Seasonal characteristics of the functioning of the hypophysis-gonad system in the suslik Citellus parryi

In experiments on the arctic ground squirrel C. parryi, studies have been made on seasonal changes in the weight of testes, follicular diameter in the ovaries and the content of sex and gonadotropic hormones in the peripheral blood. Testicular involution and arrest of follicular development were observed in prehibernation period. During hibernation, follicular growth and the increase in the weight of testes take place. The level of LH decreases during hibernation. In sleeping animals, its level is higher as compared to that in active animals during short periods of arousal. The increase in LH level takes place both in males and females in April. FSH can not be detected in males during the first half of hibernation, appearing in the peripheral blood only in March and April. In females, FSH was found in the blood in October, being absent from November to January; beginning from February, it may be found both in sleeping and active animals. Testosterone was found in hibernating males and females, its level significantly increased in March in males, being approximately constant in hibernating females. Estradiol secretion was noted in hibernating females, whereas progesterone was found in the blood only in May.     

Decreases in pineal melatonin content during the hibernation bout in the golden-mantled group squirrel, Spermophilus lateralis

The role of the pineal gland in modulating the rhythmic bouts of hibernation in the golden-mantled ground squirrel (S. lateralis) was explored by comparing pineal melatonin content in hibernating animals with that of euthermic animals at the same time of year. Significant decreases in pineal melatonin content were found in hibernating versus euthermic animals. In addition, significantly lower values for pineal melatonin were observed in hibernating animals that were sacrificed in the late bout period, just prior to expected spontaneous arousal, as compared to hibernating animals that were sacrificed on the first day of their respective bouts. A strong correlation was evident between pineal melatonin content and the duration of the individual hibernation bout. These data suggest that pineal melatonin may be important in determining the duration of individual bouts of hibernation in this species.     

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.     

Thyroid hormone levels in the peripheral blood of the ground squirrel Citellus parryi during winter hibernation

In the Arctic ground squirrel Citellus parryi, thyroxine and triiodothyronine concentrations in the peripheral blood have been determined by RIA in various seasons and during a short hibernation period. It was shown that during hibernation (December-February), hormonal concentration is higher than during homoiothermal period (August-October). The highest concentration of the thyroid hormones was found in hibernating animals in January. The data obtained indicate that thyroid hormones are involved in control of hibernation of the species investigated.     

Metabolic Hormone FGF21 Is Induced in Ground Squirrels during Hibernation but Its Overexpression Is Not Sufficient to Cause Torpor

Hibernation is a natural adaptation that allows certain mammals to survive physiological extremes that are lethal to humans. Near freezing body temperatures, heart rates of 3–10 beats per minute, absence of food consumption, and depressed metabolism are characteristic of hibernation torpor bouts that are periodically interrupted by brief interbout arousals (IBAs). The molecular basis of torpor induction is unknown, however starved mice overexpressing the metabolic hormone fibroblast growth factor 21 (FGF21) promote fat utilization, reduce body temperature, and readily enter torpor–all hallmarks of mammalian hibernation. In this study we cloned FGF21 from the naturally hibernating thirteen-lined ground squirrel (Ictidomys tridecemlineatus) and found that levels of FGF21 mRNA in liver and FGF21 protein in serum are elevated during hibernation torpor bouts and significantly elevated during IBAs compared to summer active animals. The effects of artificially elevating circulating FGF21 concentrations 50 to 100-fold via adenoviral-mediated overexpression were examined at three different times of the year. This is the first time that a transgenic approach has been used in a natural hibernator to examine mechanistic aspects of hibernation. Surgically implanted transmitters measured various metrics of the hibernation phenotype over a 7-day period including changes in motor activity, heart rate and core body temperature. In April fed-state animals, FGF21 overexpression decreased blood insulin and free fatty acid concentrations, effects similar to those seen in obese mice. However, elevated FGF21 concentrations did not cause torpor in these fed-state animals nor did they cause torpor or affect metabolic parameters in fasted-state animals in March/April, August or October. We conclude that FGF21 is strongly regulated during torpor and IBA but that its overexpression is not sufficient to cause torpor in naturally hibernating ground squirrels.     

Diuretic treatment affects the length of torpor bouts in hibernating European ground squirrels (Spermophilus citellus)

During the hibernation season, hibernating mammals show a sequence of torpor bouts that are interrupted by periodic arousals and brief normothermic periods. The functional significance of periodic arousals is still uncertain. We hypothesized that the imbalances in water economy may play a role in the timing of periodic arousals in hibernating species. We applied furosemide, a diuretic drug, to assess whether hibernating European ground squirrels respond to elevated urine production by shortening their torpor bouts. Urine production in the treated squirrels increased and led to more frequent arousals, presumably to restore water balance by recovery of lost water from blood and tissues. The length of the subsequent normothermic phase was not affected by the diuretic treatment. Body mass change correlated primarily with the amount of voided urine. Although our study did not identify the underlying mechanism, our results support the view that water economy, and water loss may play a role in the timing of periodic arousals.     

