The tolerance to hypoxia is defined by a time-sensitive response of the gene regulatory network in sea urchin embryos.

Deoxygenation, the reduction of oxygen level in the oceans induced by global warming and anthropogenic disturbances, is a major threat to marine life. This change in oxygen level could be especially harmful to marine embryos that use endogenous hypoxia and redox gradients as morphogens during normal development. Here, we show that the tolerance to hypoxic conditions changes between different developmental stages of the sea urchin embryo, possibly due to the structure of the gene regulatory networks (GRNs). We demonstrate that during normal development, the bone morphogenetic protein (BMP) pathway restricts the activity of the vascular endothelial growth factor (VEGF) pathway to two lateral domains and this restriction controls proper skeletal patterning. Hypoxia applied during early development strongly perturbs the activity of Nodal and BMP pathways that affect the VEGF pathway, dorsal-ventral (DV) and skeletogenic patterning. These pathways are largely unaffected by hypoxia applied after DV-axis formation. We propose that the use of redox and hypoxia as morphogens makes the sea urchin embryo highly sensitive to environmental hypoxia during early development, but the GRN structure provides higher tolerance to hypoxia at later stages.

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.

Hypoxia regulates the degradation of non-nuclear organelles during lens differentiation through activation of HIF1a.

Formation of the eye lens depends on the continuous differentiation of lens epithelial cells into lens fiber cells. To attain their mature structure and transparent function, nascent lens fiber cells must complete a precise cellular remodeling program hallmarked by the complete elimination of organelles to form the core lens organelle-free zone (OFZ). Lacking a blood supply, the lens resides in a hypoxic environment that results in a decreasing oxygen concentration from the lens surface to the lens core. This oxygen gradient results in a hypoxic microenvironment in the region of the lens where immature lens fiber cells initiate loss of organelles to form the core OFZ. These features of the lens suggest a potential role for low lens oxygen levels in the regulation of organelle degradation and other events critical for mature lens fiber cell formation. Hypoxia activates the master regulator of the hypoxic response, hypoxia-inducible factor 1a (HIF1a) that regulates hypoxia-responsive genes. To identify a potential role for hypoxia and HIF1a in the elimination of organelles during lens fiber cell maturation, we tested the requirement for hypoxia in the degradation of non-nuclear organelles in ex vivo cultured embryonic chick lenses by monitoring the degradation of mitochondria (MT), Golgi apparatus (GA) and endoplasmic reticulum (ER) under conditions of low (1% O2) and high (21% O2) oxygen. We also examined the requirement for HIF1a activation for elimination of these organelles under the same conditions using a specific HIF1a activator (DMOG) and a specific HIF1a inhibitor (chetomin) and examined the requirements for hypoxia and HIF1a for regulating transcription of BNIP3L that we previously showed to be required for elimination of non-nuclear lens organelles. We used ChIP-qPCR to confirm direct binding of HIF1a to the 5' untranslated region of the BNIP3L gene. Finally, we examined the effects of expressing an oxygen insensitive mutant form of HIF1a (P402A/P565A) and BNIP3L on non-nuclear organelle degradation. Our data demonstrate that hypoxia and HIF1a are required for the degradation of non-nuclear organelles during lens fiber cell formation and that they regulate this process by governing BNIP3L transcription. Our results also provide evidence that hypoxia and HIF1a are essential for achieving mature lens structure.

Hypoxia affects the ontogeny of the hypothalamus-pituitary-interrenal axis functioning in the lake whitefish (Coregonus clupeaformis).

