Differential regulation of cell proliferation in neurogenic zones in mice lacking cystine transport by xCT

The cystine/glutamate exchanger (xCT) supplies intracellular cyst(e)ine for the production of glutathione, a major cellular anti-oxidant. xCT is enriched in brain regions associated with neurogenesis. Previous studies have shown that the malfunction of this protein greatly attenuates cell proliferation in vitro and is associated with brain atrophy in vivo. Using mice that are homozygous for a function-blocking deletion in xCT (Sut mice), we examined in vivo the role of xCT in cell proliferation in neurogenic regions of the subventricular zone (SVZ) and denate gyrus (DG) in the adult brain. Our results indicate that a high level of cellular proliferation in the adult brain persists even in the absence of functional xCT. Furthermore, in both young adult and middle-aged mice (3 and 11 months old), rates of SVZ cell proliferation were comparable between Sut and wild-type controls, although there was trend towards reduced proliferation in Sut mice (12% and 9% reduction, respectively). To our surprise, rates of cell proliferation in the DG were elevated in both 3- and 11-month-old Sut mice relative to controls (22% and 28% increase, respectively). These results demonstrate that xCT expression plays a role in regulating cellular proliferation in the DG, but not the SVZ of adult mice. Furthermore, unlike previous in vitro studies, our in vivo observations clearly indicate that xCT is not essential for ongoing cellular proliferation.     

Exposure to N-ethyl-N-nitrosourea in adult mice alters structural and functional integrity of neurogenic sites

Background

Previous studies have shown that prenatal exposure to the mutagen N-ethyl-N-nitrosourea (ENU), a N-nitroso compound (NOC) found in the environment, disrupts developmental neurogenesis and alters memory formation. Previously, we showed that postnatal ENU treatment induced lasting deficits in proliferation of neural progenitors in the subventricular zone (SVZ), the main neurogenic region in the adult mouse brain. The present study is aimed to examine, in mice exposed to ENU, both the structural features of adult neurogenic sites, incorporating the dentate gyrus (DG), and the behavioral performance in tasks sensitive to manipulations of adult neurogenesis.

Methodology/Principal Findings

2-month old mice received 5 doses of ENU and were sacrificed 45 days after treatment. Then, an ultrastructural analysis of the SVZ and DG was performed to determine cellular composition in these regions, confirming a significant alteration. After bromodeoxyuridine injections, an S-phase exogenous marker, the immunohistochemical analysis revealed a deficit in proliferation and a decreased recruitment of newly generated cells in neurogenic areas of ENU-treated animals. Behavioral effects were also detected after ENU-exposure, observing impairment in odor discrimination task (habituation-dishabituation test) and a deficit in spatial memory (Barnes maze performance), two functions primarily related to the SVZ and the DG regions, respectively.

Conclusions/Significance

The results demonstrate that postnatal exposure to ENU produces severe disruption of adult neurogenesis in the SVZ and DG, as well as strong behavioral impairments. These findings highlight the potential risk of environmental NOC-exposure for the development of neural and behavioral deficits.     

Abca7 deletion does not affect adult neurogenesis in the mouse

ATP-binding cassette transporter A7 (ABCA7) is highly expressed in the brain. Recent genome-wide association studies (GWAS) have identified ABCA7 single nucleotide polymorphisms (SNPs) that increase Alzheimer''s disease (AD) risk, however, the mechanisms by which ABCA7 may control AD risk remain to be fully elucidated. Based on previous research suggesting that certain ABC transporters may play a role in the regulation of neurogenesis, we conducted a study of cell proliferation and neurogenic potential using cellular bromodeoxyuridine (BrdU) incorporation and doublecortin (DCX) immunostaining in adult Abca7 deficient mice and wild-type-like (WT) littermates. In the present study counting of BrdU-positive and DCX-positive cells in an established adult neurogenesis site in the dentate gyrus (DG) indicated there were no significant differences when WT and Abca7 deficient mice were compared. We also measured the area occupied by immunohistochemical staining for BrdU and DCX in the DG and the subventricular zone (SVZ) of the same mice and this confirmed that ABCA7 does not play a significant role in the regulation of cell proliferation or neurogenesis in the adult mouse.     