Testis development and testosterone secretion in captive European ground squirrels before, during, and after hibernation

In some hibernating species, an extended euthermic period before spring emergence has been reported during which testicular maturation occurred. In this study, we investigated whether male European ground squirrels Spermophilus citellus (Linnaeus, 1766) develop scrotal testes before or after the termination of hibernation. The course of testis development and testosterone concentrations were examined in young-of-year male ground squirrels (n = 4) before, during, and after their first hibernation. In the environmental chamber, all animals showed regular hibernation patterns with decreasing torpor bout lengths prior to the end of hibernation. Four weeks before hibernation ended, testosterone levels were elevated during spontaneous arousals. Testosterone concentrations peaked during the first 2 months post heterothermy and decreased thereafter. In 2 experimental males, testes descended from the inguinal region into the scrotum shortly before the end of heterothermy. The state of testis development and testosterone secretion corresponded to that found in semi-free-living males at spring emergence. Testis size increased in active animals to maximum widths during weeks 3 and 4 post heterothermy. Scrotal skin pigmentation was absent until heterothermy ended and developed during the first month thereafter. The data suggest that male S. citellus do not need a pre-emergence euthermic period for reproductive development.     

Periodic arousal from hibernation is necessary for initiation of immune responses in ground squirrels

Golden-mantled ground squirrels (Spermophilus lateralis) undergo seasonal hibernation during which core body temperature (T(b)) values are maintained 1-2 degrees C above ambient temperature. Hibernation is not continuous. Squirrels arouse at approximately 7-day intervals, during which T(b) increases to 37 degrees C for approximately 16 h; thereafter, they return to hibernation and sustain low T(b)s until the next arousal. Over the course of the hibernation season, arousals consume 60-80% of a squirrel's winter energy budget, but their functional significance is unknown and disputed. Host-defense mechanisms appear to be downregulated during the hibernation season and preclude normal immune responses. These experiments assessed immune function during hibernation and subsequent periodic arousals. The acute-phase response to bacterial lipopolysaccharide (LPS) was arrested during hibernation and fully restored on arousal to normothermia. LPS injection (ip) resulted in a 1-1.5 degrees C fever in normothermic animals that was sustained for > 8 h. LPS was without effect in hibernating squirrels, neither inducing fever nor provoking arousal, but a fever did develop several days later, when squirrels next aroused from hibernation; the duration of this arousal was increased sixfold above baseline values. Intracerebroventricular infusions of prostaglandin E(2) provoked arousal from hibernation and induced fever, suggesting that neural signaling pathways that mediate febrile responses are functional during hibernation. Periodic arousals may activate a dormant immune system, which can then combat pathogens that may have been introduced immediately before or during hibernation.     

Increased oxidative stress and decreased activities of Ca(2+)/Mg(2+)-ATPase and Na(+)/K(+)-ATPase in the red blood cells of the hibernating black bear

During hibernation, animals undergo metabolic changes that result in reduced utilization of glucose and oxygen. Fat is known to be the preferential source of energy for hibernating animals. Malonyldialdehyde (MDA) is an end product of fatty acid oxidation, and is generally used as an index of lipid peroxidation. We report here that peroxidation of lipids is increased in the plasma and in the membranes of red blood cells in black bears during hibernation. The plasma MDA content was about four fold higher during hibernation as compared to that during the active, non-hibernating state (P < 0.0001). Similarly, MDA content of erythrocyte membranes was significantly increased during hibernation (P < 0.025). The activity of Ca(2+)/Mg(2+)-ATPase in the erythrocyte membrane was significantly decreased in the hibernating state as compared to the active state. Na(+)/K(+)-ATPase activity was also decreased, though not significant, during hibernation. These results suggest that during hibernation, the bears are under increased oxidative stress, and have reduced activities of membrane-bound enzymes such as Ca(2+)/Mg(2+)-ATPase and Na(+)/K(+)-ATPase. These changes can be considered part of the adaptive for survival process of metabolic depression.     

