Embryonic Hypoxia Research Articles

Carryover effects of embryonic hypoxia exposure on adult fitness of the Pacific abalone

The widespread and severe drop in dissolved oxygen concentration in the open ocean and coastal waters has attracted much attention, but assessments of the impacts of environmental hypoxia on aquatic organisms have focused primarily on responses to current exposure. Past stress exposure might also affect the performance of aquatic organisms through carryover effects, and whether these effects scale from positive to negative based on exposure degree is unknown. We investigated the carryover effects of varying embryonic hypoxia levels (mediate hypoxia: 3.0−3.1 mg O2/L; severe hypoxia: 2.0−2.1 mg O2/L) on the fitness traits of adult Pacific abalone (Haliotis discus hannai), including growth, hypoxia tolerance, oxygen consumption, ammonia excretion rate, and biochemical responses to acute hypoxia. Moderate embryonic hypoxia exposure significantly improved the hypoxia tolerance of adult Pacific abalone without sacrificing growth and survival. Adult abalone exposed to embryonic hypoxia exhibited physiological plasticity, including decreased oxygen consumption rates under environmental stress, increased basal methylation levels, and a more active response to acute hypoxia, which might support their higher hypoxia tolerance. Thus, moderate oxygen declines in early life have persistent effects on the fitness of abalone even two years later, further affecting population dynamics. The results suggested that incorporating the carryover effects of embryonic hypoxia exposure into genetic breeding programs would be an important step toward rapidly improving the hypoxia tolerance of aquatic animals. The study also inspires the protection of endangered wild animals and other vulnerable species under global climate change.

Teratogenicity and Reactive Oxygen Species after transient embryonic hypoxia: Experimental and clinical evidence with focus on drugs causing failed abortion in humans

Teratogenicity and Reactive Oxygen Species after transient embryonic hypoxia: Experimental and clinical evidence with focus on drugs with human abortive potential.Reactive Oxygen Species (ROS) can be harmful to embryonic tissues. The adverse embryonic effects are dependent on the severity and duration of the hypoxic event and when during organongenesis hypoxia occurs. The vascular endothelium of recently formed arteries in the embryo is highly susceptible to ROS damage. Endothelial damage results in vascular disruption, hemorrhage and maldevelopment of organs, which normally should have been supplied by the artery. ROS can also induce irregular heart rhythm in the embryo resulting in alterations in blood flow and pressure from when the tubular heart starts beating. Such alterations in blood flow and pressure during cardiogenesis can result in a variety of cardiovascular defects, for example transpositions and ventricular septal defects. One aim of this article is to review and compare the pattern of malformations produced by transient embryonic hypoxia of various origins in animal studies with malformations associated with transient embryonic hypoxia in human pregnancy due to a failed abortion process. The results show that transient hypoxia and compounds with potential to cause failed abortion in humans, such as misoprostol and hormone pregnancy tests (HPTs) like Primodos, have been associated with a similar spectrum of teratogenicity. The spectrum includes limb reduction-, cardiovascular- and central nervous system defects. The hypoxia-ROS related teratogenicity of misoprostol and HPTs, is likely to be secondary to uterine contractions and compression of uterinoplacental/embryonic vessels during organogenesis.

Mir152 hypomethylation as a mechanism for non-syndromic cleft lip and palate

ABSTRACT Non-syndromic cleft lip with or without cleft palate (NSCLP), the most common human craniofacial malformation, is a complex disorder given its genetic heterogeneity and multifactorial component revealed by genetic, epidemiological, and epigenetic findings. Epigenetic variations associated with NSCLP have been identified; however, functional investigation has been limited. Here, we combined a reanalysis of NSCLP methylome data with genetic analysis and used both in vitro and in vivo approaches to dissect the functional effects of epigenetic changes. We found a region in mir152 that is frequently hypomethylated in NSCLP cohorts (21–26%), leading to mir152 overexpression. mir152 overexpression in human neural crest cells led to downregulation of spliceosomal, ribosomal, and adherens junction genes. In vivo analysis using zebrafish embryos revealed that mir152 upregulation leads to craniofacial cartilage impairment. Also, we suggest that zebrafish embryonic hypoxia leads to mir152 upregulation combined with mir152 hypomethylation and also analogous palatal alterations. We therefore propose that mir152 hypomethylation, potentially induced by hypoxia in early development, is a novel and frequent predisposing factor to NSCLP.

