Diet Supplementation with Pomegranate Peel Improves Embryonic Survival in a Mouse Model of Early Pregnancy Loss.

The peel of pomegranate fruit is a rich source of polyphenolic compounds with powerful antioxidant properties. We evaluated the therapeutic potential of pomegranate peel (PP) in the prevention of early pregnancy loss in a mouse model of embryonic mortality and abortion (female CBA/J x male DBA/2). CBA/J mice were divided into 3 groups: mice in control group (CONT group) were fed a standard diet, whereas mice in groups 2 and 3 were fed a standard diet supplemented with 1% PP (PP1% group) and 5% PP (PP5% group), respectively. All the mice were fed their diets for 10 days before mating and continued with the same diets for a further 14 days after mating. At day 14 of pregnancy the female mice were sacrificed and the placentas and maternal livers were harvested for measurement of the content of thiols and thiobarbituric acid reactive substances (TBARS), as biomarkers of oxidative stress, and the enzymatic activities of total superoxide dismutase (TSOD), copper/zinc SOD (SOD1), manganese SOD (SOD2), selenium glutathione peroxidase (GPX) and glutathione reductase (GR). Diet supplemented with 5% PP improved embryonic survival and reduced embryonic mortality from 28.2% (CONT) to 8.5% (PP5%). This was accompanied by increased activities of placental TSOD, SOD1 and SOD2, and thiol content. Diet supplemented with 5% PP also reduced placental oxidative stress as demonstrated by a decrease of placental TBARS content. This study highlights the potential of interventions with PP-supplemented diet before and during early pregnancy, in order to ameliorate embryonic survival and prevent early pregnancy loss.

Pomegranate peel attenuates dextran sulfate sodium-induced lipid peroxidation in rat small intestine by enhancing the glutathione/glutathione disulfide redox potential.

BACKGROUND: The peel of the pomegranate fruit is rich in polyphenols with antioxidant properties. We investigated the preventive effect of pomegranate peel (PP) powder against dextran sulfate sodium (DSS)-induced lipid peroxidation in the small intestine of rats. Rats were allocated to four groups: CONT group, fed a standard rodent diet; DSS group, fed a standard rodent diet and treated with DSS; as well as PP1%+DSS and PP5%+DSS groups, fed a standard rodent diet supplemented with either 1% or 5% of PP powder and treated with DSS. Rats of the four groups consumed their diets for 25 days. Lipid peroxidation was determined by measuring malondialdehyde (MDA) concentrations in plasma and MDA contents in the small intestine and liver. Glutathione/glutathione disulfide (GSH/GSSG) redox status and antioxidant enzyme activities were determined in the small intestine and liver. RESULTS: MDA content was higher (P < 0.001) in the small intestines of the DSS group compared to the CONT group. MDA content was reduced (P < 0.001) in the small intestines of the PP1%+DSS and PP5%+DSS groups compared to the DSS group. GSH contents and GSH/GSSG ratios were higher (P < 0.001) in the small intestines of the PP5%+DSS group compared to the CONT, DSS and PP1%+DSS groups. CONCLUSION: The present study demonstrates that PP powder protects the small intestine against DSS-induced lipid peroxidation by enhancing the GSH/GSSG redox potential. Powder of PP is a promising agricultural by-product containing a mixture of bioactive polyphenols that can be used for the production of functional foods aimed at the prevention of oxidative stress-induced small intestine pathogenesis. © 2021 Society of Chemical Industry.

Shedding light on fibered confocal fluorescence microscopy: Applications in biomedical imaging and therapies.

Discoveries of major importance in life sciences and preclinical research are linked to the invention of microscopes that enable imaging of cells and their microstructures. Imaging technologies involving in vivo procedures using fluorescent dyes that permit labelling of cells have been developed over the last two decades. Fibered confocal fluorescence microscopy (FCFM) is an imaging technology equipped with fiber-optic probes to deliver light to organs and tissues of live animals. This enables not only in vivo detection of fluorescent signals and visualization of cells, but also the study of dynamic processes, such cell proliferation, apoptosis and angiogenesis, under physiological and pathological conditions. This will allow the diagnosis of diseased organs and tissues and the evaluation of the efficacy of new therapies in animal models of human diseases. The aim of this report is to shed light on FCFM and its potential medical applications and discusses some factors that compromise the reliability and reproducibility of monitoring biological processes by FCFM. This report also highlights the issues concerning animal experimentation and welfare, and the contributions of FCFM to the 3Rs principals, replacement, reduction and refinement.

