Effect of ionizing radiation on the synthetic activity of blood system cells in ground squirrels in different physiological states of animals

The functional (synthetic) activity of blood lymphocytes and bone marrow haemopoietic cells in ground squirrels during the annual cycle as well as in hibernating and awaken animals in winter have been studied by fluorescent microspectrometry. The effect of ionizing radiation on animals in different functional states of the hibernation-arousal bout was investigated too. It was shown that the synthetic activity (parameter alpha) in blood lymphocytes was minimal in hibernating state in winter and maximal in active euthermic spring animals, then slightly decreased in June and more considerably decreased in the prehibernating autumn period. In awake animals in winter, the values of parameter alpha reached the same values as in summer. The changes of parameter alpha in bone marrow haemopoietic cells were essentially the same: the minimal values were observed in the prehibernation autumn period and in awake animals in winter the alpha values were slightly higher than in active euthermic animals in summer. The maximal synthetic activity in bone marrow haemopoietic cells in active euthermic spring animals is due mainly to cells in G1-G2 phases of the cell cycle. The decrease of the synthetic activity in summer is a result of the cell transition from G2 to mitosis and transition of a part of cells to G0 When investigating the hibernation-arousal bout in ground squirrels in winter, during arousal, we found two stages considerably differing in both the values of parameter alpha in bone marrow haemopoietic cells and the number of blood cells. The synthetic activity and the total number of blood and bone marrow cells in ground squirrels irradiated in the state of deep hibernation did not differ significantly from the state of non-irradiated hibernating animals. The negative effect of radiation appeared upon the arousal of these animals but it was expressed to a lesser degree in comparison with the animals irradiated in the active state. It was found that the acute irradiation of animals during arousal from hibernation in the second stage caused the most pronounced functional inactivation and cell death. The physiological state of ground squirrels subjected to ionizing irradiation at different phases of the hibernation-arousal bout plays a determining role in the changes of the qualitative and quantitative characteristics of blood system cells. Thus, the hypometabolic state of ground squirrels in hibernation is a factor of protection from the action of ionizing radiation on the organism and the immune system.     

Production of tumor necrosis factor in cells of hibernating ground squirrels Citellus undulatus during annual cycle

TNF production has been studied in peritoneal macrophages and splenic T cells of Arctic Yakutian ground squirrel (Citellus Undulatus Pallas) in hibernating and awake animals in winter and in prehibernating autumn as well as in active euthermic spring-summer animals. A high level of TNF production in macrophages of ground squirrel is observed over the active period and during arousals in winter. There are no significant season variations in TNF production in splenic T lymphocytes of ground squirrels. This suggests the major role of activated macrophages in the arousals of hibernating animals. T lymphocyte proliferation in ground squirrels in the active period is higher than in winter, and the most significant seasonal variations are found in T cell mitogenic response, which increases in spring-summer period. Evidence is presented that functional activity of macrophages of squirrel in autumn has much in common with that in winter rather than in spring-summer period.     

The effect of hibernation on lipids of the liver mitochondrial fraction in the Yakut ground squirrel <Emphasis Type="Italic">Spermophilus undulatus</Emphasis>

We demonstrated that the level of phospholipids in the liver mitochondrial fraction is increased by 60% during the winter hibernation season in the Yakut ground squirrel S. undulatus; the phospholipid composition in sleeping animals is characterized by an increase in phosphatidylethanolamine compared with summer animals. A sharp increase in the level of cholesterol, as well as fatty acid, monoglycerides, and diglycerides was found in the mitochondrial fraction of hibernating ground squirrels in relation to summer ground squirrels. Functional changes during hibernation concern the level of phosphatidylserine (the growth in sleeping animals compared with active animals). Seasonal modification of the lipid composition of the liver mitochondria (particularly, an increase in the level of cholesterol) can play a role in the resistance of mitochondria to the seasonal increase in the level of fatty acids in the liver. Lipids of the liver mitochondrial fraction are involved in the ground squirrel adaptation to the hibernation season.     

