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Hypoxia may influence normal and different pathological processes. Low oxygenation activates a variety of responses, many of them regulated by hypoxia-inducible factor 1 complex, which is mostly involved in cellular control of O2 consumption and delivery, inhibition of growth and development, and promotion of anaerobic metabolism. Hypoxia plays a significant physiological role in fetal development; it is involved in different embryonic processes, for example, placentation, angiogenesis, and hematopoiesis. More recently, fetal hypoxia has been associated directly or indirectly with fetal programming of heart, brain, and kidney function and metabolism in adulthood. In this review, the role of hypoxia in fetal development, placentation, and fetal programming is summarized. Hypoxia is a basic mechanism involved in different pregnancy disorders and fetal health developmental complications. Although there are scientific data showing that hypoxia mediates changes in the growth trajectory of the fetus, modulates gene expression by epigenetic mechanisms, and determines the health status later in adulthood, more mechanistic studies are needed. Furthermore, if we consider that intrauterine hypoxia is not a rare event, and can be a consequence of unavoidable exposures to air pollution, nutritional deficiencies, obesity, and other very common conditions (drug addiction and stress), the health of future generations may be damaged and the incidence of some diseases will markedly increase as a consequence of disturbed fetal programming. Birth Defects Research 109:1377-1385, 2017.© 2017 Wiley Periodicals, Inc.

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Brooding in invertebrates serves to protect embryos from stressful external conditions by retaining progeny inside the female body, effectively reducing the risk of pelagic stages being exposed to predation or other environmental stressors, but with accompanying changes in pallial fluid characteristics, including reduced oxygen availability. Brooded embryos are usually immobile and often encapsulated, but in some Ostrea species the embryos move freely inside the female pallial cavity in close association with the mother's gills for as long as eight weeks. We used endoscopic techniques to characterize the circulation pattern of embryos brooded by females of the oyster, Ostrea chilensis. Progeny at embryonic and veliger stages typically circulated in established patterns that included the use of dorsal and ventral food grooves (DFG, VFG) to move anteriorly on the gills. Both embryos and veligers accumulated around the mother's palps, and remained there until an active maternal countercurrent moved them to the gill inhalant area. Both food grooves were able to move embryos, veligers, and food-particle aggregates anteriorly, but the DFG was more important in progeny transport; early embryos were moved more rapidly than veligers in the DFG. A microcirculation pattern of embryos was apparent when they were moved by gill lamellae: when they were close to the VFG, most embryos lost gill contact and "fell" down to the DFG. Those that actually reached the DFG moved anteriorly, but others came into contact with the base of the lamellae and again moved towards the VFG. The circulation pattern of the progeny appears well-suited for both cleaning them and directing them posteriorly to an area where there is more oxygen and food than in the palp region. This process for actively circulating progeny involves the feeding structures (gill and palps) and appears to be energetically costly for the female. It also interferes with feeding, which could explain the poor energy balance previously documented for brooding females of this species.

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This comparative study of gill morphometrics in near-term embryos of freshwater stingray potamotrygonids examines gill dimensions in relation to neonatal lifestyle and habitat. In embryos of the potamotrygonids Paratrygon aiereba, Plesiotrygon iwamae, Potamotrygon motoro, Potamotrygon orbignyi, and cururu ray Potamotrygon sp. the number and length of filaments, total gill surface area, mass-specific surface area, water-blood diffusion distance, and anatomical diffusion factor were analysed. In all potamotrygonids, the 3rd branchial arch possessed a larger respiratory surface than the other gill arches. Larger embryos had more gill surface area and large spiracles, which are necessary to maintain the high oxygen uptake needed due to their larger body size. However, the higher mass-specific gill surface area observed in near-term embryos may be advantageous because neonates can use hypoxic environments as refuges against predators, as well as catch small prey that inhabit the same environment. As expected from their benthic mode of life, freshwater stingrays are sluggish animals compared to pelagic fishes. However, based on gill respiratory morphometry (such as gill area, mass-specific gill area, the water-blood diffusion barrier, anatomical diffusion factor, and relative opening of the spiracle), subtypes of lifestyles can be observed corresponding to: active, intermediate, and sluggish species according to Gray's scale.