The effect of phenytoin on embryonic heart rate in Vivo

Phenytoin is a known human teratogen with unknown etiology. Several mechanisms have been proposed including disturbances in folate metabolism, induction of embryonic hypoxia following phenytoin-induced bradycardia, free radical formation following re-oxygenation and phenytoin-induced maternal hyperglycemia.Using high frequency ultrasound, we demonstrated that phenytoin induced a dramatic decrease in the heart rate of embryos. This coincided with a moderate transient decrease in maternal heart rate and blood glucose levels. Embryonic heart rate had not fully recovered 24 h later in some embryos despite normal maternal physiological parameters. In a separate study, extent of hypoxia was measured using the marker pimonidazole. Phenytoin-exposed embryos did not demonstrate increased hypoxia compared to control embryos at 2, 4, 8 or 24 h dosing. Together our results show that phenytoin induces malformations as a result of a combination of insults: embryonic bradycardia, maternal bradycardia and maternal hyperglycemia. However, this does not appear to result in measurable embryonic hypoxia in our animal model.

Common Postzygotic Mutational Signatures in Healthy Adult Tissues Related to Embryonic Hypoxia

Postzygotic mutations are acquired in normal tissues throughout an individual’s lifetime and hold clues for identifying mutagenic factors. Here, we investigated postzygotic mutation spectra of healthy individuals using optimized ultra-deep exome sequencing of the time-series samples from the same volunteer as well as the samples from different individuals. In blood, sperm, and muscle cells, we resolved three common types of mutational signatures. Signatures A and B represent clock-like mutational processes, and the polymorphisms of epigenetic regulation genes influence the proportion of signature B in mutation profiles. Notably, signature C, characterized by C>T transitions at GpCpN sites, tends to be a feature of diverse normal tissues. Mutations of this type are likely to occur early during embryonic development, supported by their relatively high allelic frequencies, presence in multiple tissues, and decrease in occurrence with age. Almost none of the public datasets for tumors feature this signature, except for 19.6% of samples of clear cell renal cell carcinoma with increased activation of the hypoxia-inducible factor 1 (HIF-1) signaling pathway. Moreover, the accumulation of signature C in the mutation profile was accelerated in a human embryonic stem cell line with drug-induced activation of HIF-1α. Thus, embryonic hypoxia may explain this novel signature across multiple normal tissues. Our study suggests that hypoxic condition in an early stage of embryonic development is a crucial factor inducing C>T transitions at GpCpN sites; and individuals’ genetic background may also influence their postzygotic mutation profiles.

Developmental programming of DNA methylation and gene expression patterns is associated with extreme cardiovascular tolerance to anoxia in the common snapping turtle