Multiple exposures to environmental pollutants and oxidative stress: Is there a sex specific risk of developmental complications for fetuses?

Medically assisted procreation significantly contributes to an increase in twin pregnancies. One of the major factors contributing to more twin births is the use of fertility treatments. Twin pregnancy is not without a risk for fetal organ development and the health outcome of new-borns, children, and adults. Multiple pregnancies are associated with an increased risk of developmental complications, such as perinatal mortality, premature births, and low birth weight. Oxidative stress is involved in pregnancy disorders such as abortion, intrauterine growth retardation, and prenatal mortality. The link between oxidative stress and prenatal development, poorly perceived in the medical community, is a major problem in human reproductive medicine and health outcomes. The sex-based considerations and analyses are also, often neglected in biomedical research. In addition, fetal sexual dimorphism in antioxidant pathways following intrauterine exposure to environmental pollutants has not been explored. This is an important area of research because sexually dimorphic antioxidant adaptive responses to early life exposure-induced oxidative stress may have long-term effects on offspring health outcome and increase the risk of non-communicable diseases in men and women. This concept is useful, since it may open the avenue to develop antenatal antioxidant therapeutic strategies to developmental disorders and complications related to multiple pregnancies, and in association with acute or chronic environmental exposure. This article reviews the status of research, supporting data, possible pathogenic mechanisms, and future perspectives in the proposed area. Birth Defects Research (Part C) 108:351-364, 2016. © 2016 Wiley Periodicals, Inc.

The sheep conceptus modulates proteome profiles in caruncular endometrium during early pregnancy.

The stage-specific expression of functional proteins within the endometrium, and their regulation by conceptus-derived signals, are crucial for conceptus development and successful establishment of pregnancy. Accurate knowledge of endometrium-conceptus interactions is key for the development of effective strategies to improve conceptus implantation rates both following natural conception and/or assisted reproductive technologies. The unilateral pregnant ewe provides a powerful experimental model for the study of endometrial function in the presence or absence of conceptuses during the peri-implantation period. Two-dimensional gel electrophoresis and mass spectrometry-based proteomics were used to compare and identify differentially expressed proteins in caruncular endometrium collected from the gravid uterine horns and the non-gravid uterine horns at the time of conceptus attachment (day 16 of pregnancy) and early post-implantation period (day 20 of pregnancy). Fifty seven protein spots were up-regulated in the gravid horn at day 16 of pregnancy and twenty seven protein spots were up-regulated in the gravid horn at day 20 of pregnancy. Sixteen proteins with different functions such as protein metabolism, cholesterol and ion transport and cell adhesion were identified. In conclusion, the use of the unilaterally pregnant ewe model provides evidence that the early implantation and post-implanting conceptus-derived signals up-regulate caruncle endometrial proteins, including carbonic anhydrase 2 (CA-II) and apolipoprotein A-1 (APOA1) and down-regulate caruncle endometrial proteins, including adenosylhomocysteinase (AHCY) and heat shock 60kDa protein 1 (HSP60). These regulated proteins are likely involved in providing a suitable intra-uterine environment required for conceptus attachment, implantation, early post-implantation development and the successful establishment of pregnancy in sheep.

Sex-specific divergence of antioxidant pathways in fetal brain, liver, and skeletal muscles.