Akhirin regulates the proliferation and differentiation of neural stem cells/progenitor cells at neurogenic niches in mouse brain

Specialized microenvironment, or neurogenic niche, in embryonic and postnatal mouse brain plays critical roles during neurogenesis throughout adulthood. The subventricular zone (SVZ) and the dentate gyrus (DG) of hippocampus in the mouse brain are two major neurogenic niches where neurogenesis is directed by numerous regulatory factors. Now, we report Akhirin (AKH), a stem cell maintenance factor in mouse spinal cord, plays a pivotal regulatory role in the SVZ and in the DG. AKH showed specific distribution during development in embryonic and postnatal neurogenic niches. Loss of AKH led to abnormal development of the ventricular zone and the DG along with reduction of cellular proliferation in both regions. In AKH knockout mice (AKH^−/−), quiescent neural stem cells (NSCs) increased, while proliferative NSCs or neural progenitor cells decreased at both neurogenic niches. In vitro NSC culture assay showed increased number of neurospheres and reduced neurogenesis in AKH^−/−. These results indicate that AKH, at the neurogenic niche, exerts dynamic regulatory role on NSC self-renewal, proliferation and differentiation during SVZ and hippocampal neurogenesis.     

Extracellular Signal-regulated Kinase 5 (ERK5) Mediates Prolactin-stimulated Adult Neurogenesis in the Subventricular Zone and Olfactory Bulb

Prolactin-stimulated adult neurogenesis in the subventricular zone (SVZ) and olfactory bulb (OB) mediates several reproductive behaviors including mating/pregnancy, dominant male pheromone preference in females, and paternal recognition of offspring. However, downstream signaling mechanisms underlying prolactin-induced adult neurogenesis are completely unknown. We report here for the first time that prolactin activates extracellular signal-regulated kinase 5 (ERK5), a MAP kinase that is specifically expressed in the neurogenic regions of the adult mouse brain. Knockdown of ERK5 by retroviral infection of shRNA attenuates prolactin-stimulated neurogenesis in SVZ-derived adult neural stem/progenitor cells (aNPCs). Inducible erk5 deletion in adult neural stem cells of transgenic mice inhibits neurogenesis in the SVZ and OB following prolactin infusion or mating/pregnancy. These results identify ERK5 as a novel and critical signaling mechanism underlying prolactin-induced adult neurogenesis.     

Effects of experimental ventilation and ambient Po2 on O2 uptake of the isolated cutaneous tissue in the carp,Cyprinus carpio

Effects of experimental ventilation and ambient Po₂ on cutaneous O₂ uptake in vitro were studied in the carp, Cyprinus carpio. Oxygen uptake rate of the isolated cutaneous tissue was determined by ventilating the epidermis side of the skin with normoxic water in flow-through respirometers. Oxygen uptake rate of the skin increased with ventilation rate across the skin between 2.5 and 40 ml/min and became 3.2 nmol/cm²/min at a flow rate of 40 ml/min, which corresponds to an apparent water velocity of 1.1 cm/sec. At a ventilation rate of 10 ml/min, oxygen uptake rate of the skin increased with the ambient Po₂ between 115 and 230 Torr and became constant (3.8 nmol/cm²/min) between 230 and 295 Torr. When both sides of the skin were ventilated with normoxic water, oxygen uptake rate of the skin increased and became 3.7 nmol/cm²/min at a flow rate of 20–40 ml/min. These results suggest that the oxygen requirement of the skin is 3.7–3.8 nmol/cm²/min at 21.3°C and that cutaneous O₂ uptake in vitro depends on experimental ventilation and ambient Po₂, consistent with values measured in vitro in the carp (ref).     

Attenuation of lipopolysaccharide-induced oxidative stress and apoptosis in fetal pulmonary artery endothelial cells by hypoxia