Este estudo realizado com embriões a termo de arraias de água doce (Potamotrygonidae) compara e analisa as dimensões branquiais em relação ao estilo de vida e habitat dos neonatos. Nos embriões de Paratrygon aiereba, Plesiotrygon iwamae, Potamotrygon motoro, Potamotrygon orbignyi e Potamotrygon sp. (arraia cururu) foram analisados número e comprimento dos filamentos, área branquial, área superficial branquial massa-específica, barreira de difusão água-sangue e fator de difusão anatômico. Em todos os potamotrigonídeos estudados, o 3º arco branquial possui uma superfície respiratória maior que os demais arcos. Embriões de espécies de maior porte possuem grandes espiráculos e maior área de superfície branquial. Isso ajuda a manter a taxa de absorção de oxigênio proporcional ao requerimento do animal. No entanto, a grande área de superfície branquial massa-específica observadas nos embriões a termo pode ser vantajosa, pois os neonatos podem usar ambientes hipóxicos como refúgios contra predadores, bem como capturar pequenas presas que habitam o mesmo ambiente. Devido ao modo de vida bentônico, as arraias de água doce são nadadoras lentas comparadas aos peixes pelágicos. No entanto, com base na morfometria branquial (área de superfície branquial, área branquial massa-específica, barreira de difusão água-sangue, fator de difusão anatômico e abertura relativa do espiráculo), subtipos de estilos de vida podem ser observados: ativas, intermediárias e lentas, conforme escala definida por Gray.

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This comparative study of gill morphometrics in near-term embryos of freshwater stingray potamotrygonids examines gill dimensions in relation to neonatal lifestyle and habitat. In embryos of the potamotrygonids Paratrygon aiereba, Plesiotrygon iwamae, Potamotrygon motoro, Potamotrygon orbignyi, and cururu ray Potamotrygon sp. the number and length of filaments, total gill surface area, mass-specific surface area, water-blood diffusion distance, and anatomical diffusion factor were analysed. In all potamotrygonids, the 3rd branchial arch possessed a larger respiratory surface than the other gill arches. Larger embryos had more gill surface area and large spiracles, which are necessary to maintain the high oxygen uptake needed due to their larger body size. However, the higher mass-specific gill surface area observed in near-term embryos may be advantageous because neonates can use hypoxic environments as refuges against predators, as well as catch small prey that inhabit the same environment. As expected from their benthic mode of life, freshwater stingrays are sluggish animals compared to pelagic fishes. However, based on gill respiratory morphometry (such as gill area, mass-specific gill area, the water-blood diffusion barrier, anatomical diffusion factor, and relative opening of the spiracle), subtypes of lifestyles can be observed corresponding to: active, intermediate, and sluggish species according to Gray's scale.(AU)

Este estudo realizado com embriões a termo de arraias de água doce (Potamotrygonidae) compara e analisa as dimensões branquiais em relação ao estilo de vida e habitat dos neonatos. Nos embriões de Paratrygon aiereba, Plesiotrygon iwamae, Potamotrygon motoro, Potamotrygon orbignyi e Potamotrygon sp. (arraia cururu) foram analisados número e comprimento dos filamentos, área branquial, área superficial branquial massa-específica, barreira de difusão água-sangue e fator de difusão anatômico. Em todos os potamotrigonídeos estudados, o 3º arco branquial possui uma superfície respiratória maior que os demais arcos. Embriões de espécies de maior porte possuem grandes espiráculos e maior área de superfície branquial. Isso ajuda a manter a taxa de absorção de oxigênio proporcional ao requerimento do animal. No entanto, a grande área de superfície branquial massa-específica observadas nos embriões a termo pode ser vantajosa, pois os neonatos podem usar ambientes hipóxicos como refúgios contra predadores, bem como capturar pequenas presas que habitam o mesmo ambiente. Devido ao modo de vida bentônico, as arraias de água doce são nadadoras lentas comparadas aos peixes pelágicos. No entanto, com base na morfometria branquial (área de superfície branquial, área branquial massa-específica, barreira de difusão água-sangue, fator de difusão anatômico e abertura relativa do espiráculo), subtipos de estilos de vida podem ser observados: ativas, intermediárias e lentas, conforme escala definida por Gray.(AU)

