. Protein concentrations were determined by Lowry’s method
2.3. Antioxidant enzyme activity assays
PT ED
[24].
activity was measured using the pyrogallol assay based on the competition between pyrogallol oxidation by •O2− radicals and •O2− dismutation by SOD [28]. Enzymatic activity of SOD2 was determined by assaying for SOD activity in the presence of sodium cyanide, which selectively inhibits SOD1 but not SOD2 [29]. SOD1 activity was calculated by
CE
subtracting SOD2 activity from total SOD activity. The rate of auto-oxidation is taken from the increase in the absorbance at 420 nm. CAT activity was determined as described previously [30]. 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
AC
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 [30] in which oxidation of GSH was coupled to NADPH oxidation catalyzed by GR. The rate of GSH oxidized by tertiary butyl
ST
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. GSR activity was assayed by the standard method of NADPH oxidation. In this assay, GSSG is reduced to GSH by GR which oxidizes NADPH to NADP+. NADPH consumption was
JU
Free Radic Res Downloaded from informahealthcare.com by University of Newcastle on 10/06/14 For personal use only.
Enzyme activities were determined as described previously in detail [25-27]. Total SOD
determined at 340 nm.
2.4. Glutathione and glutathione disulfide determination Total GSH (GSH + GSSG) was determined as described previously according [31] based on the spectrophotometric evaluation of the reduction rate of 5,5’-dithiobis-2nitrobenzoic acid (DTNB, Sigma, France) into 5-thio-2-nitrobenzoic acid (TNB).
6
Values were determined by comparing the reduction rate against a standard curve of GSH. Briefly, NADPH (4 mg/ml) and DTNB (1.5 mg/ml) were freshly prepared in NaHCO3 (0.5%). GR (5 UI/ml) was also freshly prepared in a stock buffer consisting of 3-[N-morpholine]propanesulfonic acid (MOPS, 0.4 M) and EDTA (2 mM) adjusted to pH 6.75. Tissue extract aliquots were mixed with MOPS
PT ED
buffer, NADPH, DTNB and GR. The rate of reduction of DTNB to TNB was recorded at 412 nm. GSSG was determined under the same conditions after
pyridine added to the sample. Tissue content of GSH was calculated by subtracting GSSG content from total GSH content. 2.5. Malondialdehyde determination
The determination of MDA in biological materials is based on its reaction with thiobarbituric
CE
acid (TBA). Reversed-phase high performance liquid chromatography (HPLC) technique in which the MDA-TBA adducts are separated from interfering substances [32] was used for determining MDA. The breakdown product of 1,1,3,3-tetraethoxypropane (TEP) was used as
AC
standard. TEP undergoes hydrolysis to liberate stoichiometric amounts of MDA. Stock standard solution (480 µl of TEP in 100 ml ethanol) was prepared and this primary solution was diluted to the concentrations of 0, 1, 2, 3, 4, 5 and 6 µM. Tissue extract aliquots or standards were mixed with TBA (0.8%). The tubes were placed in a water bath at 95°C for 1 hour, and then they were cooled. Samples were neutralized with methanol-NaOH mixture
ST
(pH 6). After centrifugation, protein-free supernatant were chromatographed in the HPLC system. The column used for the separation was Adsorbosphere C18 (5 μm particle diameter, 250 mm x 4.6 mm ID). The MDA-TBA adduct is eluted from the column with potassium dihydrogen phosphate buffer (10 mM, pH 6.0)-acetonitrile (17%). The quantification of
JU
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adjusting of the pH with ethanolamine and trapping the reduced GSH with 2-vinyl
MDA derivative was established by comparing the absorption to the standard curve of MDA equivalents generated by acid-catalyzed hydrolysis of TEP.