Early life stages are sensitive to environmental insults and changes during critical developmental periods; this can often result in altered adult behaviour and physiology. Examining the development of the hypothalamus-pituitary-interrenal (HPI) axis and its responsiveness, or lack thereof, during development are important for understanding the short- and long-term impacts of stressors on embryonic and larval fish. We examined the ontogeny of the HPI axis in embryonic (21, 38, 63, 83 and 103 days post-fertilisation (dpf)) and larval (1, 2, 3 and 4 weeks post-hatch (wph)) lake whitefish (Coregonus clupeaformis) by quantifying changes in mRNA levels of several genes associated with HPI axis functioning and whole animal cortisol levels throughout development and in response to a severe or mild hypoxic stress. Cortisol, and crh, crhbp1, pomc and star transcripts were detected from the earliest embryonic age studied. Cortisol levels in control embryos decreased between 21 and 63 dpf, suggesting the utilisation of maternal cortisol deposits. However, by 83 dpf (70% developed) endogenous de novo synthesis had generated a 4.5-fold increase in whole embryo cortisol. Importantly, we provide novel data showing that the HPI axis can be activated even earlier. Whole body cortisol increased in eyed lake whitefish embryos (38 dpf; ~32% developed) in response to hypoxia stress. Coincident with this hypoxia-induced increase in cortisol in 38 dpf embryos were corresponding increases in crh, crhbp1, pomc and star transcript levels. Beyond 38 dpf, the HPI axis in lake whitefish embryos was hyporesponsive to hypoxia stress at all embryonic ages examined (63, 83 and 103 dpf; 54, 72 and 85% developed, respectively). Post-hatch, larvae responded to hypoxia with an increase in cortisol levels and HPI axis genes at 1 wph, but this response was lost and larvae appeared hyporesponsive at subsequent ages (2, 3 and 4 wph). Collectively our work demonstrates that during fish embryogenesis and the larval stage there are windows where the HPI axis is responsive and windows where it is truly hyporesponsive; both could be beneficial in ensuring undisrupted development particularly in the face of increasing environmental changes.

Impact of uteroplacental insufficiency on ovarian follicular pool in the rat.

BACKGROUND: A low oxygen supply to the fetus causes intrauterine growth restriction and can affect gonadal development of the offspring, having a potential impact on fertility. We investigated histology and gene expression in the postnatal rat ovary after fetal hypoxia induced by uterine artery ligation. METHODS: Sprague-Dawley rats underwent uterine artery ligation at day 19 of gestation. Offspring were sacrificed at 5, 20 and 40 days post-partum. Follicles were counted and classified in hematoxylin-eosin stained sections. Gene expression of 90 genes was analyzed by TaqMan® Low Density Array. RESULTS: A significantly lower number of total and primordial follicles was detected in 20 days post-partum intrauterine growth restricted animals. Follicle density was not different at 40 days post-partum, suggesting that compensatory mechanisms occurred during the pre-pubertal window. Uterine artery ligation modified the expression of 24 genes involved in different cellular functions, among which proliferation, apoptosis and metabolism. CONCLUSION: Ovarian follicle pool was affected by fetal hypoxia in early life, but this effect did not persist in puberty. Genes involved in cellular processes were affected at all ages, potentially implying long-term genetic alterations. Further analyses are needed to elucidate later effects of fetal hypoxia on ovarian function and fertility.

Perinatal changes in cardiac geometry and function in growth-restricted fetuses at term.

OBJECTIVE: To evaluate the effect of fetal growth restriction (FGR) at term on fetal and neonatal cardiac geometry and function. METHODS: This was a prospective study of 87 pregnant women delivering at term, comprising 54 normally grown and 33 FGR pregnancies. Fetal and neonatal conventional and spectral tissue Doppler and two-dimensional speckle tracking echocardiography were performed a few days before and within hours after birth. Fetal cardiac geometry, global myocardial deformation and performance and systolic and diastolic function were compared between normal and FGR pregnancies before and after birth. RESULTS: Compared with normally grown fetuses, FGR fetuses exhibited more globular ventricular geometry and poorer myocardial deformation and cardiac function (left ventricular (LV) sphericity index (SI), 0.54 vs 0.49; right ventricular (RV) SI, 0.60 vs 0.54; LV torsion, 1.2 °/cm vs 3.0 °/cm; LV isovolumetric contraction time normalized by cardiac cycle length, 121 ms vs 104 ms; interventricular septum early diastolic myocardial peak velocity/atrial contraction myocardial diastolic peak velocity ratio, 0.60 vs 0.71; P < 0.01 for all). The poorest perinatal outcomes occurred in FGR fetuses with the most impaired cardiac functional indices. When compared with normally grown neonates, FGR neonates showed persistent alteration in cardiac parameters (LV-SI, 0.53 vs 0.50; RV-SI, 0.54 vs 0.44; LV torsion, 1.1 °/cm vs 1.4 °/cm; LV myocardial performance index (MPI'), 0.52 vs 0.42; P < 0.01 for all). Paired comparison of fetal vs neonatal cardiac indices in FGR demonstrated that birth was associated with a significant improvement in some, but not all, cardiac indices (RV-SI, 0.60 vs 0.54; RV-MPI', 0.49 vs 0.39; P < 0.001 for all). CONCLUSIONS: Compared with normal pregnancies, FGR fetuses and neonates at term exhibit altered cardiac indices indicative of myocardial impairment that reflect adaptation to placental hypoxemia and alterations in hemodynamic load around the time of birth. Elucidating potential mechanisms that contribute to the alterations in perinatal cardiac adaptation in FGR could improve management and aid the development of better therapeutic strategies to reduce the risk of adverse pregnancy outcome. Copyright © 2018 ISUOG. Published by John Wiley & Sons Ltd.