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To analyze the interconnection between erythropoiesis and iron metabolism, one of the issues raised in this study was to know iron bioavailability under physiopathological conditions. Our aim was to understand the functional axis response composed of erythropoietin (Epo)-hepcidin-ferroportin (FPN), when 2 dysfunctional states coexist, using an animal model of iron overload followed by hypoxia. FPN and prohepcidin were assessed by immunohistochemistry using rabbit anti-mouse FPN polyclonal and prohepcidin monoclonal antibodies. Goat-labeled polymer - horseradish peroxidase anti-rabbit EnVision + System (DAB) was used as the secondary antibody. Epo levels were measured by ELISA. Tissue iron was studied by Prussian blue iron staining. Erythropoietic response was assessed using conventional hematological tests. Iron overload increased prohepcidin that remained high in hypoxia, coexisting with high levels of Epo in hypoxia, with or without iron overload. In hypoxia, FPN was clearly evident in reticuloendothelial macrophages, more than in hypoxia with iron overload. Interestingly, duodenal FPN was clearly identified on the basolateral membrane in hypoxia, with or without iron overload. Our data indicate that 2 signals could induce the cell-specific response as follows: (i) iron signal, induced prohepcidin, which reduced reticuloendothelial FPN and reduced iron availability; and (ii) hypoxia signal, stimulated Epo, which affected iron absorption by stabilizing duodenal FPN and allowed iron supply to erythropoiesis independently of store size.

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Com o intuito de investigar se a hipóxia representa um dos desencadeadores da regressão luteínica em cadelas não prenhes, o presente estudo foi delineado para analisar a expressão do fator transcricional indutível por hipóxia HIF1A e a de seus genes-alvo relacionados à angiogênese, como o fator de crescimento endotelial vascular (VEGFA) e à captação de glicose, como as proteínas transportadoras facilitadoras GLUT1/SLC2A1 e GLUT4/SLC2A4 no corpo lúteo canino ao longo do diestro (dias 10 a 70 após a ovulação). Para tal, utilizou-se imuno-histoquímica e western blotting para localizar e quantificar as proteínas do HIF1A, GLUT1 e GLUT4 e PCR em tempo real para quantificar a expressão do RNAm de HIF1A, SLC2A1, SLC2A4, VEGFA, FLT1 e KDR. Além disso, células luteínicas nas fases inicial (dia 10), média (dia 30) e final (dia 60) foram submetidas ao tratamento com cloreto de cobalto (CoCl2) a 500 µM para avaliar os efeitos da hipóxia sobre a expressão gênica dos fatores acima citados, bem como sobre a produção de progesterona e 17β-estradiol. Nossos resultados demonstraram que o HIF1A é expresso pelo corpo lúteo canino de maneira tempo-dependente ao longo do diestro, e que a expressão de seu RNAm está diretamente correlacionada a expressão gênica de SLC2A1, SLC2A4, VEGFA, FLT1 e KDR e com as concentrações de progesterona periférica. No cultivo primário de células luteínicas, a hipóxia induzida pelo CoCl2 diminuiu a produção de progesterona e de 17β-estradiol e estimulou significativamente a expressão de HIF1A, SLC2A1, SLC2A4 e VEGFA. Esses resultados sugerem que o HIF1A constitui um dos fatores regulatórios da função do corpo lúteo canino participando da modulação de processos como esteroidogêne, angiogênese e da captação de glicose, atuando como fator luteolítico