BackgroundEnvironmental fluctuation during embryonic and fetal development can permanently alter an organism’s morphology, physiology, and behaviour. This phenomenon, known as developmental plasticity, is particularly relevant to reptiles that develop in subterranean nests with variable oxygen tensions. Previous work has shown hypoxia permanently alters the cardiovascular system of snapping turtles and may improve cardiac anoxia tolerance later in life. The mechanisms driving this process are unknown but may involve epigenetic regulation of gene expression via DNA methylation. To test this hypothesis, we assessed in situ cardiac performance during 2 h of acute anoxia in juvenile turtles previously exposed to normoxia (21% oxygen) or hypoxia (10% oxygen) during embryogenesis. Next, we analysed DNA methylation and gene expression patterns in turtles from the same cohorts using whole genome bisulfite sequencing, which represents the first high-resolution investigation of DNA methylation patterns in any reptilian species.ResultsGenome-wide correlations between CpG and CpG island methylation and gene expression patterns in the snapping turtle were consistent with patterns observed in mammals. As hypothesized, developmental hypoxia increased juvenile turtle cardiac anoxia tolerance and programmed DNA methylation and gene expression patterns. Programmed differences in expression of genes such as SCN5A may account for differences in heart rate, while genes such as TNNT2 and TPM3 may underlie differences in calcium sensitivity and contractility of cardiomyocytes and cardiac inotropy. Finally, we identified putative transcription factor-binding sites in promoters and in differentially methylated CpG islands that suggest a model linking programming of DNA methylation during embryogenesis to differential gene expression and cardiovascular physiology later in life. Binding sites for hypoxia inducible factors (HIF1A, ARNT, and EPAS1) and key transcription factors activated by MAPK and BMP signaling (RREB1 and SMAD4) are implicated.ConclusionsOur data strongly suggests that DNA methylation plays a conserved role in the regulation of gene expression in reptiles. We also show that embryonic hypoxia programs DNA methylation and gene expression patterns and that these changes are associated with enhanced cardiac anoxia tolerance later in life. Programming of cardiac anoxia tolerance has major ecological implications for snapping turtles, because these animals regularly exploit anoxic environments throughout their lifespan.

Msx1 deficiency interacts with hypoxia and induces a morphogenetic regulation during mouse lip development.

Nonsyndromic clefts of the lip and palate are common birth defects resulting from gene-gene and gene-environment interactions. Mutations in human MSX1 have been linked to orofacial clefting and we show here that Msx1 deficiency causes a growth defect of the medial nasal process (Mnp) in mouse embryos. Although this defect alone does not disrupt lip formation, Msx1-deficient embryos develop a cleft lip when the mother is transiently exposed to reduced oxygen levels or to phenytoin, a drug known to cause embryonic hypoxia. In the absence of interacting environmental factors, the Mnp growth defect caused by Msx1 deficiency is modified by a Pax9-dependent 'morphogenetic regulation', which modulates Mnp shape, rescues lip formation and involves a localized abrogation of Bmp4-mediated repression of Pax9 Analyses of GWAS data revealed a genome-wide significant association of a Gene Ontology morphogenesis term (including assigned roles for MSX1, MSX2, PAX9, BMP4 and GREM1) specifically for nonsyndromic cleft lip with cleft palate. Our data indicate that MSX1 mutations could increase the risk for cleft lip formation by interacting with an impaired morphogenetic regulation that adjusts Mnp shape, or through interactions that inhibit Mnp growth.

Embryonic developmental oxygen preconditions cardiovascular functional response to acute hypoxic exposure and maximal β-adrenergic stimulation of anesthetized juvenile American alligators (Alligator mississippiensis).

The effects of the embryonic environment on juvenile phenotypes are widely recognized. We investigated the effect of embryonic hypoxia on the cardiovascular phenotype of 4-year-old American alligators (Alligator mississippiensis). We hypothesized that embryonic 10% O2 preconditions cardiac function, decreasing the reduction in cardiac contractility associated with acute 5% O2 exposure in juvenile alligators. Our findings indicate that dobutamine injections caused a 90% increase in systolic pressure in juveniles that were incubated in 21% and 10% O2, with the 10% O2 group responding with a greater rate of ventricular relaxation and greater left ventricle output compared with the 21% O2 group. Further, our findings indicate that juvenile alligators that experienced embryonic hypoxia have a faster rate of ventricular relaxation, greater left ventricle stroke volume and greater cardiac power following β-adrenergic stimulation, compared with juvenile alligators that did not experience embryonic hypoxia. When juveniles were exposed to 5% O2 for 20 min, normoxic-incubated juveniles had a 50% decline in left ventricle maximal rate of pressure development and maximal pressure; however, these parameters were unaffected and decreased less in the hypoxic-incubated juveniles. These data indicate that embryonic hypoxia in crocodilians alters the cardiovascular phenotype, changing the juvenile response to acute hypoxia and β-adrenergic stimulation.