Fetal heart rate monitoring and neonatal outcome in a population of early- and late-onset intrauterine growth restriction.

AIM: The early-onset intrauterine growth restriction (IUGR) is associated with severe placental insufficiency and Doppler abnormalities. The late-onset IUGR is associated with mild placental insufficiency and normal Doppler velocimetry. The computerized cardiotocographic (cCTG) monitoring is used to evaluate the fetal well-being in pregnancies complicated by IUGR. Our aim was to investigate the cardiotocographic characteristics of IUGR fetuses and to identify every cCTG difference between Healthy and IUGR fetuses. METHODS: Four hundred thirty pregnant women were enrolled starting from the 28th week of gestation until the time of delivery: 200 healthy and 230 IUGR fetuses. Fetal heart rate (FHR) baseline (FHR), short-term variability (STV), long-term irregularity (LTI), delta, interval index (II), approximate entropy (ApEn), high frequency (HF), low frequency (LF), movement frequency (MF), LF/(HF + MF) ratio (LF/(HF + MF)) and number of decelerations were examined. Newborn baby data were also collected. RESULTS: The parameters of short- and medium-term variability discriminate between IUGR and healthy fetuses before 36 weeks including FHR, STV, LTI and delta discriminate between each subgroup of IUGR were compared to each one of the other two (P < 0.05). CONCLUSION: cCTG is a useful tool for the evaluation of chronic hypoxemia, which causes a delay in the maturation of all components of the autonomic and central nervous system. However, cCTG requires integration with fetal ultrasound and Doppler vessels evaluation to improve the ability to predict the neonatal outcome.

The highs and lows of programmed cardiovascular disease by developmental hypoxia: studies in the chicken embryo.

It is now established that adverse conditions during pregnancy can trigger a fetal origin of cardiovascular dysfunction and/or increase the risk of heart disease in later life. Suboptimal environmental conditions during early life that may promote the development of cardiovascular dysfunction in the offspring include alterations in fetal oxygenation and nutrition as well as fetal exposure to stress hormones, such as glucocorticoids. There has been growing interest in identifying the partial contributions of each of these stressors to programming of cardiovascular dysfunction. However, in humans and in many animal models this is difficult, as the challenges cannot be disentangled. By using the chicken embryo as an animal model, science has been able to circumvent a number of problems. In contrast to mammals, in the chicken embryo the effects on the developing cardiovascular system of changes in oxygenation, nutrition or stress hormones can be isolated and determined directly, independent of changes in the maternal or placental physiology. In this review, we summarise studies that have exploited the chicken embryo model to determine the effects on prenatal growth, cardiovascular development and pituitary-adrenal function of isolated chronic developmental hypoxia.

Reduced fetal movements and cerebroplacental ratio: evidence for worsening fetal hypoxemia.