Mammalian hibernation as a model of disuse osteoporosis: the effects of physical inactivity on bone metabolism, structure, and strength

Reduced skeletal loading typically leads to bone loss because bone formation and bone resorption become unbalanced. Hibernation is a natural model of musculoskeletal disuse because hibernating animals greatly reduce weight-bearing activity, and therefore, they would be expected to lose bone. Some evidence suggests that small mammals like ground squirrels, bats, and hamsters do lose bone during hibernation, but the mechanism of bone loss is unclear. In contrast, hibernating bears maintain balanced bone remodeling and preserve bone structure and strength. Differences in the skeletal responses of bears and smaller mammals to hibernation may be due to differences in their hibernation patterns; smaller mammals may excrete calcium liberated from bone during periodic arousals throughout hibernation, leading to progressive bone loss over time, whereas bears may have evolved more sophisticated physiological processes to recycle calcium, prevent hypercalcemia, and maintain bone integrity. Investigating the roles of neural and hormonal control of bear bone metabolism could give valuable insight into translating the mechanisms that prevent disuse-induced bone loss in bears into novel therapies for treating osteoporosis.     

Protein synthesis in cardiac cells and the ultrastructural dynamics of cardiomyocytes of hibernating animals during the hibernation cycle

A significant increase in protein synthesis correlating with ultrastructural dynamics of cardiomyocyte organelle convertions has been demonstrated in cardiomyocytes of ground squirrel during arousal from hibernation. In hibernating ground squirrels, the ultrastructure of protein-synthesizing organelles and of the cardiomyocyte nucleus points out to the readiness of cells to active synthesis of proteins. In the perinuclear area of cardiomyocytes abundant ribosomes, elements of endoplasmic reticulum and Golgi complex, mitochondria and high-energy substrates--glycogen and lipid inclusions--are seen. The cardiomyocyte nuclei are large, with highly convoluted borders and abundant pores, their nucleolar structure is granular, the chromatin is mainly diffuse. The potency of cardiomyocyte protein-synthesizing system of hibernating ground squirrels is realized every time at periodical arousals during hibernation. The role of cyclic changes of protein synthesis rate in adaptation of cells of hibernating mammals to functioning at various temperatures is discussed.     

Spin label evidence for the role of lysoglycerophosphatides in cellular membranes of hibernating mammals.

The phospholipid composition of ground squirrel heart muscle changes during hibernation: more lysoglycerophosphatides are found in the hibernating state than in the active state. Phase transitions inferred from spin label motion occur in the usual manner typical of mammalian mitochondria for the mitochondria and mitochondrial lipids from active squirrels. However, a conspicuous absence of a spin label-detectable phase transition is observed in equivalent preparations from hibernating animals. The addition of lysolecithin to preparations from active squirrels removes the break and induces a straight line in the Arrhenius plot. The lack of a spin label-detectable phase transition in hibernating animals, therefore, is attributed to an increased content of lysoglycerophosphatides present in the phospholipids during hibernation.     

脑室注射6—羟多巴胺对黄鼠科眠入眠的影响

The effect of forced depletion of brain norepinephrine (NE) on the onset of hibernation was observed in the ground squirrel (Citellus dauricus) by intraventricular injection of 6-hydroxydopamine (6-OHDA). The results showed: (1) Intraventricular injection of 100-200 micrograms 6-OHDA, which depleted 50-60% NE, markedly facilitated the onset of hibernation, i.e. the average induction period for hibernation in the treated animals was significantly shorter than that of the natural hibernating animals. (2) The average total torpor time in the treated animals was longer than that of natural hibernating animals. (3) All hibernating animals treated with 6-OHDA were able to wake up from deep hibernation spontaneously and undergo normal hibernation bouts. The results indicate the decrease of NE system activity in brain is one important factor in triggering the onset of hibernation.     

Intraspecific differences in the pattern of hibernation in the ground squirrelSpermophilus beldingi

Summary Intraspecific differences in the patterns of heterothermy were found in captive Belding's ground squirrels that hibernated undisturbed at ambient temperatures of 5°C, 10°C, and 15°C. The timing of all entrances into and arousals from hibernation was determined from records of copper-constantan thermocouples that were mounted on the floor of each animal's nest box and connected to continuously recording potentiometers. In the absence of food, large adult males terminated hibernation spontaneously in the spring. In contrast, females and small non-breeding males (yearlings) did not stop hibernating but instead they shortened their bouts of torpor in the spring so that they aroused every three or four days. This interval of frequent arousals, termed the emergence period, lastel until the squirrels became emaciated, and it was only in the 2 or 3 weeks preceeding death (starvation period) that arousal frequency once again decreased towards midwinter values (Fig. 3). These animals terminated hibernation when fed during the emergence or starvation periods, but they were able to resume torpor if that feeding lasted less than a week.Termination of hibernation and reproductive development in males were related to the size of the animals, not their age. Males two years and older which did not deposit normal quantities of fat were like yearlings in that they had open-ended hibernation seasons and showed little testicular enlargement. Likewise, juvenile males that grew to near adult size in their first summer spontaneously terminated hibernation and had well developed testes like most older individuals. In addition, both the frequency and duration of arousals in the emergence period were related, in part, to the extent of the animals' fat reserves, such that large squirrels spent more time at high body temperatures than small individuals (Figs. 5, 6). This trend was most pronounced at high ambient temperatures.These intraspecific differences in hibernation physiology are consistent with the sex and agerelated differences in the timing of emergence above ground in nature. The increase in the time spent euthermic in the spring isinterpreted as an adaptation for increasing the opportunities for environmental assessment. The intraspecific differences in the extent of this euthermia appear to be associated with differences in the balance between the energy supplies available to an animal and its need to accelerate the use of that energy (i.e., arouse) in order to achieve an early and accurately-time emergence from the hibernaculum.     