Seasonal and age changes in the thymus gland of the Red fox, Vulpes vulpes,

Seasonal and age changes in thymus weight and histological structure were examined in the Red fox ( Vulpes vulpes ). Growth in the fox thymus slowed down after birth compared with the last third of foetal existence, but the gland still grew rapidly to reach a peak first year weight when the cubs were 20 weeks of age. From this point the thymus in both sexes decreased markedly in weight to reach a low point by the beginning of the first breeding season. During this involution lobule structure broke down and adipose tissue and connective tissue was laid down in the gland. Recovery of the thymus towards the second year weight maximum was accompanied by the regaining of lobule structure and the gland resembled that of the juvenile again. The male thymus increased in weight from the middle of the mating season, but recovery in the female thymus was delayed until the end of lactation. Involution occurred prior to the second breeding season. Thereafter, the gland never attained the high weights seen in the first two years of life, but histological changes still occurred even in old animals. The thymus gland of animals infected with sarcoptic mange is described.     

Antioxidant defenses in the ground squirrel Citellus citellus. 2. The effect of hibernation

In spring and autumn, the ground squirrel Citellus citellus is awake and active but in winter it usually hibernates. Reawakening from hibernation involves intense metabolic activity in the interscapular brown adipose tissue (IBAT). The IBAT of hibernation animals showed significant increases in the activities of superoxide dismutase (both copper-zinc and manganese-containing enzymes), glutathione peroxidase, and in the amount of ascorbate present. Glutathione peroxidase also increased in the liver, as did ascorbate in the plasma. These changes were not merely a consequence of exposure to low environmental temperatures. It is proposed that antioxidant defenses are increased in the IBAT of ground squirrels at the onset of hibernation in order to protect the tissue from reactive oxygen species generated as a result of the intense metabolic activity sustained by this tissue during reawakening.     

Study of the testis in the active and hibernating hedgehog

The structure and ultrastructure of the hedgehog's testis has been studied during active and hibernating states. The maximum activity of the hedgehog's testis manifest itself in spring and summer. In autumn they enter in a seasonal resting, in which, the interstitial cells atrophy and it can observe primary spermatocytes resting next to spermatogonia and supporting cells in the seminiferous tubules. These have a minimal diameter and contain only primary spermatocytes in hibernating period also the seminiferous epithelium that compound the walls of the tubules are about 72 microns thick in the active period and about 47 microns in hibernation. The Sertoli cells shows a small diameter in hibernation than in the active season. The nucleus has one or two nucleoli with distinguish themselves from those observed in the active season because of their closer union with the inner nuclear envelope. In autumn (resting period) the interstitial tissue regress and the Leydig cells are aggregated in blocks with little cytoplasm, minimum size nuclei and 1 or 2 nucleolus closely to the inner nuclear membrane. Like then in the ovary, the hedgehog's testis follows a seasonal cycle determined by the temperature and environmental changes and it have a power of physiological adaptation inherent of a typical hibernating animal.     

Comparison of Brain Transcriptome of the Greater Horseshoe Bats (Rhinolophus ferrumequinum) in Active and Torpid Episodes

Hibernation is an energy-saving strategy which is widely adopted by heterothermic mammals to survive in the harsh environment. The greater horseshoe bat (Rhinolophus ferrumequinum) can hibernate for a long period in the hibernation season. However, the global gene expression changes between hibernation and non-hibernation season in the greater horseshoe bat remain largely unknown. We herein reported a comprehensive survey of differential gene expression in the brain between winter hibernating and summer active greater horseshoe bats using next-generation sequencing technology. A total of 90,314,174 reads were generated and we identified 1,573 differentially expressed genes between active and torpid states. Interestingly, we found that differentially expressed genes are over-represented in some GO categories (such as metabolic suppression, cellular stress responses and oxidative stress), which suggests neuroprotective strategies might play an important role in hibernation control mechanisms. Our results determined to what extent the brain tissue of the greater horseshoe bats differ in gene expression between summer active and winter hibernating states and provided comprehensive insights into the adaptive mechanisms of bat hibernation.     

The hibernation cycle and related changes in the brown fat tissue of Citellus lateralis

The hibernating habits of Citellus lateralis under standard laboratory conditions are described. It has been shown that it is possible to use a biopsy technique to investigate the brown fat tissues without serious disturbance to the hibernation pattern. This technique was used to sample brown and white fat tissues in the same animal at various seasons of the year. Histological studies did not reveal any changes in morphology or lipid composition which could be related purely to the hibernating season, however, during the short periods of arousal a large proportion of the brown fat cells appeared to be partially depleted of neutral fat. At this time a large increase in Luxol fast blue staining was always observed, which may be due to unmasking of tissue phosphatides.     

