Plasma androgen and gonadotropin levels during hibernation and testicular maturation in golden-mantled ground squirrels

The 4-5-mo hibernation season of golden-mantled ground squirrels consists of extended torpor bouts interspersed with brief, periodic intervals of normothermic arousal. Plasma levels of testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) and degree of scrotal pigmentation were measured in torpid and aroused male ground squirrels throughout a season of hibernation and in active animals after the termination of torpor. T was basal in torpid animals; beginning 3 weeks before torpor ended, T was elevated in normothermic males during the first half of periodic arousals but returned to basal levels before animals reentered torpor. After the last (terminal) arousal from torpor, T levels were moderately elevated for 4 wk and maximal for the next 6 wk before they returned to basal values. LH patterns were similar to those of T; however, levels of T and LH were positively correlated only in aroused or posthibernation males. FSH levels remained constant and low during most of the heterothermic season but increased in several torpid males within 3 days of terminal arousal. FSH levels peaked 2 wk after the end of heterothermy. Scrotal pigmentation developed over the first 4 wk after terminal arousal. Maturation of reproductive function occurs during the 4 wk after termination of heterothermy, but elevated levels of T during arousals and variable levels of FSH in the last days of torpor suggest that activation or increased sensitivity of the hypothalamic-pituitary-gonadal axis is important in the termination of heterothermy in ground squirrels.     

The influence of hibernation on testis growth and spermatogenesis in the golden-mantled ground squirrel, Spermophilus lateralis

Testis size and spermatogenesis were monitored serially in individual golden-mantled ground squirrels before, during, and after the hibernation season. During hibernation, animals spent 81% of days in torpor at body temperatures of 3-4 degrees C. Torpor bouts of 6 days duration were interspersed with brief arousals from torpor during which animals were normothermic. In the 5 mo between December (when animals entered hibernation) and April (when torpor was spontaneously terminated), the estimated mass of testes increased gradually from 500 to 1100 mg, but spermatogenesis did not advance beyond pachytene spermatocytes, which were present before hibernation began. In contrast, during the month after torpor was terminated, testes increased rapidly to 3500 mg and after 31 days, spermatozoa were found in the epididymides. We suggest that the limited testis growth that occurred during the hibernation season was restricted to intervals during which squirrels were aroused from torpor. The major portion of gonadal growth and spermatogenesis in the laboratory, and presumably in the field, occurs after ground squirrels have regained the normothermic state. Since males are reproductively mature when first trapped in spring, these findings suggest that males are normothermic for several weeks before they emerge from their hibernacula in the spring.     

Staying cold through dinner: cold-climate bats rewarm with conspecifics but not sunset during hibernation

For temperate endotherms (i.e., mammals and birds) energy costs are highest during winter but food availability is lowest and many mammals depend on hibernation as a result. Hibernation is made up of energy-saving torpor bouts [periods of controlled reduction in body temperature (T _b)], which are interrupted by brief periodic arousals to normothermic T _b. What triggers these arousals in free-ranging hibernators is not well understood. Some temperate bats with intermittent access to flying insects during winter synchronize arousals with sunset, which suggests that, in some species, feeding opportunities influence arousal timing. We tested whether hibernating bats from a cold climate without access to food during winter also maintain a circadian rhythm for arousals or whether cues from conspecifics in the same cluster are more important. We used temperature telemetry to monitor skin temperature (T _sk) of free-ranging little brown bats (Myotis lucifugus) hibernating in central Manitoba, Canada, where temperatures from 22 October to 22 March were too cold for flying insects. We found no evidence bats synchronized arousals with photoperiod but they did arouse synchronously with other bats in the same cluster. Thus, in the northern part of their range where flying insects are almost never available during winter, little brown bats exhibit no circadian pattern to arousals. Warming synchronously with others could reduce the energetic costs of arousal for individuals or could reflect disturbance of torpid bats by cluster-mates.     

Hibernation by tree-roosting bats

In summer, long-eared bats (Nyctophilus spp.) roost under bark and in tree cavities, where they appear to benefit from diurnal heating of roosts. In contrast, hibernation is thought to require a cool stable temperature, suggesting they should prefer thermally insulated tree cavities during winter. To test this prediction, we quantified the winter thermoregulatory physiology and ecology of hibernating tree-roosting bats, Nyctophilus geoffroyi and N. gouldi in the field. Surprisingly, bats in winter continued to roost under exfoliating bark (65%) on the northern, sunny side of trees and in shallow tree cavities (35%). Despite passive re-warming of torpid bats by 10-20 degrees C per day, torpor bouts lasted up to 15 days, although shorter bouts were also common. Arousals occurred more frequently and subsequent activity lasted longer on warmer nights, suggesting occasional winter foraging. We show that, because periodic arousals coincide with maximum roost temperatures, when costs of rewarming and normothermic thermoregulation are minimal, exposure to a daily temperature cycle could largely reduce energy expenditure during hibernation. Our study provides further evidence that models of torpor patterns and energy expenditure from hibernators in cold temperate climates are not directly applicable in milder climates, where prolonged torpor can be interspersed with more frequent arousals and occasional foraging.     