OBJECTIVE: To investigate the fetal cerebroplacental ratio (CPR) in women presenting with reduced fetal movements (RFM). METHODS: This was a retrospective cohort study of data collected over an 8-year period at a fetal medicine unit at a tertiary referral center. The cohort comprised 4500 singleton pregnancies presenting with RFM at or after 36 weeks' gestation and 1527 control pregnancies at a similar gestational age without RFM. Fetal biometry and Doppler parameters were recorded and converted into centiles and multiples of the median (MoM). CPR was defined as the ratio between the fetal middle cerebral artery (MCA) pulsatility index (PI) and the umbilical artery (UA) PI. Subgroup analysis for fetal size and for single vs multiple episodes of RFM was performed. RESULTS: Compared with controls, pregnancies with RFM had lower MCA-PI MoM (median, 0.95 vs 0.97; P < 0.001) and CPR MoM (median, 0.97 vs 0.99; P = 0.018). Compared with women presenting with single episodes of RFM, pregnancies with multiple episodes (≥ 2 episodes) had lower CPR MoM (median, 0.94 vs 0.98; P = 0.003). On subgroup analysis for fetal size, compared with controls, appropriate-for-gestational-age fetuses in the RFM group had lower MCA-PI MoM (median, 0.96 vs 0.97; P = 0.003) and higher rate of CPR below the 5th centile (5.3% vs 3.6%; P = 0.015). Logistic regression analysis demonstrated an association of risk of recurrent RFM with maternal age (OR, 0.96; 95% CI, 0.93-0.99), non-Caucasian ethnicity (OR, 0.72; 95% CI, 0.53-0.97), estimated fetal weight centile (OR, 1.01; 95% CI, 1.00-1.02) and CPR MoM (OR, 0.24; 95% CI, 0.12-0.47). CONCLUSION: Pregnancies complicated by multiple episodes of RFM show significantly lower CPR MoM and MCA-PI MoM compared with those with single episodes and controls. This is likely to be due to worsening fetal hypoxemia in women presenting with recurrent RFM. Copyright © 2017 ISUOG. Published by John Wiley & Sons Ltd.

Cardiovascular susceptibility to in vivo ischemic myocardial injury in male and female rat offspring exposed to prenatal hypoxia.

Intrauterine growth restriction (IUGR) following prenatal hypoxia exposure leads to a higher risk of developing cardiovascular disease (CVD) in later life. Our aim was to evaluate cardiac susceptibility and its pathophysiological mechanisms following acute myocardial infarction (MI) in adult rat offspring exposed to prenatal hypoxia. Male and female rat offspring, which experienced normoxia (21% O2) or hypoxia (11% O2) in utero underwent sham or MI surgery at 12 weeks of age. Echocardiographic data revealed that both sexes had systolic dysfunction following MI surgery, independent of prenatal hypoxia. Male offspring exposed to prenatal hypoxia, however, had left ventricular dilatation, global dysfunction, and signs of diastolic dysfunction following MI surgery as evident by increased left ventricular internal diameter (LVID) during diastole (MI effect, P<0.01), Tei index (MI effect, P<0.001), and E/E' ratio (prenatal hypoxia or MI effect, P<0.01). In contrast, diastolic dysfunction in female offspring was not as evident. Cardiac superoxide levels increased only in prenatal hypoxia exposed male offspring. Cardiac sarcoendoplasmic reticulum Ca2+-ATPase2a (SERCA2a) levels, a marker of cardiac injury and dysfunction, decreased in both male and female MI groups independent of prenatal hypoxia. Prenatal hypoxia increased cardiac ryanodine receptor 2 (RYR2) protein levels, while MI reduced RYR2 in only male offspring. In conclusion, male offspring exposed to prenatal hypoxia had an increased susceptibility to ischemic myocardial injury involving cardiac phenotypes similar to heart failure involving diastolic dysfunction in adult life compared with both offspring from healthy pregnancies and their female counterparts.

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.

Suppression of vascular network formation by chronic hypoxia and prolyl-hydroxylase 2 (phd2) deficiency during vertebrate development.

In the adult, new vessels and red blood cells form in response to hypoxia. Here, the oxygen-sensing system (PHD-HIF) has recently been put into focus, since the prolyl-hydroxylase domain proteins (PHD) and hypoxia-inducible factors (HIF) are considered as potential therapeutic targets to treat ischemia, cancers or age-related macula degeneration. While the oxygen-sensing system (PHD-HIF) has been studied intensively in this respect, only little is known from developing vertebrate embryos since mutations within this pathway led to an early decease of embryos due to placental defects. During vertebrate embryogenesis, a progenitor cell called hemangioblast is assumed to give rise to blood cells and blood vessels in a process called hematopoiesis and vasculogenesis, respectively. Xenopus provides an ideal experimental system to address these processes in vivo, as its development does not depend on a functional placenta and thus allows analyzing the role of oxygen directly. To this end, we adopted a computer-controlled four-channel system, which allowed us to culture Xenopus embryos under defined oxygen concentrations. Our data show that the development of vascular structures and blood cells is strongly impaired under hypoxia, while general development is less compromised. Interestingly, suppression of Phd2 function using specific antisense morpholinos or a chemical inhibitor resulted in mostly overlapping vascular defects; nevertheless, blood cell was formed almost normally. Our results provide the first evidence that oxygen via Phd2 has a decisive influence on the formation of the vascular network during vertebrate embryogenesis. These findings may be considered in certain potential treatment concepts.