The sex-specific divergence of antioxidant pathways in fetal organs of opposite-sex twin is unknown and remains urgently in need of investigation. Such study faces many challenges, mainly the ethical impossibility of obtaining human fetal organs. Opposite-sex sheep twins represent a unique model for studying a sex dimorphism for antioxidant systems. The activity of total superoxide dismutase (SOD), SOD1, SOD2, glutathione peroxidase (GPX), glutathione reductase (GR) and catalase (CAT), the content of total glutathione, reduced glutathione (GSH), and oxidized glutathione (GSSG) were measured in brain, lung, liver, kidney, and skeletal muscles of female and male fetuses collected from sheep twin pregnancies at day 65 of gestation. Lipid peroxidation was assessed by measuring melondialdehyde (MDA) tissue content. Male brain has greater total SOD and SOD1 activities than female brain. Female liver has greater SOD2 activity than male liver. Male liver has greater GR activity than female liver. Male liver has higher total GSH and GSSG content than female liver. Male skeletal muscles have higher total GSH, GSH, and GSSG content than female skeletal muscles. Female brain and liver have higher MDA content than male brain and liver. This is the first report of a sex dimorphism for fetal organ antioxidative pathways. Brain, liver, and skeletal muscles of male and female fetuses display distinct antioxidant pathways. Such sexually dimorphic responses to early life oxidative stress might be involved in the sex-related difference in fetal development that may have a long-term effect on offspring. Our study urges researchers to take into consideration the importance of sex as a biologic variable in their investigations.

Fatty acid binding protein 7 and n-3 poly unsaturated fatty acid supply in early rat brain development.

Fatty acid binding protein 7 (FABP7), abundant in the embryonic brain, binds with the highest affinity to docosahexaenoic acid (DHA) and is expressed in the early stages of embryogenesis. Here, we have examined the consequences of the exposure to different DHA levels and of the in utero depletion of FABP7 on early rat brain development. Neurodevelopment was evaluated through the contents of two proteins, connexin 43 (Cx43) and cyclin-dependent kinase 5 (CDK5), both involved in neuroblast proliferation, differentiation, and migration. The dams were fed with diets presenting different DHA contents, from deficiency to supplementation. DHA brain embryos contents already differed at embryonic day 11.5 and the differences kept increasing with time. Cx43 and CDK5 contents were positively associated with the brain DHA levels. When FABP7 was depleted in vivo by injections of siRNA in the telencephalon, the enhancement of the contents of both proteins was lost in supplemented animals, but FABP7 depletion did not modify phospholipid compositions regardless of the diets. Thus, FABP7 is a necessary mediator of the effect of DHA on these proteins synthesis, but its role in DHA uptake is not critical, although FABP7 is localized in phospholipid-rich areas. Our study shows that high contents of DHA associated with FABP7 are necessary to promote early brain development, which prompted us to recommend DHA supplementation early in pregnancy.

Pomegranate peel extract decreases small intestine lipid peroxidation by enhancing activities of major antioxidant enzymes.

BACKGROUND: Pomegranate peel extract (PPE) contains several compounds with antioxidative properties. PPE added to foods may interact with endogenous antioxidants and promote health. However, little is known about the biochemical mechanisms by which PPE exerts their actions on tissues of biological systems in vivo. The purpose of this study was to determine the effects of PPE on activities of antioxidant enzymes. Mice were used to investigate the effects of PPE on plasma levels of malondialdehyde (MDA), tissue MDA content and activities of superoxide dismutase 1 (SOD1), SOD2 and glutathione peroxidase (GPX) in the small intestine, liver and skeletal muscle - different tissues involved in the digestion, absorption and metabolism of dietary nutrients. Control mice were fed a standard diet, whereas treated mice were fed for 40 days with the standard diet containing 5% or 10% PPE. RESULTS: Mice fed the 10% PPE diet exhibited lower plasma MDA concentrations, reduced content of MDA in the small intestine and liver and higher levels of SOD1 and GPX activities in the small intestine compared to mice fed the control diet. CONCLUSIONS: These findings demonstrate that intake of PPE in diet attenuates small intestine lipid peroxidation and strengthens the first line of small intestine antioxidant defense by enhancing enzymatic antioxidative pathways. PPE is worthy of further study as a therapeutic approach to prevent peroxidative stress-induced gut pathogenesis. © 2015 Society of Chemical Industry.

Environmental pollutants and lifestyle factors induce oxidative stress and poor prenatal development.