Membrane lipids and morphology of brain cortex synaptosomes isolated from hibernating Yakutian ground squirrel

Synaptosomes were isolated from Yakutian ground squirrel brain cortex of summer and winter hibernating animals in active and torpor states. Synaptosomal membrane cholesterol and phospholipids were determined. The seasonal changes of synaptosomal lipid composition were found. Synaptosomes isolated from hibernating Yakutian ground squirrel brain cortex maintained the cholesterol sphingomyelin, phosphatidylethanolamine, lysophosphatidylcholine, cardiolipin, phosphatidylinositol and phosphatidylserine contents 2.5, 1.8, 2.6, 1.8, 1.6, and 1.3 times less, respectively, and the content of phosphatidylcholine twice as much as the one in summer season. The synaptosomal membrane lipid composition of summer animals was shown to be markedly different from that as hibernating ground squirrels and non-hibernating rodents. It is believed that phenotypic changes of synaptosomal membrane lipid composition in summer Yakutian ground squirrel are the important preparation step for hibernation. The phosphatidylethanolamine content was increased in torpor state compared with winter-active state and the molar ratio of cholesterol/phospholipids in synaptosomal membrane of winter torpor ground squirrels was lower than that in active winter and summer animals. These events were supposed to lead to increase of the synaptosomal membrane fluidity during torpor. Synaptosomes isolated from torpor animals have larger sizes and contain a greater number of synaptic vesicles on the synaptosomal profile area. The synaptosomal membrane lipid composition and synaptosome morphology were involved in phenotypic adaptation of Yakutian ground squirrel to hibernation.     

Specifics of monoaminergic regulation of the central nervous system of hibernating animals (Citellus undulatus)

Seasonal features of open-field behavior of Yakut ground squirrels (Citellus undulatus) and changes in the brain MAO A activity were studied. It was found that all parameters of exploratory activity in the open field and holeboard test reached the values characteristic of summer animals very rapidly, within a few days or (in some cases) even within the first 24 hours after the arousal from hibernation in the middle of April. In autumn these parameters decreased to their minimum values 1.5-2 months prior to hibernation. Biochemical analysis showed that in April the activity of MAO A measured with serotonin as a substrate in the hippocampus was 1.8 times (p < 0.05) higher than the activity of MAO A with respect to noradrenaline. In contrast, in autumn the MAO A activity determined with noradrenaline as a substrate was 2.5 (p < 0.05) times higher than the activity a MAO A with respect to serotonin. Taken together, these data indicate that the seasonal features of the higher nervous activity of hibernating animals depend on the balance between serotonergic and noradrenergic systems in different periods of the annual cycle.     

Activation of protein synthesis in tissues of gophers after arousal from hibernation

Intensity of the protein synthesis is studied in cells of different organs and tissues of hibernating, arousing and active gophers and rats. It is established that during hibernation a level of labelled aminoacids incorporation into proteins of all organs under study (ventricle, auricle, liver, spleen, diencephalon, adrenal gland, brown fatty tissue) is inconsiderable. The protein synthesis intensity essentially grows after animals arousing and their body temperature elevation, ten times exceeding the level of the studied process in hibernating gophers. Rate of precursors' incorporation in cell proteins of active gophers and rats is 3-4 times lower than in arousing rodents. The same regularity is revealed when studying rates of labelled amino acids incorporation into slices of the above organs and tissues.     

Seasonal changes in critical enzymes of lipogenesis and triacylglycerol synthesis in the marmot (Marmota flaviventris)

Fatty acid metabolism and triacylglycerol synthesis are critical processes for the survival of hibernating mammals that undergo a prolonged fasting period. Fatty acid synthase, fatty-acid-CoA ligase, diacylglycerol acyltransferase, and monoacylglycerol acyltransferase activities were measured in liver and in white and brown adipose tissue, in order to determine whether enzymes of lipogenesis and triacylglycerol synthesis vary seasonally during hibernation in the yellow-bellied marmot (Marmota flaviventris). Compared with mid-winter hibernation, fatty acid synthase activity was higher in all three tissues during early spring when marmots emerged from hibernation and in mid-summer when they were feeding, consistent with the synthesis of fatty acids from the carbohydrate-rich summer diet. Fatty-acid-CoA ligase and diacylglycerol acyltransferase activities were highest in summer in white adipose tissue when triacylglycerol synthesis would be expected to be high; diacylglycerol acyltransferase activity was also high in brown adipose tissue during spring and summer. In liver, however, diacylglycerol acyltransferase specific activity was highest during hibernation, suggesting that triacylglycerol synthesis may be prominent in liver in winter. Monoacylglycerol acyltransferase activity, which may aid in the retention of essential fatty-acids, was 80-fold higher in liver than in white or brown adipose tissue, but did not vary seasonally. Its dependence on palmitoyl-CoA suggests that a divalent cation might play a role in enzyme activation. The high hepatic diacylglycerol acyltransferase activity during hibernation suggests that the metabolism of very low density lipoprotein may be important in the movement of adipose fatty acids to brown adipose tissue and muscle during the rewarming that occurs periodically during hibernation. These studies suggest that enzymes of lipid metabolism vary seasonally in the marmot, consistent with requirements of this hibernator for triacylglycerol synthesis and metabolism.Abbreviations BAT brown adipose tissue - DGAT diacylglycerol acyltransferase - FAS fatty acid synthase - K _m Michaelis constant - MGAT monoacylglycerol acyltransferase - RQ respiratory quotiant - VLDL very low density lipoprotein - WAT white adipose tissue     