Periods of cardiovascular susceptibility to hypoxia in embryonic american alligators (Alligator mississippiensis).

During embryonic development, environmental perturbations can affect organisms' developing phenotype, a process known as developmental plasticity. Resulting phenotypic changes can occur during discrete, critical windows of development. Critical windows are periods when developing embryos are most susceptible to these perturbations. We have previously documented that hypoxia reduces embryo size and increases relative heart mass in American alligator, and this study identified critical windows when hypoxia altered morphological, cardiovascular function and cardiac gene expression of alligator embryos. We hypothesized that incubation in hypoxia (10% O2) would increase relative cardiac size due to cardiac enlargement rather than suppression of somatic growth. We exposed alligator embryos to hypoxia during discrete incubation periods to target windows where the embryonic phenotype is altered. Hypoxia affected heart growth between 20 and 40% of embryonic incubation, whereas somatic growth was affected between 70 and 90% of incubation. Arterial pressure was depressed by hypoxic exposure during 50-70% of incubation, whereas heart rate was depressed in embryos exposed to hypoxia during a period spanning 70-90% of incubation. Expression of Vegf and PdgfB was increased in certain hypoxia-exposed embryo treatment groups, and hypoxia toward the end of incubation altered ß-adrenergic tone for arterial pressure and heart rate. It is well known that hypoxia exposure can alter embryonic development, and in the present study, we have identified brief, discrete windows that alter the morphology, cardiovascular physiology, and gene expression in embryonic American alligator.

Developmental plasticity of mitochondrial function in American alligators, Alligator mississippiensis.

The effect of hypoxia on cellular metabolism is well documented in adult vertebrates, but information is entirely lacking for embryonic organisms. The effect of hypoxia on embryonic physiology is particularly interesting, as metabolic responses during development may have life-long consequences, due to developmental plasticity. To this end, we investigated the effects of chronic developmental hypoxia on cardiac mitochondrial function in embryonic and juvenile American alligators (Alligator mississippiensis). Alligator eggs were incubated in 21% or 10% oxygen from 20 to 90% of embryonic development. Embryos were either harvested at 90% development or allowed to hatch and then reared in 21% oxygen for 3 yr. Ventricular mitochondria were isolated from embryonic/juvenile alligator hearts. Mitochondrial respiration and enzymatic activities of electron transport chain complexes were measured with a microrespirometer and spectrophotometer, respectively. Developmental hypoxia induced growth restriction and increased relative heart mass, and this phenotype persisted into juvenile life. Embryonic mitochondrial function was not affected by developmental hypoxia, but at the juvenile life stage, animals from hypoxic incubations had lower levels of Leak respiration and higher respiratory control ratios, which is indicative of enhanced mitochondrial efficiency. Our results suggest developmental hypoxia can have life-long consequences for alligator morphology and metabolic function. Further investigations are necessary to reveal the adaptive significance of the enhanced mitochondrial efficiency in the hypoxic phenotype.

Phenotypic plasticity in the common snapping turtle (Chelydra serpentina): long-term physiological effects of chronic hypoxia during embryonic development.