Developmental toxicity caused by exposure to a mixture of environmental pollutants has become a major health concern. Human-made chemicals, including xenoestrogens, pesticides and heavy metals, as well as unhealthy lifestyle behaviours, mainly tobacco smoking, alcohol consumption and medical drug abuse, are major factors that adversely influence prenatal development and increase susceptibility of offspring to diseases. There is evidence to suggest that the developmental toxicological mechanisms of chemicals and lifestyle factors involve the generation of reactive oxygen species (ROS) and cellular oxidative damage. Overproduction of ROS induces oxidative stress, a state where increased ROS generation overwhelms antioxidant protection and subsequently leads to oxidative damage of cellular macromolecules. Data on the involvement of oxidative stress in the mechanism of developmental toxicity following exposure to environmental pollutants are reviewed in an attempt to provide an updated basis for future studies on the toxic effect of such pollutants, particularly the notion of increased risk for developmental toxicity due to combined and cumulative exposure to various environmental pollutants. The aims of such studies are to better understand the mechanisms by which environmental pollutants adversely affect conceptus development and to elucidate the impact of cumulative exposures to multiple pollutants on post-natal development and health outcomes. Developmental toxicity caused by exposure to mixture of environmental pollutants has become a major health concern. Human-made chemicals, including xenoestrogens, pesticides and heavy metals, as well as unhealthy lifestyle behaviors, mainly tobacco smoking, alcohol consumption and medical drug abuse, are major factors that adversely influence prenatal development and increase the susceptibility of offspring to development complications and diseases. There is evidence to suggest that the developmental toxicological mechanisms of human-made chemicals and unhealthy lifestyle factors involve the generation of reactive oxygen species (ROS) and cellular oxidative damage. Overproduction of ROS induces oxidative stress, a state where increased generation of ROS overwhelms antioxidant protection and subsequently leads to oxidative damage of cellular macromolecules. Exposure to various environmental pollutants induces synergic and cumulative dose-additive adverse effects on prenatal development, pregnancy outcomes and neonate health. Data from the literature on the involvement of oxidative stress in the mechanism of developmental toxicity following in vivo exposure to environmental pollutants will be reviewed in an attempt to provide an updated basis for future studies on the toxic effect of such pollutants, particularly the notion of increased risk for developmental toxicity due to combined and cumulative exposure to various environmental pollutants. The aims of such studies are to better understand the mechanisms by which environmental pollutants adversely affect conceptus development and to elucidate the impact of cumulative exposures to multiple pollutants on postnatal development and health outcomes.

Roles of antioxidant enzymes in corpus luteum rescue from reactive oxygen species-induced oxidative stress.

Progesterone produced by the corpus luteum (CL) regulates the synthesis of various endometrial proteins required for embryonic implantation and development. Compromised CL progesterone production is a potential risk factor for prenatal development. Reactive oxygen species (ROS) play diverse roles in mammalian reproductive biology. ROS-induced oxidative damage and subsequent adverse developmental outcomes constitute important issues in reproductive medicine. The CL is considered to be highly exposed to locally produced ROS due to its high blood vasculature and steroidogenic activity. ROS-induced apoptotic cell death is involved in the mechanisms of CL regression that occurs at the end of the non-fertile cycle. Luteal ROS production and propagation depend upon several regulating factors, including luteal antioxidants, steroid hormones and cytokines, and their crosstalk. However, it is unknown which of these factors have the greatest contribution to the maintenance of CL integrity and function during the oestrous/menstrual cycle. There is evidence to suggest that antioxidants play important roles in CL rescue from luteolysis when pregnancy ensues. As luteal phase defect impacts fertility by preventing implantation and early conceptus development in livestock and humans, this review attempts to address the importance of ROS-scavenging antioxidant enzymes in the control of mammalian CL function and integrity. The corpus luteum (CL) is a transient endocrine organ that develops after ovulation from the ovulated follicle during each reproductive cycle. The main function of the CL is the production and secretion of progesterone which is necessary for embryonic implantation and development. Compromised CL progesterone production is a potential risk factor for prenatal development and pregnancy outcomes. Reactive oxygen species (ROS), which are natural by-products of cellular respiration and metabolism, play diverse roles in mammalian reproductive biology. ROS-induced oxidative damage and subsequent development of adverse pregnancy outcomes constitute important issues in reproductive medicine. Before the end of the first trimester, a high rate of human and animal conceptions end in spontaneous abortion and most of these losses occur at the time of implantation in association with ROS-induced oxidative damage. Every cell in the body is normally able to defend itself against the oxidative damage caused by the ROS. The cellular antioxidant enzymes constitute the first line of defence against the toxic effects of ROS. The CL is considered to be highly exposed to locally produced ROS due to its high blood vasculature and metabolic activity. There is now evidence to suggest that cellular antioxidants play important roles in CL rescue from regression when pregnancy ensues. As defective CL function impacts fertility by preventing implantation and early conceptus development in livestock and humans, this review attempts to address the importance of antioxidant enzymes in the control of mammalian CL function and integrity.

