Quantification of heat shock protein mRNA expression in warm and cold anoxic turtles (Trachemys scripta) using an external RNA control for normalization

Stecyk, Jonathan A. W.; Couturier, Christine S.; Fagernes, Cathrine E.; Ellefsen, Stian; Nilsson, Göran

doi:10.1016/j.cbd.2011.11.001

Cited by 29 publications

(25 citation statements)

References 62 publications

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“…Also surprising was the readily detectable normoxic levels of Hsp72, because in mammals it is essentially undetectable under control conditions (Snoeckx et al, 2001). Additional work has shown high basal levels of numerous HSPs in the brain ) and other organs (Stecyk et al, 2012) and their rapid upregulation in anoxia. Some HSPs continue to increase over 24 h anoxia in the brain; in mammals these are associated primarily with glia, leading to the speculation that in the turtle brain astrocytes may play a significant role throughout anoxia even if neurons shut down much of their function .…”

Section: Neuroprotection At the Molecular Levelmentioning

confidence: 99%

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No oxygen? No problem! Intrinsic brain tolerance to hypoxia in vertebrates

Larson

Drew

Folkow

et al. 2014

Journal of Experimental Biology

134

102

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Many vertebrates are challenged by either chronic or acute episodes of low oxygen availability in their natural environments. Brain function is especially vulnerable to the effects of hypoxia and can be irreversibly impaired by even brief periods of low oxygen supply. This review describes recent research on physiological mechanisms that have evolved in certain vertebrate species to cope with brain hypoxia. Four model systems are considered: freshwater turtles that can survive for months trapped in frozen-over lakes, arctic ground squirrels that respire at extremely low rates during winter hibernation, seals and whales that undertake breath-hold dives lasting minutes to hours, and naked mole-rats that live in crowded burrows completely underground for their entire lives. These species exhibit remarkable specializations of brain physiology that adapt them for acute or chronic episodes of hypoxia. These specializations may be reactive in nature, involving modifications to the catastrophic sequelae of oxygen deprivation that occur in non-tolerant species, or preparatory in nature, preventing the activation of those sequelae altogether. Better understanding of the mechanisms used by these hypoxiatolerant vertebrates will increase appreciation of how nervous systems are adapted for life in specific ecological niches as well as inform advances in therapy for neurological conditions such as stroke and epilepsy.KEY WORDS: Arctic ground squirrel, Cetacean, Hypoxia, Naked mole-rat, Seal, Turtle IntroductionEnvironmental conditions vary enormously for vertebrates, both with respect to the extreme conditions tolerated by a given species at different times and with respect to average living conditions tolerated by different species. Temperature is perhaps the most obvious example: from the poles to the equator, average ambient temperatures vary widely and have been accompanied by physiological adaptations appropriate to resident species; seasonal variations in temperature and resource variability can induce dramatic changes in physiological and/or behavioral patterns including migration and hibernation. Oxygen levels also vary widely, with animals adapted to sea level, high-altitude, underground and aquatic habitats. Oxygen levels can also change dramatically on a shorter-term basis, as can occur in tidal pools or in breath-hold divers. Approximately 20% of the oxygen consumed by the human body is used by the brain. The greater part of this oxygen is used to produce the ATP required to maintain the membrane potentials necessary for electrical signaling with synaptic and action potentials (Harris et al., 2012). In many vertebrates, including adult humans, interruption of the oxygen supply to the brain for more than a few minutes leads to irreversible neurological damage, including neuronal death. Without oxidative phosphorylation, ATP-dependent neuronal processes including ion transport and neurotransmitter reuptake decline sharply. Without pumping, ion gradients fail and neurons depolarize, releasing excessive levels...

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Section: Neuroprotection At the Molecular Levelmentioning

confidence: 99%

“…Interestingly, Stecyk et al reported HSP levels are also enhanced by cold temperatures, hypothesizing that turtles will experience winter temperatures before their ponds freeze over, and thus cold may further prepare them for anoxia before oxygen levels are fully depleted (Stecyk et al, 2012).…”

Section: Neuroprotection At the Molecular Levelmentioning

confidence: 99%

No oxygen? No problem! Intrinsic brain tolerance to hypoxia in vertebrates

Larson

Drew

Folkow

et al. 2014

Journal of Experimental Biology

134

102

View full text Add to dashboard Cite

show abstract

“…wesenbergi that shows no sequence homology to known vertebrate mRNA species, was recently developed as a more reliable method for the normalization of real-time RT-PCR data from tissues of anoxia-tolerant species than other commonly employed normalization techniques (Ellefsen et al, 2008). Additionally, the methodology allows for inter-tissue comparisons of target gene expression (Stecyk et al, 2012). Samples were then chilled on ice, vortexed, incubated at room temperature for 15 min and vortexed again.…”