Studies of embryonic and hatchling reptiles have revealed marked plasticity in morphology, metabolism, and cardiovascular function following chronic hypoxic incubation. However, the long-term effects of chronic hypoxia have not yet been investigated in these animals. The aim of this study was to determine growth and postprandial O2 consumption (V̇o2), heart rate (fH), and mean arterial pressure (Pm, in kPa) of common snapping turtles (Chelydra serpentina) that were incubated as embryos in chronic hypoxia (10% O2, H10) or normoxia (21% O2, N21). We hypothesized that hypoxic development would modify posthatching body mass, metabolic rate, and cardiovascular physiology in juvenile snapping turtles. Yearling H10 turtles were significantly smaller than yearling N21 turtles, both of which were raised posthatching in normoxic, common garden conditions. Measurement of postprandial cardiovascular parameters and O2 consumption were conducted in size-matched three-year-old H10 and N21 turtles. Both before and 12 h after feeding, H10 turtles had a significantly lower fH compared with N21 turtles. In addition, V̇o2 was significantly elevated in H10 animals compared with N21 animals 12 h after feeding, and peak postprandial V̇o2 occurred earlier in H10 animals. Pm of three-year-old turtles was not affected by feeding or hypoxic embryonic incubation. Our findings demonstrate that physiological impacts of developmental hypoxia on embryonic reptiles continue into juvenile life.

Effects of chronic hypoxia on cardiac function measured by pressure-volume catheter in fetal chickens.

Hypoxia is a common component of many developmental insults and has been studied in early-stage chicken development. However, its impact on cardiac function and arterial-ventricular coupling in late-stage chickens is relatively unknown. To test the hypothesis that hypoxic incubation would reduce baseline cardiac function but protect the heart during acute hypoxia in late-stage chickens, white Leghorn eggs were incubated at 21% O2 or 15% O2. At 90% of incubation (19 days), hypoxic incubation caused growth restriction (-20%) and increased the LV-to-body ratio (+41%). Left ventricular (LV) pressure-volume loops were measured in anesthetized chickens in normoxia and acute hypoxia (10% O2). Hypoxic incubation lowered the maximal rate of pressure generation (ΔP/ΔtMax; -22%) and output (-57%), whereas increasing end-systolic elastance (ELV; +31%) and arterial elastance (EA; +122%) at similar heart rates to normoxic incubation. Both hypoxic incubation and acute hypoxia lengthened the half-time of relaxation (τ; +24%). Acute hypoxia reduced heart rate (-8%) and increased end-diastolic pressure (+35%). Hearts were collected for mRNA analysis. Hypoxic incubation was marked by decreased mRNA expression of sarco(endo)plasmic reticulum Ca(2+)-ATPase 2, Na(+)/Ca(2+) exchanger 1, phospholamban, and ryanodine receptor. In summary, hypoxic incubation reduces LV function in the late-stage chicken by slowing pressure generation and relaxation, which may be driven by altered intracellular excitation-contraction coupling. Cardiac efficiency is greatly reduced after hypoxic incubation. In both incubation groups acute hypoxia reduced diastolic function.

A hypoxic episode during cardiogenesis downregulates the adenosinergic system and alters the myocardial anoxic tolerance.

To what extent hypoxia alters the adenosine (ADO) system and impacts on cardiac function during embryogenesis is not known. Ectonucleoside triphosphate diphosphohydrolase (CD39), ecto-5'-nucleotidase (CD73), adenosine kinase (AdK), adenosine deaminase (ADA), equilibrative (ENT1,3,4), and concentrative (CNT3) transporters and ADO receptors A1, A2A, A2B, and A3 constitute the adenosinergic system. During the first 4 days of development chick embryos were exposed in ovo to normoxia followed or not followed by 6 h hypoxia. ADO and glycogen content and mRNA expression of the genes were determined in the atria, ventricle, and outflow tract of the normoxic (N) and hypoxic (H) hearts. Electrocardiogram and ventricular shortening of the N and H hearts were recorded ex vivo throughout anoxia/reoxygenation ± ADO. Under basal conditions, CD39, CD73, ADK, ADA, ENT1,3,4, CNT3, and ADO receptors were differentially expressed in the atria, ventricle, and outflow tract. In H hearts ADO level doubled, glycogen decreased, and mRNA expression of all the investigated genes was downregulated by hypoxia, except for A2A and A3 receptors. The most rapid and marked downregulation was found for ADA in atria. H hearts were arrhythmic and more vulnerable to anoxia-reoxygenation than N hearts. Despite downregulation of the genes, exposure of isolated hearts to ADO 1) preserved glycogen through activation of A1 receptor and Akt-GSK3ß-GS pathway, 2) prolonged activity and improved conduction under anoxia, and 3) restored QT interval in H hearts. Thus hypoxia-induced downregulation of the adenosinergic system can be regarded as a coping response, limiting the detrimental accumulation of ADO without interfering with ADO signaling.