Pulmonary vascular endothelial injury resulting from lipopolysaccharide (LPS) and oxygen toxicity contributes to vascular simplification seen in the lungs of premature infants with bronchopulmonary dysplasia. Whether the severity of endotoxin-induced endothelial injury is modulated by ambient oxygen tension (hypoxic intrauterine environment vs. hyperoxic postnatal environment) remains unknown. We posited that ovine fetal pulmonary artery endothelial cells (FPAEC) will be more resistant to LPS toxicity under hypoxic conditions (20–25 Torr) mimicking the fetal milieu. LPS (10 μg/ml) inhibited FPAEC proliferation and induced apoptosis under normoxic conditions (21% O₂) in vitro. LPS-induced FPAEC apoptosis was attenuated in hypoxia (5% O₂) and exacerbated by hyperoxia (55% O₂). LPS increased intracellular superoxide formation, as measured by 2-hydroxyethidium (2-HE) formation, in FPAEC in normoxia and hypoxia. 2-HE formation in LPS-treated FPAEC increased in parallel with the severity of LPS-induced apoptosis in FPAEC, increasing from hypoxia to normoxia to hyperoxia. Differences in LPS-induced apoptosis between hypoxia and normoxia were abolished when LPS-treated FPAEC incubated in hypoxia were pretreated with menadione to increase superoxide production. Apocynin decreased 2-HE formation, and attenuated LPS-induced FPAEC apoptosis under normoxic conditions. We conclude that ambient oxygen concentration modulates the severity of LPS-mediated injury in FPAEC by regulating superoxide levels produced in response to LPS.     

Neurogenesis in the aged and neurodegenerative brain

It has been well established that adult neurogenesis occurs throughout life in the subventricular (SVZ) and subgranular (SGZ) zones. However, the exact role of this type of brain plasticity is not yet clear. Many studies have shown that neurogenesis is involved in learning and memory. This has led to a hypothesis which suggests that impairment in memory during aging and neurodegenerative diseases such as Alzheimer’s disease (AD) may involve abnormal neurogenesis. Indeed, during aging, there is an age-related decline in adult neurogenesis. This decline is mostly related to decreased proliferation, associated to decreased stimulation to proliferate in an aging brain. In AD, there is also evidence for decreased neurogenesis, that accompanies the neuronal loss characteristic of the disease. Interestingly in AD, there is increased proliferation, that may be caused by increasing amounts of soluble amyloid ß42-protein (Aβ₄₂). However, most of these new neurons die, and fibrillar Aβ₄₂ seems to be involved in generating an inappropriate environment for these neurons to mature. These findings open prospects for new strategies that can increase neurogenesis in normal or pathological processes in the aging brain, and by that decrease memory deficits.     

The HDAC inhibitor, sodium butyrate, stimulates neurogenesis in the ischemic brain

In the healthy adult brain, neurogenesis normally occurs in the subventricular zone (SVZ) and hippocampal dentate gyrus (DG). Cerebral ischemia enhances neurogenesis in neurogenic and non-neurogenic regions of the ischemic brain of adult rodents. This study demonstrated that post-insult treatment with a histone deacetylase inhibitor, sodium butyrate (SB), stimulated the incorporation of bromo-2'-deoxyuridine (BrdU) in the SVZ, DG, striatum, and frontal cortex in the ischemic brain of rats subjected to permanent cerebral ischemia. SB treatment also increased the number of cells expressing polysialic acid–neural cell adhesion molecule, nestin, glial fibrillary acidic protein, phospho-cAMP response element-binding protein (CREB), and brain-derived neurotrophic factor (BDNF) in various brain regions after cerebral ischemia. Furthermore, extensive co-localization of BrdU and polysialic acid–neural cell adhesion molecule was observed in multiple regions after ischemia, and SB treatment up-regulated protein levels of BDNF, phospho-CREB, and glial fibrillary acidic protein. Intraventricular injection of K252a, a tyrosine kinase B receptor antagonist, markedly reduced SB-induced cell proliferation detected by BrdU and Ki67 in the ipsilateral SVZ, DG, and other brain regions, blocked SB-induced nestin expression and CREB activation, and attenuated the long-lasting behavioral benefits of SB. Together, these results suggest that histone deacetylase inhibitor-induced cell proliferation, migration and differentiation require BDNF–tyrosine kinase B signaling and may contribute to long-term beneficial effects of SB after ischemic injury.     