Mitochondria: omega-3 in the route of mitochondrial reactive oxygen species.

Mitochondria are the main organelles that produce reactive oxygen species (ROS). Overproduction of ROS induces oxidative damage to macromolecules, including lipids, and can damage cellular membrane structure and functions. Mitochondria, the main target of ROS-induced damage, are equipped with a network of antioxidants that control ROS production. Dietary intake of omega-3 polyunsaturated fatty acids (ω3PUFAs) and consequently the increase in ω3PUFA content of membrane lipids may be disadvantageous to the health because ROS-induced oxidative peroxidation of ω3PUFAs within membrane phospholipids can lead to the formation of toxic products. Mitochondrial control of lipid peroxidation is one of the mechanisms that protect cell against oxidative damage. This review discusses the role of mitochondria in ROS generation and the mechanisms by which it regulates ROS production. The susceptibility to peroxidation of PUFAs by ROS raises the question of the adverse effects of ω3PUFA dietary supplementation on embryonic development and prenatal developmental outcomes.

Antioxidative signalling pathways regulate the level of reactive oxygen species at the endometrial-extraembryonic membranes interface during early pregnancy.

Conceptus (embryo and associated extraembryonic membranes) implantation and development require a reciprocal biochemical and physical interactions between the extraembryonic membranes and the endometrium. However, the enzymatic antioxidative pathways controlling reactive oxygen species production at the endometrial-extraembryonic membrane interface early in pregnancy are not known. We aimed therefore to determine the content of malondialdehyde, as biomarkers of lipid peroxidation, and the activities of the major antioxidant enzymes, copper-zinc containing and manganese containing superoxide dismutases, catalase and glutathione peroxidase, in sheep extraembryonic membranes, caruncular and intercaruncular endometrium zones sampled at specific stages of pregnancy corresponding to the conceptus implantation (day 16) and the early post-implantation period (day 21). Malondialdehyde content in caruncular, intercaruncular and extraembryonic tissues was not different between stages of the pregnancy. Extraembryonic membranes demonstrated increased manganese containing superoxide dismutase and glutathione peroxidase activities, whereas catalase activity in these tissues decreased from day 16 to day 21. Caruncular tissues demonstrated increased manganese containing superoxide dismutase activity from day 16 to day 21. Intercaruncular tissues demonstrated increased copper-zinc containing superoxide dismutase, manganese containing superoxide dismutase and catalase activities from day 16 to day 21. The ovine extraembryonic membranes exhibit dynamic changes in enzymatic antioxidative pathways different from those of endometrial tissues during the transition from implantation to post-implantation period. This biochemical data provides novel insights into the developmental changes in antioxidative pathways of extraembryonic membranes and endometrium during early conceptus development.

Influence of gender on DHA synthesis: the response of rat liver to low dietary α-linolenic acid evidences higher ω3 ∆4-desaturation index in females.

PURPOSE: The conversion rate of α-linolenic acid (ALA) into docosahexaenoic acid (DHA) is determined by dietary and non-dietary factors. Higher capacity of DHA synthesis has been evidenced in females, indicating that sex factors influence the conversion pathway. To evaluate the extent to which sexual dimorphism of DHA synthesis is subordinated to nutritional handling, we measured the ω3 ∆4-desaturation index in male and female rats receiving adequate or inadequate amounts of ALA. The ω3 ∆4-desaturation index was drawn from the DHA to docosapentaenoic acid (ω3DPA) ratio in liver phospholipids. METHODS: Male and female rats born to ω3-deficient dams were fed a supplemented diet supplying low, inadequate, intermediate, or adequate ALA (5, 20, 100, or 300 mg ALA/100 g diet, respectively). Control rats from both gender received the adequate diet from fetal life. RESULTS: Compared with control, low ALA feeding induced the ω3 ∆4-desaturation index to increase by 38 and 70% in the phosphatidylethanolamine fraction of males and females, respectively, and by 67% in phosphatidylcholine in females only. Supplementations with increased doses of ALA progressively smoothed this gender effect. Moreover, the analysis of our data from a previous study shows that ovariectomy decreased, whereas estradiol treatment increased the ω3 index to values comparable with those of diet-matched males and intact females, respectively. CONCLUSION: Females are more prone than males to increase their index of ω3 ∆4-desaturation, especially in response to low supplies in ALA. Estradiol supports the ω3 index, suggesting that this hormone plays a role in the effect of gender on DHA synthesis.