This study was designed to investigate if hypoxia is one of the triggers of luteal regression in non-pregnant bitches. For that, we analyzed the hypoxia- inducible factor (HIF1A) expression as well as the expression of its target genes related to angiogenesis (vascular endothelial growth factor VEGFA) and to glucose uptake (glucose transporters GLUT/SLC2A 1 and 4) in canine corpus luteum throughout diestrus (days 10 to 70 after ovulation). We used immunohistochemistry and western blotting to localize and quantify the protein expression of HIF1A, GLUT1 and GLUT4, respectively, and real time PCR to analyze HIF1A, SLC2A1, SLC2A4, VEGFA, FLT1 and KDR gene expression. Moreover, luteal cells from early (day 10), mid (day 30) and late luteal phase (day 60) were submitted to 500 µM cobalt chloride (CoCl2) treatment to verify hypoxia effects on gene expression of the above cited genes and on progesterone and 17β-estradiol production. Our results showed that luteal cells expressed HIF1A in a time-dependent manner over diestrus and that its expression was directly correlated to both SLC2A1, SLC2A4, VEGFA, FLT1 and KDR gene expression and progesterone production. The protein expression of the studied genes also changed over diestrus and was correlated with the respective gene expression. In primary luteal cells culture, cobalt chloride-induced hypoxia downregulated progesterone and 17β-estradiol production, but upregulated HIF1A, SLC2A1, SLC2A4 and VEGFA gene expression. These findings suggest that HIF1A is one of the factors regulating canine luteal function by modulating important process as steroidogenesis, angiogenesis and glucose uptake, acting as a pro-survival factor

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The molecular pathways underlying the neuroprotective effects of preconditioning are promising, potentially drugable targets to promote cell survival. However, these pathways are complex and are not yet fully understood. In this study we have established a paradigm of hypoxic preconditioning based on a chick embryo model of normobaric acute hypoxia previously developed by our group. With this model, we analyzed the role of hypoxia-inducible factor-1α (HIF-1α) stabilization during preconditioning in HIF-1 signaling after the hypoxic injury and in the development of a neuroprotective effect against the insult. To this end, we used a pharmacological approach, based on the in vivo administration of positive (Fe(2+), ascorbate) and negative (CoCl(2)) modulators of the activity of HIF-prolyl hydroxylases (PHDs), the main regulators of HIF-1. We have found that preconditioning has a reinforcing effect on HIF-1 accumulation during the subsequent hypoxic injury. In addition, we have also demonstrated that HIF-1 induction during hypoxic preconditioning is necessary to obtain an enhancement in HIF-1 accumulation and to develop a tolerance against a subsequent hypoxic injury. We provide in vivo evidence that administration of Fe(2+) and ascorbate modulates HIF accumulation, suggesting that PHDs might be targets for neuroprotection in the CNS.

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The pregnant llama (Lama glama) has walked for millions of years through the thin oxygen trail of the Andean altiplano. We hypothesize that a pool of genes has been selected in the llama that express efficient mechanisms to withstand this low-oxygen milieu. The llama fetus responds to acute hypoxia with an intense peripheral vasoconstriction that is not affected by bilateral section of the carotid sinus nerves. Moreover, the increase in fetal plasma concentrations of vasoconstrictor hormones, such as catecholamines, neuropeptide Y, and vasopressin, is much greater in the llama than in the sheep fetus. Furthermore, treatment of fetal llamas with an alpha-adrenergic antagonist abolished the peripheral vasoconstriction and resulted in fetal cardiovascular collapse and death during acute hypoxia, suggesting an indispensable upregulation of alpha-adrenergic mechanisms in this high altitude species. Local endothelial factors such as nitric oxide (NO) also play a key role in the regulation of fetal adrenal blood flow and in the adrenal secretion of catecholamines and cortisol. Interestingly, in contrast to the human or sheep fetus, the llama fetus showed a small increase in brain blood flow during acute hypoxia, with no increase in oxygen extraction across the brain, and thereby a decrease in brain oxygen consumption. These results suggest that the llama fetus responds to acute hypoxia with hypometabolism. How this reduction in metabolism is produced and how the cells are preserved during this condition remain to be elucidated.