Parabronchial remodeling in chicks in response to embryonic hypoxia.

The embryonic development of parabronchi occurs mainly during the second half of incubation in precocious birds, which makes this phase sensitive to possible morphological modifications induced by O2 supply limitation. Thus, we hypothesized that hypoxia during the embryonic phase of parabronchial development induces morphological changes that remain after hatching. To test this hypothesis, chicken embryos were incubated entirely (21 days) under normoxia or partially under hypoxia (15% O2 during days 12 to 18). Lung structures, including air capillaries, blood capillaries, infundibula, atria, parabronchial lumen, bronchi, blood vessels larger than capillaries and interparabronchial tissue, in 1- and 10-day-old chicks were analyzed using light microscopy-assisted stereology. Tissue barrier and surface area of air capillaries were measured using electron microscopy-assisted stereology, allowing for calculation of the anatomical diffusion factor. Hypoxia increased the relative volumes of air and blood capillaries, structures directly involved in gas exchange, but decreased the relative volumes of atria in both groups of chicks, and the parabronchial lumen in older chicks. Accordingly, the surface area of the air capillaries and the anatomical diffusion factor were increased under hypoxic incubation. Treatment did not alter total lung volume, relative volumes of infundibula, bronchi, blood vessels larger than capillaries, interparabronchial tissue or the tissue barrier of any group. We conclude that hypoxia during the embryonic phase of parabronchial development leads to a morphological remodeling, characterized by increased volume density and respiratory surface area of structures involved in gas exchange at the expense of structures responsible for air conduction in chicks up to 10 days old.

Early Gestational Hypoxia and Adverse Developmental Outcomes.

Hypoxia is a normal and essential part of embryonic development. However, this state may leave the embryo vulnerable to damage when oxygen supply is disturbed. Embryofetal response to hypoxia is dependent on duration and depth of hypoxia, as well as developmental stage. Early postimplantation rat embryos were resilient to hypoxia, with many surviving up to 1.5 hr of uterine clamping, while most mid-gestation embryos were dead after 1 hour of clamping. Survivors were small and many had a range of defects, principally terminal transverse limb reduction defects. Similar patterns of malformations occurred when embryonic hypoxia was induced by maternal hypoxia, interruption of uteroplacental flow, or perfusion and embryonic bradycardia. There is good evidence that high altitude pregnancies are associated with smaller babies and increased risk of some malformations, but these results are complicated by increased risk of pre-eclampsia. Early onset pre-eclampsia itself is associated with small for dates and increased risk of atrio-ventricular septal defects. Limb defects have clearly been associated with chorionic villus sampling, cocaine, and misoprostol use. Similar defects are also observed with increased frequency among fetuses who are homozygous for thalassemia. Drugs that block the potassium current, whether as the prime site of action or as a side effect, are highly teratogenic in experimental animals. They induce embryonic bradycardia, hypoxia, hemorrhage, and blisters, leading to transverse limb defects as well as craniofacial and cardiovascular defects. While evidence linking these drugs to birth defects in humans is not compelling, the reason may methodological rather than biological. Birth Defects Research 109:1358-1376, 2017.© 2017 Wiley Periodicals, Inc.

Embryonic hypoxia programmes postprandial cardiovascular function in adult common snapping turtles (Chelydra serpentina).