2.6. Statistical analysis Student t-test (maternal body weight, foetal and placental weights, number of viable foetuses, placental MDA contents, placental enzyme activities, GSH and GSSG
contents)
and
χ2
7
analysis
(% foetal/placental
resorptions)
were
performed to test the significance difference between groups. Differences were considered significant at P < 0.05. 3. Results At day 14.5 of pregnancy, the abortion-prone mating showed a higher rate of foetal-
PT ED
placental resorptions (Figure 1B) when compared with the non abortion-prone mating (Figure 1A). Viable 14.5-day foetuses and their placentas derived from the
compared with foetuses and placentas of the non abortion-prone mating (Figure 1C). Maternal body weight at day 14.5 of pregnancy was lower (P
Abortion-prone mating influences placental antioxidant status and adversely affects placental and foetal development Kaïs H. Al-Gubory, Krawiec Angele, Sandra Grange, Patrice Faure and Catherine Garrel Doi: 10.3109/10715762.2014.967690
Free Radic Res Downloaded from informahealthcare.com by University of Newcastle on 10/06/14 For personal use only.
Abstract Oxidative stress is associated with decreased female fertility and adversely affects prenatal development. Mammalian cells have developed a network of enzymatic and non-enzymatic antioxidant defence systems to prevent oxidative stress. Little attention has been paid to the antioxidative pathways in placentas of normal and disturbed pregnancies, leaving a gap in our knowledge about the role of antioxidants in the control of foeto-placental development. The challenges in studying early human pregnancy can partly be overcome by designing animal models of abnormal pregnancy. We aimed to determine whether the antioxidant status of placentas from the CBA/J x DBA/2 abortion-prone pregnant mice differed from that of normal pregnant mice. The foetal/placental weight ratio was lower in abortion-prone matings compared with non abortion-prone matings. The increased placental lipid peroxidation, the end products of lipid peroxidation, with concomitants alterations in placental antioxidants, namely copper-zinc containing superoxide dismutase (SOD1), manganese containing (SOD2), glutathione peroxidases (GPX), glutathione reductase (GR) and catalase (CAT) activities may be involved in placental and foetal growth restriction. We show that placental oxidative stress is linked with poor prenatal development and pregnancy losses in CBA/J x DBA/2 mice matings. This animal model may be useful in the evaluation of nutritional antioxidant therapies of oxidative stress and associated prenatal developmental disorders.
© 2014 Informa UK, Ltd. This provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. DISCLAIMER: The ideas and opinions expressed in the journal’s Just Accepted articles do not necessarily reflect those of Informa Healthcare (the Publisher), the Editors or the journal. The Publisher does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of the material contained in these articles. The reader is advised to check the appropriate medical literature and the product information currently provided by the manufacturer of each drug to be administered to verify the dosages, the method and duration of administration, and contraindications. It is the responsibility of the treating physician or other health care professional, relying on his or her independent experience and knowledge of the patient, to determine drug dosages and the best treatment for the patient. Just Accepted articles have undergone full scientific review but none of the additional editorial preparation, such as copyediting, typesetting, and proofreading, as have articles published in the traditional manner. There may, therefore, be errors in Just Accepted articles that will be corrected in the final print and final online version of the article. Any use of the Just Accepted articles is subject to the express understanding that the papers have not yet gone through the full quality control process prior to publication.