Section: Rna Extractionmentioning

confidence: 99%

Phylogeny and effects of anoxia on hyperpolarization-activated, cyclic nucleotide-gated channel gene expression in the heart of a primitive chordate, the Pacific Hagfish (eptatretus stoutii)

Wilson

Stecyk

Couturier

et al. 2013

Journal of Experimental Biology

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SUMMARYThe aneural heart of the Pacific hagfish, Eptatretus stoutii, varies heart rate fourfold during recovery from anoxia. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which play an important role in establishing the pacemaker rate of vertebrate hearts, were postulated to be present in this ancestral vertebrate heart, and it was also theorized that changes in hagfish heart rate with oxygen availability involved altered HCN expression. Partial gene cloning revealed six HCN isoforms in the hagfish heart. Hagfish representatives of HCN2, HCN3 and HCN4 were discovered, with HCN2 and HCN3 existing as isoforms designated as HCN2a, HCN2b, HCN3a, two paralogs of HCN3b, and HCN3c. Phylogenetic analysis revealed HCN3b and HCN3c to be ancestral, followed by HCN3a, HCN4 and HCN2. Moreover, HCN3a expression was dominant in both the atrial and ventricular chambers, suggesting that the HCN4 dominance in adult mammalian hearts appeared after hagfish divergence. HCN expression was higher in the atrium than in the ventricle, as might be expected given that atrial beating rate is known to be faster than the ventricular rate. In addition, mRNA expression under normoxic conditions was compared with that following 24 h of anoxia, and either a 2-h or 36-h recovery in normoxic water. In the ventricle, anoxia decreased HCN3a but not HCN4 expression. In contrast, atrial HCN3a expression significantly increased following 2 h of recovery, before returning to control levels following 36 h of recovery, possibly contributing to heart rate changes previously observed under these conditions.

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“…Total RNA was eluted in nuclease-free water and quantified using a QUBIT v. 2.0 Fluorometer (Invitrogen). Two hundred nanograms of total RNA was reverse-transcribed into cDNA using the oligo (dT) 16 primer with MultiScribe TM MuLV Reverse Transcriptase in a High Capacity RNA-to-cDNA Kit (4387406, Invitrogen), following the manufacturer's protocol. Quantitative real-time PCR was performed in triplicate using the iTaq Universal SYBR w Green Supermix (172-5121, BioRad) on a Step-One Plus PCR System (Applied Biosystems) with the following program: 958C for 10 min; 40 cycles of 958C for 10 s, 608C for 60 s. RT-qPCR was performed in a 10 ml reaction with 10 ng of cDNA and final primer concentration of 200 nM.…”

Section: (C) Rna Extraction and Rt-qpcrmentioning

confidence: 99%

Heritable variation in heat shock gene expression: a potential mechanism for adaptation to thermal stress in embryos of sea turtles

et al. 2016

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The capacity of species to respond adaptively to warming temperatures will be key to their survival in the Anthropocene. The embryos of egg-laying species such as sea turtles have limited behavioural means for avoiding high nest temperatures, and responses at the physiological level may be critical to coping with predicted global temperature increases. Using the loggerhead sea turtle (Caretta caretta) as a model, we used quantitative PCR to characterise variation in the expression response of heat-shock genes (hsp60, hsp70 and hsp90; molecular chaperones involved in cellular stress response) to an acute non-lethal heat shock. We show significant variation in gene expression at the clutch and population levels for some, but not all hsp genes. Using pedigree information, we estimated heritabilities of the expression response of hsp genes to heat shock and demonstrated both maternal and additive genetic effects. This is the first evidence that the heat-shock response is heritable in sea turtles and operates at the embryonic stage in any reptile. The presence of heritable variation in the expression of key thermotolerance genes is necessary for sea turtles to adapt at a molecular level to warming incubation environments.

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Quantification of heat shock protein mRNA expression in warm and cold anoxic turtles (Trachemys scripta) using an external RNA control for normalization

Cited by 29 publications

References 62 publications

No oxygen? No problem! Intrinsic brain tolerance to hypoxia in vertebrates

No oxygen? No problem! Intrinsic brain tolerance to hypoxia in vertebrates

Phylogeny and effects of anoxia on hyperpolarization-activated, cyclic nucleotide-gated channel gene expression in the heart of a primitive chordate, the Pacific Hagfish (eptatretus stoutii)

Heritable variation in heat shock gene expression: a potential mechanism for adaptation to thermal stress in embryos of sea turtles

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