The effects of social environment on adult neurogenesis in the female prairie vole

In the mammalian brain, adult neurogenesis has been found to occur primarily in the subventricular zone (SVZ) and dentate gyrus of the hippocampus (DG) and to be influenced by both exogenous and endogenous factors. In the present study, we examined the effects of male exposure or social isolation on neurogenesis in adult female prairie voles (Microtus ochrogaster). Newly proliferated cells labeled by a cell proliferation marker, 5-bromo-2'-deoxyuridine (BrdU), were found in the SVZ and DG, as well as in other brain areas, such as the amygdala, hypothalamus, neocortex, and caudate/putamen. Two days of male exposure significantly increased the number of BrdU-labeled cells in the amygdala and hypothalamus in comparison to social isolation. Three weeks later, group differences in BrdU labeling generally persisted in the amygdala, whereas in the hypothalamus, the male-exposed animals had more BrdU-labeled cells than did the female-exposed animals. In the SVZ, 2 days of social isolation increased the number of BrdU-labeled cells compared to female exposure, but this difference was no longer present 3 weeks later. We have also found that the vast majority of the BrdU-labeled cells contained a neuronal marker, indicating neuronal phenotypes. Finally, group differences in the number of cells undergoing apoptosis were subtle and did not seem to account for the observed differences in BrdU labeling. Together, our data indicate that social environment affects neuron proliferation in a stimulus- and site-specific manner in adult female prairie voles.     

Adult neurogenic process in the subventricular zone‐olfactory bulb system is regulated by Tau protein under prolonged stress

ObjectivesThe area of the subventricular zone (SVZ) in the adult brain exhibits the highest number of proliferative cells, which, together with the olfactory bulb (OB), maintains constant brain plasticity through the generation, migration and integration of newly born neurons. Despite Tau and its malfunction is increasingly related to deficits of adult hippocampal neurogenesis and brain plasticity under pathological conditions [e.g. in Alzheimer''s disease (AD)], it remains unknown whether Tau plays a role in the neurogenic process of the SVZ and OB system under conditions of chronic stress, a well‐known sculptor of brain and risk factor for AD.Materials and methodsDifferent types of newly born cells in SVZ and OB were analysed in animals that lack Tau gene (Tau‐KO) and their wild‐type littermates (WT) under control or chronic stress conditions.ResultsWe demonstrate that chronic stress reduced the number of proliferating cells and neuroblasts in the SVZ leading to decreased number of newborn neurons in the OB of adult WT, but not Tau‐KO, mice. Interestingly, while stress‐evoked changes were not detected in OB granular cell layer, Tau‐KO exhibited increased number of mature neurons in this layer indicating altered neuronal migration due to Tau loss.ConclusionsOur findings suggest the critical involvement of Tau in the neurogenesis suppression of SVZ and OB neurogenic niche under stressful conditions highlighting the role of Tau protein as an essential regulator of stress‐driven plasticity deficits.     

RNA-binding protein FXR2 regulates adult hippocampal neurogenesis by reducing Noggin expression

In adult mammalian brains, neurogenesis persists in the subventricular zone of the lateral ventricles (SVZ) and the dentate gyrus (DG) of the hippocampus. Although evidence suggest that adult neurogenesis in these two regions is subjected to differential regulation, the underlying mechanism is unclear. Here, we show that the RNA-binding protein FXR2 specifically regulates DG neurogenesis by reducing the stability of Noggin mRNA. FXR2 deficiency leads to increased Noggin expression and subsequently reduced BMP signaling, which results in increased proliferation and altered fate specification of neural stem/progenitor cells in DG. In contrast, Noggin is not regulated by FXR2 in the SVZ, because Noggin expression is restricted to the ependymal cells of the lateral ventricles, where FXR2 is not expressed. Differential regulation of SVZ and DG stem cells by FXR2 may be a key component of the mechanism that governs the different neurogenic processes in these two adult germinal zones.     

Circadian Clock Genes Are Essential for Normal Adult Neurogenesis,Differentiation, and Fate Determination