1
Abortion-prone mating influences placental antioxidant status and adversely affects placental and foetal development
Kaïs H. Al-Gubory1, Krawiec Angele2, Sandra Grange2, Patrice Faure2 and Catherine
PT ED
Garrel2 1
INRA, UMR1198 Biologie du Développement et Reproduction, Département de
Physiologie Animale et Systèmes d’Elevage, 78350 Jouy-en-Josas, France, 2Unité de
Grenoble, Département de Biologie - Toxicologie - pharmacologie, 38043 Grenoble cedex 9, France
Short title: Placental antioxidant status of abortion-prone mating *
CE
Correspondence should be addressed to Kaïs H. Al-Gubory, Institut National de la Recherche Agronomique (INRA), Département de Physiologie Animale et Systèmes d’Elevage, UMR 1198 Biologie du Développement et de la Reproduction, 78352 Jouy-en-Josas cedex, France. Tel: 33 1 34652362, Fax: 33 1 34652364, Email: [email protected] Abstract
ST
AC
Oxidative stress is associated with decreased female fertility and adversely affects prenatal development. Mammalian cells have developed a network of enzymatic and non-enzymatic antioxidant defence systems to prevent oxidative stress. Little attention has been paid to the antioxidative pathways in placentas of normal and disturbed pregnancies, leaving a gap in our knowledge about the role of antioxidants in the control of foeto-placental development. The challenges in studying early human pregnancy can partly be overcome by designing animal models of abnormal pregnancy. We aimed to determine whether the antioxidant status of placentas from the CBA/J x DBA/2 abortion-prone pregnant mice differed from that of normal pregnant mice. The foetal/placental weight ratio was lower in abortion-prone matings compared with non abortion-prone matings. The increased placental lipid peroxidation, the end products of lipid peroxidation, with concomitants alterations in placental antioxidants, namely copper-zinc containing superoxide dismutase (SOD1), manganese containing (SOD2), glutathione peroxidases (GPX), glutathione reductase (GR) and catalase (CAT) activities may be involved in placental and foetal growth restriction. We show that placental oxidative stress is linked with poor prenatal development and pregnancy losses in CBA/J x DBA/2 mice matings. This animal model may be useful in the evaluation of nutritional antioxidant therapies of oxidative stress and associated prenatal developmental disorders.
JU
Free Radic Res Downloaded from informahealthcare.com by University of Newcastle on 10/06/14 For personal use only.
Biochimie Hormonale et Nutritionnelle, Centre Hospitalier Universitaire de
2
Key words: Abortion-prone mating, Oxidative stress, Antioxidants, Placenta, Foeto-placental development
1. Introduction The incidence of early pregnancy failure is high in humans, estimated to be 30% prior to conceptus implantation and 30% before 6 weeks of pregnancy [1].
PT ED
Uncontrolled reactive oxygen species (ROS) production by antioxidants can result in oxidative damage to cellular macromolecules, including lipids, proteins and
DNA lesions [2] that adversely affect prenatal developmental outcome [3,4]. ROSinduced oxidative stress has been reported to be involved in a number of pregnancy disorders such as abortion, intra-uterine growth restriction (IUGR) of foetus and
CE
prenatal mortality [5,6].
Mammalian cells have developed a network of enzymatic and non-enzymatic antioxidant defence systems to control ROS production and prevent their spread and dissemination within and out of cellular organelles. The first enzymatic
AC
antioxidative protective pathway is the dismutation of superoxide anion (•O2−) into hydrogen peroxide (H2O2) by copper-zinc containing superoxide dismutase (SOD1), which is a dimeric protein essentially located in the cytoplasm [7] and manganese containing SOD (SOD2), which is a homotetrameric protein located in the
ST
mitochondria [8]. Glutathione peroxidases (GPXs), located within the mitochondrial matrix and the cytoplasm, and catalase (CAT), found within peroxisomes, both belong to the secondary antioxidative pathway that catalyzes the conversion of H2O2 to H2O. Hence, GPXs and CAT represent the second main cellular defence
JU
Free Radic Res Downloaded from informahealthcare.com by University of Newcastle on 10/06/14 For personal use only.
nucleic acids, and consequently leads to lipid peroxidation, protein degradation and
pathways against oxidative damage [9,10] by limiting the generation and
propagation of highly reactive and toxic H2O2-derived ROS, mainly hydroxyl radical (•OH). Glutathione reductase (GR) catalyzes the reduction of glutathione
disulphide (GSSG) to glutathione (GSH) with NADPH as the reducing agent [11] and is considered to be an essential antioxidant enzyme for the glutathione redox
3
cycle that ensures adequate levels of GSH necessary for the maintenance of cells in a reduced state [12]. The placenta provides an interface for exchange of nutrients and gases between the maternal and foetal blood circulation [13] that is prerequisite for proper foetal development and successful pregnancy. However, no attention has been paid to
PT ED
enzymatic and non-enzymatic antioxidants in the placenta of normal and disturbed pregnancies, leaving a gap in our knowledge about the importance of antioxidant
many challenges, including the ethical impossibility of obtaining human placental tissue samples during pregnancy. Therefore, appropriate animal models of normal and disturbed pregnancy might help answer this important question of biochemistry, developmental biology and reproductive medicine. In addition, understanding the
CE
roles of antioxidants in the control of placental and foetal development is of great interest because it may help in the design of nutritional antioxidant therapies for treatment of prenatal developmental dysfunction and complications [14,15] and in
AC
utero programmable adulthood metabolic and endocrine disorders [16-18]. Rodents are valuable models for assessing disruption of reproduction and fertility. In the present study, the ♀CBA/J x ♂DBA/2 mice mating, which is known for its high rate of foetal-placental resorption, and the ♀C57BL/6 x ♂DBA/2 mice mating were
ST
used as models of disturbed and normal pregnancy, respectively [19]. The non-T suppressor cells that arise in mouse decidua, the maternal part of the placenta, prevent the expression of transplantation immunity at the foeto-maternal interface and ensure the success of the foetal allograft [20]. The decidua of the ♀CBA/J x
JU
Free Radic Res Downloaded from informahealthcare.com by University of Newcastle on 10/06/14 For personal use only.