Identification of qRT-PCR reference genes for analysis of opioid gene expression in a hibernator

Previous work has suggested that central and peripheral opioid signaling are involved in regulating torpor behavior and tissue protection associated with the hibernation phenotype. We used quantitative real-time PCR (qRT-PCR) to measure mRNA levels of opioid peptide precursors and receptors in the brain and heart of summer ground squirrels (Ictidomys tridecemlineatus) and winter hibernating squirrels in the torpid or interbout arousal states. The use of appropriate reference genes for normalization of qRT-PCR gene expression data can have profound effects on the analysis and interpretation of results. This may be particularly important when experimental subjects, such as hibernating animals, undergo significant morphological and/or functional changes during the study. Therefore, an additional goal of this study was to identify stable reference genes for use in qRT-PCR studies of the 13-lined ground squirrel. Expression levels of 10 potential reference genes were measured in the small intestine, liver, brain, and heart, and the optimal combinations of the most stable reference genes were identified by the GeNorm Excel applet. Based on this analysis, we provide recommendations for reference genes to use in each tissue that would be suitable for comparative studies among different activity states. When appropriate normalization of mRNA levels was used, there were no changes in opioid-related genes in heart among the three activity states; in brain, DOR expression was highest during torpor, lowest in interbout arousal and intermediate in summer. The results support the idea that changes in DOR expression may regulate the level of neuronal activity in brain during the annual hibernation cycle and may contribute to hibernation-associated tissue protection.     

Evaluation of the role of AMP-activated protein kinase and its downstream targets in mammalian hibernation

Mammalian hibernation requires an extensive reorganization of metabolism that typically includes a greater than 95% reduction in metabolic rate, selective inhibition of many ATP-consuming metabolic activities and a change in fuel use to a primary dependence on the oxidation of lipid reserves. We investigated whether the AMP-activated protein kinase (AMPK) could play a regulatory role in this reorganization. AMPK activity and the phosphorylation state of multiple downstream targets were assessed in five organs of thirteen-lined ground squirrels (Spermophilus tridecemlineatus) comparing euthermic animals with squirrels in deep torpor. AMPK activity was increased 3-fold in white adipose tissue from hibernating ground squirrels compared with euthermic controls, but activation was not seen in liver, skeletal muscle, brown adipose tissue or brain. Immunoblotting with phospho-specific antibodies revealed an increase in phosphorylation of eukaryotic elongation factor-2 at the inactivating Thr56 site in white adipose tissue, liver and brain of hibernators, but not in other tissues. Acetyl-CoA carboxylase phosphorylation at the inactivating Ser79 site was markedly increased in brown adipose tissue from hibernators, but no change was seen in white adipose tissue. No change was seen in the level of phosphorylation of the Ser565 AMPK site of hormone-sensitive lipase in adipose tissues of hibernating animals. In conclusion, AMPK does not appear to participate in the metabolic re-organization and/or the metabolic rate depression that occurs during ground squirrel hibernation.     

Environmental Influences on Mass Dynamics of the Cotton Rat (Sigmodon hispidus) Thymus Gland

The influence of season on thymus gland mass was examined relative to captivity, gender, and age in 921 cotton rats (Sigmodon hispidus) from free-ranging and laboratory populations. Age-related involution of the thymus gland was evident in free-ranging males and females and captive females. A distinct seasonal cycle in thymus mass dynamics was apparent among adult cotton rats. Mass of the thymus gland was greatest from late fall to early winter before declining 2-4 fold during spring. Thymus gland mass remained low through spring and summer in adult cotton rats when reproductive activity was maximum. No seasonal cycle in thymus mass was apparent among juveniles. Possible involvement of sex hormones in regulating thymus size is discussed.     