Proteomic analysis of the winter-protected phenotype of hibernating ground squirrel intestine

The intestine of hibernating ground squirrels is protected against damage by ischemia-reperfusion (I/R) injury. This resistance does not depend on the low body temperature of torpor; rather, it is exhibited during natural interbout arousals that periodically return hibernating animals to euthermia. Here we use fluorescence two-dimensional difference gel electrophoresis (DIGE) to identify protein spot differences in intestines of 13-lined ground squirrels in the sensitive and protected phases of the circannual hibernation cycle, comparing sham-treated control animals with those exposed to I/R. Protein spot differences distinguished the sham-treated summer and hibernating samples, as well as the response to I/R between summer and hibernating intestines. The majority of protein changes among these groups were attributed to a seasonal difference between summer and winter hibernators. Many of the protein spots that differed were unambiguously identified by high-pressure liquid chromatography followed by tandem mass spectrometry of their constituent peptides. Western blot analysis confirmed significant upregulation for three of the proteins, albumin, apolipoprotein A-I, and ubiquitin hydrolase L1, that were identified in the DIGE analysis as increased in sham-treated hibernating squirrels compared with sham-treated summer squirrels. This study identifies several candidate proteins that may contribute to hibernation-induced protection of the gut during natural torpor-arousal cycles and experimental I/R injury. It also reveals the importance of enterocyte maturation in defining the hibernating gut proteome and the role of changing cell populations for the differences between sham and I/R-treated summer animals.     

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.     

Inhibition of succinate oxidation and K+ transport in mitochondria during hibernation

Respiration of liver mitochondria of ground squirrels changes with physiological state. The inhibition of respiration at the level of dehydrogenases occurs during hibernation which is spontaneously removed during arousal. The main mechanism causing a decrease in respiration during hibernation seems to be the inhibition of succinate oxidation, induced by oxaloacetic acid. This is evidenced by the removal of the inhibition by glutamic and isocitric acids. A close correlation between the changes of K+ transport in mitochondria and of the physiological state of hibernator is observed. During hibernation the K+ transport rate decreases 3 times and during arousal it increases 1.5-fold in comparison with the active animals. The K+ content in mitochondria of hibernating and active ground squirrels is the same, whereas during arousal it increases 2-fold.     

Circannual fluctuations of the serum cortisol in the European ground squirrel, Citellus citellus L

1. Serum cortisol levels were measured throughout an annual cycle in male European ground squirrels, Citellus citellus L. 2. A circannual rhythm of these levels in euthermic animals was found: the highest being in late October, November and December, the lowest in May, early October and March. 3. The levels varied in the hibernating period as well: strongly reduced during the first part (October-December) and significantly elevated in the second one (January-March). 4. A significant elevation of the cortisol levels was marked 18-20 hr after full arousal from hibernation in October.     

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 characteristics of the structural-functional state of the erythrocytes from homoiothermic and heterothermic animals during hypothermic storage]

Haemoglobin leakage and permeability for 86Rb and K ions during storage at normal and hypothermic conditions have been investigated in the erythrocytes of the ground squirrel Citellus undulatus in hibernating, arousing and awake animals, as well as in rats. During hibernation, stabilization of the barrier properties and a decrease in passive ionic permeability of erythrocyte membrane were observed. Preservation of ionic homeostasis of the erythrocytes in hibernating animals is favoured by activation of Na(+)-pump. By means of radioautography of electrophoregrams of the blood serum proteins, appearance of a rapidly labeling low-molecular protein was noted at the beginning of the baut and its disappearance before arousal. The data obtained are discussed in relation to the role of the blood plasma components in modification of erythrocyte membranes in hibernating animals.