mechanisms in the control of placental and foetal development. Such studies face
♂DBA/2 mating is deficient in non-T suppressor cell activity before the onset of foetal death [21]. The antioxidant status of placentas in relation to foeto-placental development has been determined in these models of non abortion-prone and abortion-prone matings. The activities of the antioxidant enzymes, SOD1, SOD2, GPX, GR, CAT, the content of the non-enzymatic antioxidant, GSH and GSSG, and
4
the content of malondialdehyde (MDA), a
commonly
used
biomarker
of
peroxidative stress, were determined in placental tissues collected at day 14.5 of pregnancy. 2. Materials and Methods 2.1. Animals and experimental design
PT ED
Virgin females CBA/J and C57Bl/6, and males DBA/2 mice were obtained from Charles River Laboratories France (L’arbresle, France) at 8 weeks of age. They
before mating. They were breed overnight in a 2:1 female to male ratio. Mating was confirmed by detection of a vaginal plug and the morning of sighting was designed as day 0.5 of pregnancy. Throughout the experiment, animals were individually housed in plastic cages in an environmentally controlled room with constant
CE
temperature (22 °C) and a 12:12 h light-dark cycle. Diet and water were freely available. All procedures were approved by the institutional animal care and use committee according to the French regulation for animal experimentation
AC
(authorisation no° 78-34).
2.2. Tissue collection and extraction
The animals were euthanized by cervical dislocation at day 14.5 of pregnancy since the placenta is yet formed and after day 12 of pregnancy there is a clear difference
ST
between resorbing foetal-placental units and normal units [22]. For each mouse, the total number of implantation sites and number of foetal resorption was recorded. The rate of post-implantation foetal-placental resorption (%R) was determined by the following formula: %R = 100 x RF/(RF + VF) where RF, number of resorbed
JU
Free Radic Res Downloaded from informahealthcare.com by University of Newcastle on 10/06/14 For personal use only.
were kept for two weeks in our rodent experimental unit facility (Jouy-en-Josas)
foetuses and VF, number of viable foetuses [23]. The viable foetus with the placenta and yolk sac attached and intact is removed and then the placentas were separated from the foetus and extra-foetal membranes. Each foetus and its placenta obtained were weighed individually. Placentas were pooled separately for each litter prior to
biochemical analysis. The pooled placental tissue samples were snap frozen in liquid
5
nitrogen then stored at -80°C. Placentas were homogenized separately in cold phosphate buffer (50 mM, pH 7.4) and then the homogenates were centrifuged at 15,000 x g for 30 min at 4 °C. The resulting supernatant were stored at -80°C until processed for the activities of SOD1, SOD2, CAT, GPX, GR and the contents of GSH, GSSG and MDA. Protein concentrations were determined by Lowry’s method
2.3. Antioxidant enzyme activity assays
PT ED
[24].