Adult neurogenesis creates new neurons and glia from stem cells in the human brain throughout life. It is best understood in the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ). Circadian rhythms have been identified in the hippocampus, but the role of any endogenous circadian oscillator cells in hippocampal neurogenesis and their importance in learning or memory remains unclear. Any study of stem cell regulation by intrinsic circadian timing within the DG is complicated by modulation from circadian clocks elsewhere in the brain. To examine circadian oscillators in greater isolation, neurosphere cultures were prepared from the DG of two knockout mouse lines that lack a functional circadian clock and from mPer1::luc mice to identify circadian oscillations in gene expression. Circadian mPer1 gene activity rhythms were recorded in neurospheres maintained in a culture medium that induces neurogenesis but not in one that maintains the stem cell state. Although the differentiating neural stem progenitor cells of spheres were rhythmic, evidence of any mature neurons was extremely sparse. The circadian timing signal originated in undifferentiated cells within the neurosphere. This conclusion was supported by immunocytochemistry for mPER1 protein that was localized to the inner, more stem cell-like neurosphere core. To test for effects of the circadian clock on neurogenesis, media conditions were altered to induce neurospheres from BMAL1 knockout mice to differentiate. These cultures displayed unusually high differentiation into glia rather than neurons according to GFAP and NeuN expression, respectively, and very few BetaIII tubulin-positive, immature neurons were observed. The knockout neurospheres also displayed areas visibly devoid of cells and had overall higher cell death. Neurospheres from arrhythmic mice lacking two other core clock genes, Cry1 and Cry2, showed significantly reduced growth and increased astrocyte proliferation during differentiation, but they generated normal percentages of neuronal cells. Neuronal fate commitment therefore appears to be controlled through a non-clock function of BMAL1. This study provides insight into how cell autonomous circadian clocks and clock genes regulate adult neural stem cells with implications for treating neurodegenerative disorders and impaired brain functions by manipulating neurogenesis.     

Partial pressure of oxygen in brain and peripheral nerve during damaging electrical stimulation

The studies were performed to elucidate the mechanism underlying the neural damage which may occur during prolonged electrical stimulation of either brain tissue or peripheral nerve. The partial pressure of oxygen (pO₂) was measured in the sciatic nerve and the cerebral cortex of adult cats before and during direct, local electrical stimulation of these neural tissues, using stimulus parameters capable of inducing neural injury. pO₂ was monitored by the polarographic method, employing a platinum microelectrode inserted into the tissue adjacent to or beneath the stimulating electrode. In the sciatic nerve there was no marked change in intrafascicular pO₂ in three cats upon initiation of the electrical stimulation. In a fourth animal intraneural pO₂ increased briefly upon intitiation of the stimulation. In no case did the intrafascicular compartment of nerves become significantly hypoxic. In the cerebral cortex, the start of stimulation was accompanied by a significant increase (approximately 12–15 Torr) in intracortical pO₂ beneath the stimulating electrode, and pO₂ remained at or above the pre-stimulus value for the duration of the stimulation. These results show that extracellular hypoxia is unlikely to be a significant factor in the neural injury induced in brain or peripheral nerve by prolonged electrical stimulation.     

The effects of hypoxia and temperature on metabolic aspects of embryonic development in the annual killifish Austrofundulus limnaeus

Embryos of Austrofundulus limnaeus are exceptional in their ability to tolerate prolonged bouts of complete anoxia. Hypoxia and anoxia are a normal part of their developmental environment. Here, we exposed embryos to a range of PO₂ levels at two different temperatures (25 and 30 °C) to study the combined effects of reduced oxygen and increased temperature on developmental rate, heart rate, and metabolic enzyme capacity. Hypoxia decreased overall developmental rate and caused a stage-specific decline in heart rate. However, the rate of early development prior to the onset of organogenesis is insensitive to PO₂. Increased incubation temperature caused an increase in the developmental rate at high PO₂s, but hindered developmental progression under severe hypoxia. Embryonic DNA content in pre-hatching embryos was positively correlated with PO₂. Citrate synthase, lactate dehydrogenase, and phosphoenolpyruvate carboxykinase capacity were all reduced in embryos developing under hypoxic conditions. Embryos of A. limnaeus are able to develop normally across a wide range of PO₂s and contrary to most other vertebrates severe hypoxia is not a teratogen. Embryos of A. limnaeus do not respond to hypoxia through an increase in the capacity for enzymatic activity of the metabolic enzymes lactate dehydrogenase, citrate synthase, or phosphoenolpyruvate carboxykinase. Instead they appear to adjust whole-embryo metabolic capacity to match oxygen availability. However, decreased DNA content in hypoxia-reared embryos suggests that cellular enzymatic capacity may remain unchanged in response to hypoxia, and the reduced capacity may rather indicate reduced cell number in hypoxic embryos.     