Dynamics of blood viscosity regulation during hypoxic challenges in the chicken embryo (Gallus gallus domesticus).

Hypoxia in chicken embryos increases hematocrit (Hct), blood O2 content, and blood viscosity. The latter may limit O2 transport capacity (OTC) via increased peripheral resistance. Hct increase may result from increased nucleated red blood cell concentration ([RBC]) and mean corpuscular volume (MCV) or reduced plasma volume. We hypothesized changes in Hct, hemoglobin concentration ([Hb]), [RBC] and MCV and their effects on viscosity would reduce OTC. Five experimental treatments that increase Hct were conducted on day 15 embryos: 60min water submergence with 60min recovery in air; exposure to 15% O2 with or without 5% CO2 for 24 h with 6 h recovery; or exposure to 10% O2 with or without 5% CO2 for 120 min with 120 min recovery. Control Hct, [Hb], [RBC], MCV, and viscosity were approximately 26%, 9g%, 2.0 10(6)µL(-1), 130µm(3), and 1.6mPas, respectively. All manipulations increased Hct and blood viscosity without changing blood osmolality (276mmolkg(-1)). Increased viscosity was attributed to increased [RBC] and MCV in submerged embryos, but solely MCV in embryos experiencing 10% O2 regardless of CO2. Blood viscosity in embryos exposed to 15% O2 increased via increased MCV alone, and viscosity was constant during recovery despite increased [RBC]. Consequently, blood viscosity was governed by MCV and [RBC] during submergence, while MCV was the strongest determinant of blood viscosity in extrinsic hypoxia with or without hypercapnia. Increased Hct and blood O2 content did not compensate for the effect of increased viscosity on OTC during these challenges.

Activity of Phosphate-Dependent Glutaminase in the Brain of Rats Exposed to Prenatal Hypoxia during Organogenesis.

We studied the effect of hypoxia (days 9-15 of pregnancy) on phosphate-dependent glutaminase activity in the brain of rat offspring aging 18 days and 1, 3, and 6 months. Activity of glutaminase significantly increased in mitochondria from the orbital, visual, and limbic cortex, hypothalamus, and midbrain of 17-day-old offspring. Activity of this enzyme in all brain regions increased at the age of 1 month, but significantly decreased in animals aging 3 and 6 months and prenatally exposed to hypoxia (during organogenesis). Changes in glutaminase activity in various structures of the brain are probably associated with activation of the glutamatergic and GABAergic systems and serve as an adaptive and compensatory reaction of the brain.

Transcriptomics of the fetal hypothalamic response to brachiocephalic occlusion and estradiol treatment.

Estradiol (E2) is a well-known modulator of fetal neuroendocrine activity and has been proposed as a critical endocrine signal readying the fetus for birth and postnatal life. To investigate the modulatory role of E2 on fetal stress responsiveness and the response of the fetal brain to asphyxic stress, we subjected chronically catheterized fetal sheep to a transient (10 min) brachiocephalic artery occlusion (BCO) or sham occlusion. Half of the fetuses received subcutaneous pellets that increased plasma E2 concentrations within the physiological range. Hypothalamic mRNA was analyzed using the Agilent 8x15k ovine array (019921), processed and annotated as previously reported by our laboratory. Analysis of the data by ANOVA revealed that E2 differentially regulated (DR) 561 genes, and BCO DR 894 genes compared with control and E2+BCO DR 1,153 genes compared with BCO alone (all P < 0.05). E2 upregulated epigenetic pathways and downregulated local steroid biosynthesis but did not significantly involve genes known to directly respond to the estrogen receptor. Brachiocephalic occlusion upregulated kinase pathways as well as genes associated with lymphocyte infiltration into the brain and downregulated neuropeptide synthesis. E2 upregulated immune- and apoptosis-related pathways after BCO and reduced kinase and epigenetic pathway responses to the BCO. Responses to BCO are different from responses to hypoxic hypoxia suggesting that mechanisms of responses to these two forms of brain hypoxia are distinct. We conclude that cerebral ischemia caused by BCO might stimulate lymphocyte infiltration into the brain and that this response appears to be modified by estradiol.