Reduced oxygen availability (hypoxia) is a potent stressor during embryonic development, altering the trajectory of trait maturation and organismal phenotype. We previously documented that chronic embryonic hypoxia has a lasting impact on the metabolic response to feeding in juvenile snapping turtles (Chelydra serpentina). Turtles exposed to hypoxia as embryos [10% O2 (H10)] exhibited an earlier and increased peak postprandial oxygen consumption rate, compared with control turtles [21% O2 (N21)]. In the current study, we measured central blood flow patterns to determine whether the elevated postprandial metabolic response in H10 turtles is linked to lasting impacts on convective transport. Five years after hatching, turtles were instrumented to quantify systemic ([Formula: see text]) and pulmonary ([Formula: see text]) blood flows and heart rate (fH) before and after a ∼5% body mass meal. In adult N21 and H10 turtles, fH was increased significantly by feeding. Although total stroke volume (VS,tot) remained at fasted values, this tachycardia contributed to an elevation in total cardiac output ([Formula: see text]). However, there was a postprandial reduction in a net left-right (L-R) shunt in N21 snapping turtles only. Relative to N21 turtles, H10 animals exhibited higher [Formula: see text] due to increased blood flow through the right systemic outflow vessels of the heart. This effect of hypoxic embryonic development, reducing a net L-R cardiac shunt, may support the increased postprandial metabolic rate we previously reported in H10 turtles, and is further demonstration of adult reptile cardiovascular physiology being programmed by embryonic hypoxia.

Protective effect of maternal vitamin E supplementation on phenytoin-induced teratogenicity in rat pups

Objectives:Phenytoin teratogenicity is induced by embryonic hypoxia as a result of generation of reactive oxygen species.Antioxidants are effective in treating conditions associated with oxidative damage. This study investigated the possible protective effect of vitamin E maternal supplementation on oxidative damage, and the morphological and morphometricchanges induced by phenytoin in pups. Methods: Five groups of female rats were utilized. All the treatments were injected intraperitoneal. Groups I and II were injected with saline and olive oil, respectively. Group III was injected with vitamin E (0.5 g/kg BW/day) from day one to day 20 of gestation. Group IV was injected with phenytoin (150 mg/kg BW/day) from day 6 to day 18 of gestation. Group V was injected withphenytoin and vitamin E. The N-acetyl D-glucosaminidase, malondialdehyde, and glutathione were determined as markers of tissue damage. The skeletons of pups were examined after staining with alizarin red-S. Results: Phenytoin significantly increased oxidative stress indices in maternal plasma and tissues and in pup tissues. This wasassociated with a significant decrease in the weight, length and number of pups. Moreover, there were maxillary hypoplasia and skeletal anomalies. Co-administration of vitamin E with phenytoin reduced oxidative damage with significant increasein the weight, length and number of pups. Reduction in maxillary hypoplasia and skeletal anomalies were observed. Conclusion:Vitamin E maternal supplementation has significant effect in the reduction of the anomalies induced by phenytoin in pups

Editorial (Thematic Issues: Advances in Developmental and Reproductive Toxicology)

The manuscripts of this special issue of Current Pharmaceutical Design cover a wide spectrum of relevant and fascinating topics in the area of developmental and reproductive toxicology. Experimental studies have consistently shown that commonly prescribed therapeutic agents can decrease embryonic cardiac output, leading to periods of embryonic hypoxia causing malformations or embryonic death. The article authored by Webster and co-workers [1] describes the available experimental data on the subject and discusses the potential consequences of drug-induced reduction in cardiac output in the human embryo. Alcohol is a well established human teratogen. It has been estimated that the incidence of Fetal Alcohol Syndrome (FAS), which is the most severe manifestation within the fetal alcohol spectrum disorders, is about 1 per 1000 in industrialized countries. In their review, Brennan and Giles [2] pay specific attention on the noxious effects of alcohol exposure on eye development. Insightful discussion on the mechanistic role played by sonic hedgehog pathway disruption is also provided. The potential of foreign compounds to induce ovarian damage, leading to ovarian reserve depletion or cancer, is a topic of increasing global concern. Iorio and co-workers [3] review the noxious effects induced by some widely diffused xenobiotics and by common anticancer therapies on ovary function. Research priorities for the development of preventive measures are also highlighted. Perobelli [4] focuses on male reproductive aspects in prepubertal toxicity assays. The human prepubertal phase has been regarded as a phase of reproductive tract quiescence and reduced vulnerability to toxicants, but emerging data suggest it may represent a susceptible window to endocrine disrupting chemicals. The paper presented by Kanungo et al. [5] summarizes current perspectives on the zebrafish model, which is emerging as a promising alternative animal model for developmental toxicity screening. The elevated potentiality for high-throughput screening is one of the remarkable features offered by this model system. Glucocorticoids play important roles in skeletogenesis. Cheng et al. [6] provide an update on the effects of the synthetic corticosteroid agent dexamethasone on embryo skeletogenesis. The potential impact of glucocorticoids on key regulators of embryo skeletogenesis, including BMPs, FGFs, Hedgehog and Wnt signaling pathways, is also discussed. Histone deacetylases (HDAC) are a family of enzymes playing an important role in the modulation of the transcriptional state of many regions of the genome. Inhibitors of HDAC are promising therapeutic agents in several conditions, including solid and hematological cancers. Updated research information on the teratogenic effects elicited by this class of agents is presented by Giavini and Menegola [7]. Nitric oxide (NO) is multifunctional gaseous molecule, playing crucial roles in the mediation of a wide spectrum of biological processes. The concept that optimal NO levels are essential for normal embryonic development is supported by an increasing number of experimental studies. This topic is reviewed by Tiboni and Ponzano [8].