Omega-3 fatty acids enhance mitochondrial superoxide dismutase activity in rat organs during post-natal development.

The protection of the developing organism from oxidative damage is ensured by antioxidant defense systems to cope with reactive oxygen species (ROS), which in turn can be influenced by dietary polyunsaturated fatty acids (PUFAs). PUFAs in membrane phospholipids are substrates for ROS-induced peroxidation reactions. We investigated the effects of dietary supplementation with omega-3 PUFAs on lipid peroxidation and antioxidant enzyme activities in rat cerebrum, liver and uterus. Pups born from dams fed a diet low in omega-3 PUFAs were fed at weaning a diet supplying low α-linolenic acid (ALA), adequate ALA or enriched with eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA). Malondialdehyde (MDA), a biomarker of lipid peroxidation, and the activities of superoxide dismutase 1 (SOD1), SOD2, catalase (CAT) and glutathione peroxidase (GPX) were determined in the three target organs. Compared to low ALA feeding, supplementation with adequate ALA or with EPA+DHA did not affect the cerebrum MDA content but increased MDA content in liver. Uterine MDA was increased by the EPA+DHA diet. Supplementation with adequate ALA or EPA+DHA increased SOD2 activity in the liver and uterus, while only the DHA diet increased SOD2 activity in the cerebrum. SOD1, CAT and GPX activities were not altered by ALA or EPA+DHA supplementation. Our data suggest that increased SOD2 activity in organs of the growing female rats is a critical determinant in the tolerance to oxidative stress induced by feeding a diet supplemented with omega-3 PUFAs. This is may be a specific cellular antioxidant response to ROS production within the mitochondria.

Ovine corpus luteum proteins, with functions including oxidative stress and lipid metabolism, show complex alterations during implantation.

Progesterone (P(4)) secreted by the corpus luteum (CL) is critical for in utero embryo survival and development, although CL proteins are key regulatory factors during the luteal phase. We, therefore, characterised protein expression patterns in ovine CL of pregnancy (days 12, 16 and 20) compared with those of controls, CL of oestrous cycle (days 12 and 16), using two-dimensional gel electrophoresis (2DE) gel-based proteomics. Proteins in 24 significantly altered spots were identified by tandem mass spectroscopy. At the time of embryo implantation (day 16), 77 spots were up-regulated and 101 spots were down-regulated in CL of pregnancy compared with regressed CL. Vimentin, lamin A/C (LMNA), [Mn] superoxide dismutase (SOD2), isocitrate dehydrogenase 1, annexin A1 and elongation factor Tu, mitochondrial (TUFM) altered during CL regression, whereas glutathione S-transferase A1, apolipoprotein A-1, myxovirus resistance protein 1, ornithine aminotransferase and enoyl-CoA hydratase, mitochondrial (ECHS1) tended to be altered during CL maintenance. biliverdin reductase B (BLVRB), FDXR, guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2 (GNB2) and cytochrome b-c1 complex subunit 1, mitochondrial (UQCRC1) showed divergent expression during CL regression and maintenance. The expression of two representative proteins, SOD2 and BLVRB, by western blot increased in CL of non-pregnant ewes on day 16 compared with that on day 12. SOD2 and BLVRB were localised in the large and small luteal cells and endothelial cells of CL over peri-implantation periods. 2DE gel and mass spectrometry have been used, for the first time, to study ovine CL function. We have identified proteins involved in key pathways, including oxidative stress, steroidogenesis, signal transduction and apoptosis, which have not previously been associated with changes occurring in the CL during the peri-implantation period. These proteins are most likely involved with mechanisms allowing the CL to produce P(4) during early pregnancy.