Seasonal characteristics of the monoaminergic regulation of the behavior of hibernating animals

The stimulating effect of L-DOPA on exploratory activity of the ground squirrel in the hole-board and open field experiments was stronger in the spring than in autumn, whereas the regulating effect of the 5-HTP on adaptive behaviour of hibernating animals was stronger in autumn. Results of the biochemical analysis of the MAO A activity with serotonin or noradrenaline as substrates revealed a seasonal dependence of the substrate specific changes of this enzyme activity in hibernating animals. The data obtained suggest that the seasonal distinctions in effects of similar pharmacological agents depend on activity of the brain monoaminergic systems' activity in hibernating animals in different periods of the annual cycle.     

Myoglobin content in skeletal muscles of hibernating ground squirrels rises in autumn and winter

The content of myoglobin (Mb) in skeletal muscles of Arctic Yakutian ground squirrel (Citellus undulatus Pallas) was measured in the active euthermic summer and prehibernating autumn animals as well as in hibernating and awake animals in winter. The myoglobin content in winter, irrespective of the state of the animal, was found to be about three times higher than in summer. The content of myoglobin in autumn was also two-fold increased compared to summer, suggesting that high myoglobin level is necessary for hibernation. Analysis of biochemical data available suggests that the increase in myoglobin content in winter is probably related to a high oxygen demand of muscles at the first stage of arousal (non-shivering thermogenesis) when rectal temperature rises from 0 to 10-12 degrees C. At this stage, the oxygen-dependent processes in muscles proceed under the conditions when peripheral blood flow is blocked and anaerobic glycolysis is switched off.     

Functional analysis for gut microbes of the brown tree frog (Polypedates megacephalus) in artificial hibernation

Background

Annual hibernation is an adaptation that helps many animals conserve energy during food shortage in winter. This natural cycle is also accompanied by a remodeling of the intestinal immune system, which is an aspect of host biology that is both influenced by, and can itself influence, the microbiota. In amphibians, the bacteria in the intestinal tract show a drop in bacterial counts. The proportion of pathogenic bacteria is greater in hibernating frogs than that found in nonhibernating frogs. This suggests that some intestinal gut microbes in amphibians can be maintained and may contribute to the functions in this closed ecosystem during hibernation. However, these results were derived from culture-based approaches that only covered a small portion of bacteria in the intestinal tract.

Methods

In this study, we use a more comprehensive analysis, including bacterial appearance and functional prediction, to reveal the global changes in gut microbiota during artificial hibernation via high-throughput sequencing technology.

Results

Our results suggest that artificial hibernation in the brown tree frog (Polypedates megacephalus) could reduce microbial diversity, and artificially hibernating frogs tend to harbor core operational taxonomic units that are rarely distributed among nonhibernating frogs. In addition, artificial hibernation increased significantly the relative abundance of the red-leg syndrome-related pathogenic genus Citrobacter. Furthermore, functional predictions via PICRUSt and Tax4Fun suggested that artificial hibernation has effects on metabolism, disease, signal transduction, bacterial infection, and primary immunodeficiency.

Conclusions

We infer that artificial hibernation may impose potential effects on primary immunodeficiency and increase the risk of bacterial infections in the brown tree frog.

     

Seasonal rhythms in the functioning of the hypophyseo-thyroid system of the suslik Citellus parryi

In experiments on the Arctic ground squirrel Citellus parryi, radioimmune assay of the content of thyrotropin of the hypophysis and thyroxine and triiodothyronine of the thyroid in the peripheral blood has been made at monthly intervals from July until May. It was found that during hibernation period, thyroxine and triiodothyronine concentrations in the blood of sleeping animals are high as compared with those during pre-hibernation period in autumn and active period in May. Thyrotropin content of the blood increases from October to May, being the lowest however during the season of the deepest hibernation from December to February. It is suggested that activation of the hypophysial--thyroid system after resting period in summer and autumn begins in October. During deep sleeping, it is depressed, being recovered in March. High levels of the thyroid hormones during hibernation period may be explained by metabolic strategy of the organism in hibernating animals which is directed to optimization of energy supply of hibernation.