Effects of macrolide antibiotics on rat embryonic heart function in vitro.

The Swedish Medical Product Agency (MPA) has listed erythromycin as a suggested human teratogen, causing cardiovascular malformations. It is further suggested that this may be a class effect of macrolide antibiotics. The proposed teratogenic mechanism is blockade of the human ether-á-go-go-related (hERG)/IKr current in the embryonic heart causing bradycardia and arrhythmia resulting in altered cardiac blood flow and/or embryonic hypoxia. To test this hypothesis, we examined the effect of three macrolide antibiotics on the function of the rat embryonic heart. Gestational day 13 rat embryos in vitro were exposed to erythromycin (25-500 μM), clarithromycin (25-500 μM), or azithromycin (100 μM to 1 mM) for 3 hr. The effect on the embryonic heart was monitored every hour. The results showed that erythromycin and clarithromycin caused a concentration-dependent bradycardia. Twenty-five micromolar was a no-effect concentration for erythromycin and was close to a no-effect concentration for clarithromycin. Azithromycin only caused significant bradycardia at 1 mM. Additional studies were performed with the embryos cultured at 40°C instead of 38°C, to mimic fever. The increased temperature increased the number of arrhythmias but did not worsen the drug-induced bradycardia. The results support the concept that erythromycin and clarithromycin can adversely affect the embryonic heart but only at concentrations well outside expected embryonic exposure in the human.

Prenatal hypoxia modifies cardiac performance in adult bobwhite quail, assessed by pressure‐volume loop analysis (1150.10)

Prenatal hypoxia modifies cardiac performance in adult bobwhite quail, assessed by pressure‐volume loop analysis Josele Flores‐Santin1 joselefloressantin@my.unt.edu, Warren Burggren1 Burggren@unt.edu1University of North Texas, Biology department, 1155 Union circle, Denton Texas U.S.The consequences of early embryonic stressors and how they affect subsequent adult life reflects the concept of “fetal programming”. This programming potentially produces functional anomalies in major organs including the lungs, the kidneys and the heart, based on studies of a limited number of “traditional” animal models for fetal programming. The aim of this project is to use a non‐traditional model ‐ the rapidly maturing bobwhite quail ‐ to study the effects of embryonic stressors as they manifest themselves in adults, with an emphasis on the working heart. The fast development along with the small size of quail makes them a potentially important alternative animal model to study cardiovascular physiology and the changes that prenatal stressors produce on it. In this study we submitted quail eggs to hypoxia (15%). This treatment accelerated hatching by one day (26±0.11) in comparison to controls (27.6±.12). The treatment also induced a lower hatching mass (6.9±1.0) than controls (5.9±0.1). Pressure‐volume loop SVL catheter technology was used to evaluate cardiovascular performance in adult birds (controls and following embryonic hypoxia). Hypoxic birds presented a phenotype resembling mammalian left ventricular hypertrophy, with a decreased cardiac output (38660±817), ejection fraction (38.8±0.4) and stroke work (11750±376). We conclude that bobwhite quail are a viable alternative model to study cardiovascular fetal programming.Grant Funding Source: Supported by NSF & CoMeCyT/CONACYT