activity was measured using the pyrogallol assay based on the competition between pyrogallol oxidation by •O2− radicals and •O2− dismutation by SOD [28]. Enzymatic activity of SOD2 was determined by assaying for SOD activity in the presence of sodium cyanide, which selectively inhibits SOD1 but not SOD2 [29]. SOD1 activity was calculated by
CE
subtracting SOD2 activity from total SOD activity. The rate of auto-oxidation is taken from the increase in the absorbance at 420 nm. CAT activity was determined as described previously [30]. 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
AC
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 [30] in which oxidation of GSH was coupled to NADPH oxidation catalyzed by GR. The rate of GSH oxidized by tertiary butyl
ST
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. GSR activity was assayed by the standard method of NADPH oxidation. In this assay, GSSG is reduced to GSH by GR which oxidizes NADPH to NADP+. NADPH consumption was
JU
Free Radic Res Downloaded from informahealthcare.com by University of Newcastle on 10/06/14 For personal use only.
Enzyme activities were determined as described previously in detail [25-27]. Total SOD
determined at 340 nm.
2.4. Glutathione and glutathione disulfide determination Total GSH (GSH + GSSG) was determined as described previously according [31] based on the spectrophotometric evaluation of the reduction rate of 5,5’-dithiobis-2nitrobenzoic acid (DTNB, Sigma, France) into 5-thio-2-nitrobenzoic acid (TNB).
6
Values were determined by comparing the reduction rate against a standard curve of GSH. Briefly, NADPH (4 mg/ml) and DTNB (1.5 mg/ml) were freshly prepared in NaHCO3 (0.5%). GR (5 UI/ml) was also freshly prepared in a stock buffer consisting of 3-[N-morpholine]propanesulfonic acid (MOPS, 0.4 M) and EDTA (2 mM) adjusted to pH 6.75. Tissue extract aliquots were mixed with MOPS
PT ED
buffer, NADPH, DTNB and GR. The rate of reduction of DTNB to TNB was recorded at 412 nm. GSSG was determined under the same conditions after
pyridine added to the sample. Tissue content of GSH was calculated by subtracting GSSG content from total GSH content. 2.5. Malondialdehyde determination
The determination of MDA in biological materials is based on its reaction with thiobarbituric
CE
acid (TBA). Reversed-phase high performance liquid chromatography (HPLC) technique in which the MDA-TBA adducts are separated from interfering substances [32] was used for determining MDA. The breakdown product of 1,1,3,3-tetraethoxypropane (TEP) was used as
AC
standard. TEP undergoes hydrolysis to liberate stoichiometric amounts of MDA. Stock standard solution (480 µl of TEP in 100 ml ethanol) was prepared and this primary solution was diluted to the concentrations of 0, 1, 2, 3, 4, 5 and 6 µM. Tissue extract aliquots or standards were mixed with TBA (0.8%). The tubes were placed in a water bath at 95°C for 1 hour, and then they were cooled. Samples were neutralized with methanol-NaOH mixture
ST
(pH 6). After centrifugation, protein-free supernatant were chromatographed in the HPLC system. The column used for the separation was Adsorbosphere C18 (5 μm particle diameter, 250 mm x 4.6 mm ID). The MDA-TBA adduct is eluted from the column with potassium dihydrogen phosphate buffer (10 mM, pH 6.0)-acetonitrile (17%). The quantification of
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adjusting of the pH with ethanolamine and trapping the reduced GSH with 2-vinyl
MDA derivative was established by comparing the absorption to the standard curve of MDA equivalents generated by acid-catalyzed hydrolysis of TEP.
2.6. Statistical analysis Student t-test (maternal body weight, foetal and placental weights, number of viable foetuses, placental MDA contents, placental enzyme activities, GSH and GSSG
contents)
and
χ2
7
analysis
(% foetal/placental
resorptions)
were
performed to test the significance difference between groups. Differences were considered significant at P < 0.05. 3. Results At day 14.5 of pregnancy, the abortion-prone mating showed a higher rate of foetal-
PT ED
placental resorptions (Figure 1B) when compared with the non abortion-prone mating (Figure 1A). Viable 14.5-day foetuses and their placentas derived from the
compared with foetuses and placentas of the non abortion-prone mating (Figure 1C). Maternal body weight at day 14.5 of pregnancy was lower (P