Tauroursodeoxycholic Acid Enhances Mitochondrial Biogenesis,Neural Stem Cell Pool,and Early Neurogenesis in Adult Rats

Although neurogenesis occurs in restricted regions of the adult mammalian brain, neural stem cells (NSCs) produce very few neurons during ageing or after injury. We have recently discovered that the endogenous bile acid tauroursodeoxycholic acid (TUDCA), a strong inhibitor of mitochondrial apoptosis and a neuroprotective in animal models of neurodegenerative disorders, also enhances NSC proliferation, self-renewal, and neuronal conversion by improving mitochondrial integrity and function of NSCs. In the present study, we explore the effect of TUDCA on regulation of NSC fate in neurogenic niches, the subventricular zone (SVZ) of the lateral ventricles and the hippocampal dentate gyrus (DG), using rat postnatal neurospheres and adult rats exposed to the bile acid. TUDCA significantly induced NSC proliferation, self-renewal, and neural differentiation in the SVZ, without affecting DG-derived NSCs. More importantly, expression levels of mitochondrial biogenesis-related proteins and mitochondrial antioxidant responses were significantly increased by TUDCA in SVZ-derived NSCs. Finally, intracerebroventricular administration of TUDCA in adult rats markedly enhanced both NSC proliferation and early differentiation in SVZ regions, corroborating in vitro data. Collectively, our results highlight a potential novel role for TUDCA in neurologic disorders associated with SVZ niche deterioration and impaired neurogenesis.     

Targeted Deletion of the ERK5 MAP Kinase Impairs Neuronal Differentiation,Migration, and Survival during Adult Neurogenesis in the Olfactory Bulb

Recent studies have led to the exciting idea that adult-born neurons in the olfactory bulb (OB) may be critical for complex forms of olfactory behavior in mice. However, signaling mechanisms regulating adult OB neurogenesis are not well defined. We recently reported that extracellular signal-regulated kinase (ERK) 5, a MAP kinase, is specifically expressed in neurogenic regions within the adult brain. This pattern of expression suggests a role for ERK5 in the regulation of adult OB neurogenesis. Indeed, we previously reported that conditional deletion of erk5 in adult neurogenic regions impairs several forms of olfactory behavior in mice. Thus, it is important to understand how ERK5 regulates adult neurogenesis in the OB. Here we present evidence that shRNA suppression of ERK5 in adult neural stem/progenitor cells isolated from the subventricular zone (SVZ) reduces neurogenesis in culture. By contrast, ectopic activation of endogenous ERK5 signaling via expression of constitutive active MEK5, an upstream activating kinase for ERK5, stimulates neurogenesis. Furthermore, inducible and conditional deletion of erk5 specifically in the neurogenic regions of the adult mouse brain interferes with cell cycle exit of neuroblasts, impairs chain migration along the rostral migratory stream and radial migration into the OB. It also inhibits neuronal differentiation and survival. These data suggest that ERK5 regulates multiple aspects of adult OB neurogenesis and provide new insights concerning signaling mechanisms governing adult neurogenesis in the SVZ-OB axis.     

Embryonic rotational behaviour in the pond snail Lymnaea stagnalis: influences of environmental oxygen and development stage

Responses of freshwater organisms to environmental oxygen tensions (PO₂) have focused on adult (i.e. late developmental) stages, yet responses of embryonic stages to changes in environmental PO₂ must also have implications for organismal biology. Here we assess how the rotational behaviour of the freshwater snail Lymnaea stagnalis changes during development in response to conditions of hypoxia and hyperoxia. As rotation rate is linked to gas mixing in the fluid surrounding the embryo, we predicted that it would increase under hypoxic conditions but decrease under hyperoxia. Contrary to predictions, early, veliger stage embryos showed no change in their rotation rate under hyperoxia, and later, hippo stage embryos showed only a marginally significant increase in rotation under these conditions. Predictions for hypoxia were broadly supported, however, with both veliger and hippo stages showing a marked hypoxia-related increase in their rotation rates. There were also subtle differences between developmental stages, with hippos responding at PO₂s (50% air saturation) greater than those required to elicit a similar response in veligers (20% air saturation). Differences between developmental stages also occurred on return to normoxic conditions following hypoxia: rotation in veligers returned to pre-exposure levels, whereas there was a virtual cessation in embryos at the hippo stage, likely the result of overstimulation of oxygen sensors driving ciliary movement in later, more developed embryos. Together, these findings suggest that the spinning activity of L. stagnalis embryos varies depending on environmental PO₂s and developmental stage, increasing during hypoxia to mix capsular contents and maintain a diffusive gradient for oxygen entry into the capsule from the external environment (“stir-bar” theory of embryonic rotational behaviour).