Effects of embryonic hypoxia on lip formation

The upper lip is formed by the fusion of facial processes, a process in which many genetic and environmental factors are involved. Embryonic hypoxia is induced by uterine anemia and the administration of vasoconstrictors during pregnancy. To define the relationship between hypoxia and upper lip formation, hypoxic conditions were created in a whole embryo culture system. Hypoxic embryos showed a high frequency of impaired fusion, reflecting failure in the growth of the lateral nasal process (LNP). In hypoxic embryos, cell proliferation activity in the LNP mesenchyme was decreased following downregulation of genes that are involved in lip formation. We also observed upregulation of vascular endothelial growth factor expression along with the induction of apoptosis in the LNP. These results suggest that embryonic hypoxia during lip formation induces apoptosis in physiologically hypoxic regions, hypoxia-induced gene expression and downregulation of the genes involved in maxillofacial morphogenesis as immediate responses, followed by reduction of mesenchymal cell proliferation activity, resulting in insufficient growth of the facial processes.

Caffeine Acts via A1 Adenosine Receptors to Disrupt Embryonic Cardiac Function

BackgroundEvidence suggests that adenosine acts via cardiac A1 adenosine receptors (A1ARs) to protect embryos against hypoxia. During embryogenesis, A1ARs are the dominant regulator of heart rate, and A1AR activation reduces heart rate. Adenosine action is inhibited by caffeine, which is widely consumed during pregnancy. In this study, we tested the hypothesis that caffeine influences developing embryos by altering cardiac function.Methodology/Principal FindingsEffects of caffeine and adenosine receptor-selective antagonists on heart rate were studied in vitro using whole murine embryos at E9.5 and isolated hearts at E12.5. Embryos were examined in room air (21% O2) or hypoxic (2% O2) conditions. Hypoxia decreased heart rates of E9.5 embryos by 15.8% and in E12.5 isolated hearts by 27.1%. In room air, caffeine (200 µM) had no effect on E9.5 heart rates; however, caffeine increased heart rates at E12.5 by 37.7%. Caffeine abolished hypoxia-mediated bradycardia at E9.5 and blunted hypoxia-mediated bradycardia at E12.5. Real-time PCR analysis of RNA from isolated E9.5 and E12.5 hearts showed that A1AR and A2aAR genes were expressed at both ages. Treatment with adenosine receptor-selective antagonists revealed that SCH-58261 (A2aAR-specific antagonist) had no affects on heart function, whereas DPCPX (A1AR-specific antagonist) had effects similar to caffeine treatment at E9.5 and E12.5. At E12.5, embryonic hearts lacking A1AR expression (A1AR−/−) had elevated heart rates compared to A1AR+/− littermates, A1AR−/− heart rates failed to decrease to levels comparable to those of controls. Caffeine did not significantly affect heart rates of A1AR−/− embryos.Conclusions/SignificanceThese data show that caffeine alters embryonic cardiac function and disrupts the normal cardiac response to hypoxia through blockade of A1AR action. Our results raise concern for caffeine exposure during embryogenesis, particularly in pregnancies with increased risk of embryonic hypoxia.