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Unlike fetal animals of lowland species, the llama fetus does not increase its cerebral blood flow during an episode of acute hypoxaemia. This study tested the hypothesis that the fetal llama brain maintains cerebral hemispheric O2 consumption by increasing cerebral O2 extraction rather than decreasing cerebral oxygen utilisation during acute hypoxaemia. Six llama fetuses were surgically instrumented under general anaesthesia at 217 days of gestation (term ca 350 days) with vascular and amniotic catheters in order to carry out cardiorespiratory studies. Following a control period of 1 h, the llama fetuses underwent 3 x 20 min episodes of progressive hypoxaemia, induced by maternal inhalational hypoxia. During basal conditions and during each of the 20 min of hypoxaemia, fetal cerebral blood flow was measured with radioactive microspheres, cerebral oxygen extraction was calculated, and fetal cerebral hemispheric O2 consumption was determined by the modified Fick principle. During hypoxaemia, fetal arterial O2 tension and fetal pH decreased progressively from 24 +/- 1 to 20 +/- 1 Torr and from 7.36 +/- 0.01 to 7.33 +/- 0.01, respectively, during the first 20 min episode, to 16 +/- 1 Torr and 7.25 +/- 0.05 during the second 20 min episode and to 14 +/- 1 Torr and 7.21 +/- 0.04 during the final 20 min episode. Fetal arterial partial pressure of CO2 (P(a,CO2), 42 +/- 2 Torr) remained unaltered from baseline throughout the experiment. Fetal cerebral hemispheric blood flow and cerebral hemispheric oxygen extraction were unaltered from baseline during progressive hypoxaemia. In contrast, a progressive fall in fetal cerebral hemispheric oxygen consumption occurred during the hypoxaemic challenge. In conclusion, these data do not support the hypothesis that the fetal llama brain maintains cerebral hemispheric O2 consumption by increasing cerebral hemispheric O2 extraction. Rather, the data show that in the llama fetus, a reduction in cerebral hemispheric metabolism occurs during acute hypoxaemia.

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The Central Nervous System is known to be critically affected in the prenatal-perinatal period by hypoxic-ischemic insults, which produce several disorders such as loss of neural projections, increased susceptibility to seizures, apoptosis and an imbalance in normal activity of glutamatergic and GABAergic neurones, resulting in acute cell excitotoxicity. The aim of the present work was to establish a chick embryo model of normobaric acute hypoxic hypoxia as well as to evaluate modifications in GABA(A) receptor complex from chick optic lobe, that may result from this injury. Fertile chicken (Gallus gallus domesticus) eggs from White Leghorn were incubated and at embryonic days (ED) 12 to 18, subjected to a stream of 8%O(2)/92%N(2) during1 h, and then were either returned to their shelves in the incubator for recovery, or immediately processed for biochemical studies. Hypoxic treatment produced a significant age dependent reduction in GABA binding sites showing the greatest decrease at the earliest stages studied (ED12-ED16). Saturation curves of GABA binding performed at ED12 showed a decrease in B(max), (control, 5.48+/-0.20, hypoxic, 3.90+/-0.39 pmol/mg protein), but no significant change in K(d). Following 48 h in normoxic atmosphere post-hypoxia reduction in [3H]GABA binding was reversed. Pharmacological properties of GABA(A) receptor at ED12 showed that positive allosteric modulation effects of the steroid 3alpha-hydroxy-5alpha-pregnan-20-one and the barbiturate pentobarbital sodium were enhanced by the treatment. This model of acute prenatal hypoxic hypoxia produced marked alterations in inhibitory CNS neurotransmission that proved reversible and age dependent.

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