Ovariectomy and 17ß-estradiol alter transcription of lipid metabolism genes and proportions of neo-formed n-3 and n-6 long-chain polyunsaturated fatty acids differently in brain and liver.

Hormonal and nutritional factors regulate the metabolism of long-chain polyunsaturated fatty acids (LC-PUFA). We aimed to determine whether ovarian hormones influence the capacity of rats to synthesize the end-products 22:6n-3 (DHA) and 22:5n-6 (n-6DPA) from their respective dietary precursors (18:3n-3 and 18:2n-6), and can regulate PUFA conversion enzymes gene transcription in brain and/or liver. Females born with a low DHA status were fed from weaning to 8 weeks of age a diet providing both essential precursors, and were concurrently submitted to sham-operated control (SOC) or ovariectomy (OVX) in combination with or without 17ß-estradiol (E2) dosed at 8 or 16 µg/day. Relative to SOC, OVX increased the hepatic Δ9-, Δ6- and Δ5-desaturase transcripts and cognate transcription factors (PPARα, PPARγ, RXRα, RARα), but it did not affect LC-PUFA contents in phospholipids. In comparison with SOC and OVX groups, both E2 doses prevented the increase of transcripts, while paradoxically augmenting DHA and n-6DPA in liver phospholipids. Thus, in the liver of rats undergoing ovariectomy, changes of LC-PUFA synthesizing enzyme transcripts and of LC-PUFA proportions were not correlated. In brain, ovariectomy did not modify the transcripts of lipid metabolism genes, but it decreased DHA (-15%) and n-6DPA (-28%). In comparison with SOC and OVX groups, ovariectomized females treated with E2 preserved their status of both LC-PUFA in brain and had increased transcripts of E2 receptor ß, PPARδ, RARα and LC-PUFA synthesizing enzymes. In conclusion, E2 sustained the transcription of lipid metabolism genes and proportions of neo-formed DHA and n-6DPA differently in brain and liver.

The roles of cellular reactive oxygen species, oxidative stress and antioxidants in pregnancy outcomes.

Reactive oxygen species (ROS) are generated as by-products of aerobic respiration and metabolism. Mammalian cells have evolved a variety of enzymatic mechanisms to control ROS production, one of the central elements in signal transduction pathways involved in cell proliferation, differentiation and apoptosis. Antioxidants also ensure defenses against ROS-induced damage to lipids, proteins and DNA. ROS and antioxidants have been implicated in the regulation of reproductive processes in both animal and human, such as cyclic luteal and endometrial changes, follicular development, ovulation, fertilization, embryogenesis, embryonic implantation, and placental differentiation and growth. In contrast, imbalances between ROS production and antioxidant systems induce oxidative stress that negatively impacts reproductive processes. High levels of ROS during embryonic, fetal and placental development are a feature of pregnancy. Consequently, oxidative stress has emerged as a likely promoter of several pregnancy-related disorders, such as spontaneous abortions, embryopathies, preeclampsia, fetal growth restriction, preterm labor and low birth weight. Nutritional and environmental factors may contribute to such adverse pregnancy outcomes and increase the susceptibility of offspring to disease. This occurs, at least in part, via impairment of the antioxidant defense systems and enhancement of ROS generation which alters cellular signalling and/or damage cellular macromolecules. The links between oxidative stress, the female reproductive system and development of adverse pregnancy outcomes, constitute important issues in human and animal reproductive medicine. This review summarizes the role of ROS in female reproductive processes and the state of knowledge on the association between ROS, oxidative stress, antioxidants and pregnancy outcomes in different mammalian species.

Antioxidant adaptive responses of extraembryonic tissues from cloned and non-cloned bovine conceptuses to oxidative stress during early pregnancy.