Metabolic and ventilatory sensitivity to hypoxia in avian embryos

The article discusses the establishment of pulmonary ventilation (V˙E) in the avian embryo, the metabolic and V˙E sensitivity to hypoxia and the effects of sustained embryonic hypoxia on the hatchling's V˙E chemosensitivity. Throughout embryogenesis, hypometabolism is the common response to hypoxia, with no compensation by anaerobic energy supply. It originates primarily from the depression in body growth and, later in development, from the depression of thermogenesis. The V˙E responses to acute hypoxia or hypercapnia are clearly detectable during the internal pipping phase; their magnitude rapidly increases in the first postnatal day. Sustained prenatal hypoxia diminishes the V˙E chemosensitivity of the hatchling and reduces the hypometabolic response to an acute hypoxic episode. The former most likely originates from a disturbance in the normal development of the carotid bodies, the latter from the central action of hypoxia on thermogenesis. The avian embryo is a model suitable for the studies of the development of respiratory control and offers an alternative to mammalian models for investigations on the short- and long-term effects of prenatal hypoxia.

P113. Effects of embryonic hypoxia on maxillofacial development

It has been well known that many genetic and environmental factors are involved in maxillofacial development. Disturbance in these factors could result in congenital anomalies such as cleft lip and palate in human. Some environmental factors, administration of vasoconstrictors and uterine anemia during pregnancy, induce embryonic anemia, and it has been suggested that embryonic hypoxia inhibits development of circulatory organs, limb and maxillofacial organs. In this study we studied the effect of hypoxic condition on mouse lip formation. Lip forms by the fusion between facial processes, medial nasal, lateral nasal, and maxillary processes. We cultured mouse embryos at the beginning of facial process fusion stage by whole embryo culture system under standard oxygen supply and hypoxic conditions, and found that hypoxia induced the failure in the fusion, cleft lip, at higher frequency than standard oxygen supply. Then, we further investigated the effects of embryonic hypoxia on expression change of the genes that are associated with angiogenesis as a consequence of hypoxia and maxillofacial morphogenesis in the facial processes. Expression levels of vascular endothelial growth factor (VEGF) that is upregulated in response to hypoxic condition in vivo were significantly increased in the embryos cultured under hypoxic condition (hypoxic embryos). Transcription factors, Msx1 and Msx2, are expressed in the facial mesenchyme and required for proliferation and differentiation. These transcription factors were downregulated in the facial mesenchyme of hypoxic embryos. Active Wnt signaling has been shown to be involved in lip formation, and the amount of phosphor-Gsk3 alpha/beta, active Wnt signaling marker, were decreased in hypoxic embryos. These results suggest that embryonic hypoxia during the period of facial process fusion induces similar molecular response to adult hypoxic condition and affects expression levels of genes that are strongly associated with lip formation.

Ventilatory response to hypoxia in chicken hatchlings: A developmental window of sensitivity to embryonic hypoxia

We had reported previously [Szdzuy, K., Mortola, J.P., 2007b. Ventilatory chemosensitivity of the 1-day-old chicken hatchling after embryonic hypoxia. Am. J. Physiol. (Regul. Integr. Comp. Physiol.) 293, R1640-R1649] that hypoxia during incubation blunted ventilatory chemosensitivity in the hatchling. Because the carotid bodies become functional in the last portion of incubation, we asked whether these last days were the critical period for the effects of hypoxia on the development of ventilatory chemosensitivity. White Leghorn chicken eggs were incubated at 38 degrees C either in 21% O(2) (Controls) or in 15% O(2) for the whole 3-week incubation (HxTot) or for only the 1st (Hx1), 2nd (Hx2) or 3rd week of incubation (Hx3). Hatching time had a delay of half a day in HxTot, and was normal in the other groups. Body weight was similar in all hatchlings. Oxygen consumption ( [Formula: see text] ) and pulmonary ventilation (V e) were measured at about 20 h post-hatching. Ventilatory chemosensitivity was evaluated from the degree of hyperpnea (increase in V e) and hyperventilation (increase in [Formula: see text] ) during acute hypoxia (15 and 10% O(2), 20 min each) and acute hypercapnia (2 and 4% CO(2), 20 min each). The responses to hypoxia were similarly decreased in HxTot and in Hx3 compared to controls, and were normal in the other experimental groups; those to hypercapnia were blunted only in HxTot. The results are in agreement with the idea that prenatal hypoxia blunts V e chemosensitivity by interfering with the normal development of the carotid bodies.