Placental oxidative stress has been suggested as a key factor in early pregnancy failure. Abnormal placental development limits success in pregnancies obtained by somatic cell nuclear transfer (SCNT). Malondialdehyde (MDA) content, an index of oxidative stress, and superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) activities were determined in bovine extraembryonic tissues of SCNT or artificial insemination (AI) conceptuses. Chorionic tissues of SCNT and AI conceptuses show no difference in MDA content at day 32 of pregnancy. MDA content in chorionic tissues of SCNT and AI conceptuses decreased from day 32 to 62 of pregnancy. MDA content was lower in chorionic tissues of SCNT conceptuses than that in chorionic tissues of AI conceptuses at day 62 of pregnancy. SOD1, SOD2 and GPX activities in chorionic tissues of SCNT conceptuses were not different from those in chorionic tissues of AI conceptuses at both gestational ages. CAT activity in chorionic tissues of SCNT conceptuses was lower at day 32, and it was higher at day 62 of pregnancy than that in chorionic tissues of AI conceptuses. CAT and GPX activities increased in chorionic tissues of SCNT conceptuses with gestational age. SOD1 activity decreased while that of SOD2 and GPX increased in chorionic tissues of AI conceptuses with gestational age. At day 62 of pregnancy, MDA content and enzyme activities in cotyledonary tissues were not different between AI and SCNT conceptuses. Different antioxidant mechanisms may operate within the chorion of AI and SCNT conceptuses. Further experiments are required to elucidate this point.

Developmental changes in antioxidant enzymatic defences against oxidative stress in sheep placentomes.

Early pregnancy is susceptible to oxidative stress, and thus characterisation of antioxidant systems and pro- and anti-apoptotic pathways would improve understanding of placental development and function. We aimed, therefore, to determine the activities of the antioxidant enzymes, copper/zinc-superoxide dismutase (SOD1), manganese-SOD (SOD2), catalase (CAT), glutathione (GSH) peroxidase (GPX) and GSH reductase (GSR); and to quantify the expression of BAX and MCL1 proteins in relation to the developmental changes in antioxidant defences in sheep placentomes sampled on days 35, 55 and 80 of pregnancy. Placentome progesterone content was analyzed to determine steroidogenic capacity. Malondialdehyde (MDA) and protein carbonyl were quantified in placentomes as biomarkers of lipid peroxidation and protein damage respectively. Placentome tissues demonstrated significantly increased content of progesterone and MDA at day 80 of pregnancy and protein carbonyl as early as day 50 of pregnancy. Progesterone and MDA contents were not different between days 35 and 55 of pregnancy. While SOD1 and CAT activities did not alter significantly, SOD2 activity decreased from days 35 to 55. GPX activity increased from days 35 to 55 and increased further to day 80 of pregnancy. GSR activity increased from days 35 to 55 of pregnancy. BAX protein expression decreased, while MCL1 increased from days 35 to 55 and 80 of pregnancy. The increased GPX activity was associated with a decrease in the BAX/MCL1 protein expression ratio. Changes in the antioxidant enzymatic defences could be a part of placentome adaptation to reactive oxygen species-induced oxidative stress at specific early developmental stages of pregnancy.

Regulation of key antioxidant enzymatic systems in the sheep endometrium by ovarian steroids.

Reactive oxygen species (ROS) and their control by antioxidant enzymes are involved in the physiology of the female reproductive system. Thus, it is important to understand the regulation of key antioxidant enzymatic pathways. The roles of estrogen and progesterone in regulating the physiological functions of the endometrium have become central dogma. We examined the effects of ovarian steroids on superoxide dismutases (SOD1 and SOD2), catalase (CAT), glutathione peroxidase (GPX), and glutathione reductase (GSR) activities in the aglandular caruncular and glandular inter-caruncular endometrial tissues of ovariectomized (OVX) ewes and in OVX ewes treated with estradiol (E2), progesterone (P4), or both hormones according to schedules designed to produce physiological changes of these hormones during the estrous cycle. The activities SOD2, CAT, GPX and GSR in both endometrial tissues were unaffected by P4 treatment. The activity of SOD1 in the aglandular tissue was unaffected by P4 treatment, however this treatment decreased SOD1 activity in the glandular tissue (P < 0.01). Treatment with E2, either alone or in combination with P4, decreased SOD1 (P < 0.01), CAT (P < 0.01) and GPX (P < 0.05) activities in both endometrial tissues. The activity of GSR decreased only in the glandular tissue (P < 0.05) after E2 treatment, either alone or in combination with P4. No change in SOD2 activity was detected in both endometrial tissues after administration of E2, P4 or both hormones. This study provides the first firm evidence for the role of ovarian steroid hormones in the regulation of the activities of key antioxidant enzyme in the endometrium of female mammals.