Thermoregulatory changes anticipate hibernation onset by 45 days: data from free-living arctic ground squirrels

Hibernation is a strategy of reducing energy expenditure, body temperature (T(b)) and activity used by endotherms to escape unpredictable or seasonally reduced food availability. Despite extensive research on thermoregulatory adjustments during hibernation, less is known about transitions in thermoregulatory state, particularly under natural conditions. Laboratory studies on hibernating ground squirrels have demonstrated that thermoregulatory adjustments may occur over short intervals when animals undergo several brief, preliminary torpor bouts prior to entering multiday torpor. These short torpor bouts have been suggested to reflect a resetting of hypothalamic regions that control T(b) or to precondition animals before they undergo deep, multiday torpor. Here, we examined continuous records of T(b) in 240 arctic ground squirrels (Urocitellus parryii) prior to hibernation in the wild and in captivity. In free-living squirrels, T(b) began to decline 45 days prior to hibernation, and average T(b) had decreased 4.28 °C at the onset of torpor. Further, we found that 75 % of free-living squirrels and 35 % of captive squirrels entered bouts of multiday torpor with a single T(b) decline and without previously showing short preliminary bouts. This study provides evidence that adjustments in the thermoregulatory component of hibernation begin far earlier than previously demonstrated. The gradual reduction in T(b) is likely a component of the suite of metabolic and behavioral adjustments, controlled by an endogenous, circannual rhythm, that vary seasonally in hibernating ground squirrels.     

Dietary fatty acid composition and the hibernation patterns in free-ranging arctic ground squirrels

Laboratory experiments have demonstrated that the amount of polyunsaturated fatty acids (PUFAs) in the diet before hibernation influences patterns of mammalian torpor. The hibernation ability of ground squirrels is greatest (longest torpor bouts, greatest number of animals entering torpor) when the PUFA content of their fall diets is 33-74 mg/g, under laboratory conditions. The extent to which natural fall diets both (a) vary in PUFA content and (b) influence the torpor patterns of free-ranging populations of hibernating mammals is unknown, however. We conducted a 3-yr study on the diet PUFA contents and subsequent hibernation patterns of free-ranging arctic ground squirrels (Spermophilus parryii) in the Brooks Range of Alaska. We found that the PUFA contents of fall diets varied more than threefold among individuals. Our study also revealed that arctic ground squirrels that consumed a moderate-PUFA (33-74 mg/g) diet had (a) longer torpor bouts, (b) fewer arousals from torpor, (c) shorter arousal periods, (d) more days in torpor, and (e) greater probability of persisting in the population than those that consumed a high-PUFA (>74 mg/g) diet during the fall. No animals were demonstrated to have consumed a diet representing low-PUFA (<33 mg/g) values. Our study is therefore the first to demonstrate that estimated dietary PUFA levels of a free-ranging hibernator influence subsequent torpor patterns.     

A simple molecular mathematical model of mammalian hibernation

A simple model of the dynamics of the body temperature of a hibernating mammal is presented. Our model provides a good match to experimental data, showing the interruption of low-temperature torpor bouts with periodic interbout arousals (IBAs). In this paper we present a mathematical model of the molecules that participate in the initiation, regulation, and maintenance of the hibernating state. This model can be used to describe the role of regulatory molecules, signal transducers, downstream target enzymes, structural proteins, or metabolites. Because many of the biochemical mechanisms are unknown, this is a preliminary and largely phenomenological model that we hope will inspire further investigation.     

Increased heat loss affects hibernation in golden-mantled ground squirrels

During hibernation at ambient temperatures (T(a)) above 0 degrees C, rodents typically maintain body temperature (T(b)) approximately 1 degrees C above T(a), reduce metabolic rate, and suspend or substantially reduce many physiological functions. We tested the extent to which the presence of an insulative pelage affects hibernation. T(b) was recorded telemetrically in golden-mantled ground squirrels (Spermophilus lateralis) housed at a T(a) of 5 degrees C; food intake and body mass were measured at regular intervals throughout the hibernation season and after the terminal arousal. Animals were subjected to complete removal of the dorsal fur or a control procedure after they had been in hibernation for 3-4 wk. Shaved squirrels continued to hibernate with little or no change in minimum T(b), bout duration, duration of periodic normothermic bouts, and food intake during normothermia. Rates of rewarming from torpor were, however, significantly slower in shaved squirrels, and rates of body mass loss were significantly higher, indicating increased depletion of white adipose energy stores. An insulative pelage evidently conserves energy over the course of the hibernation season by decreasing body heat loss and reducing energy expenditure during periodic arousals from torpor and subsequent intervals of normothermia. This prolongs the hibernation season by several weeks, thereby eliminating the debilitating consequences associated with premature emergence from hibernation.     

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.     

Hibernation induces glutathione redox imbalance in ground squirrel intestine

Glutathione (GSH) is the major thiol-disulfide redox buffer in cells and is a critical component of antioxidant defense. Here we examined GSH redox balance in the intestinal mucosa during the annual cycle of 13-lined ground squirrels (Spermophilus tridecemlineatus). The ratio of reduced GSH to its oxidized form (glutathione disulfide, GSSG), which is an index of oxidative stress, was five-fold lower in hibernating compared with summer-active squirrels, an effect due primarily to elevated GSSG concentration in hibernators. During hibernation the total pool of GSH equivalents was lowest in squirrels undergoing arousal and highest in squirrels during interbout arousals. Hibernation decreased intestinal GSSG reductase activity by approximately 50%, but had no effect on activities of glutathione peroxidase or glucose-6-phosphate dehydrogenase. Within the hibernation season, expression of the stress protein HSP70 in intestinal mucosa was highest in squirrels entering torpor and early in a torpor bout, and lowest in squirrels arousing from torpor and during interbout euthermia. The results suggest that hibernation in ground squirrels is associated with a shift in intestinal GSH redox balance to a more oxidized state. Higher levels of HSP70 during the early phases of torpor may reflect induction of the stress response due to aberrations in protein folding or may be a mechanism to increase enterocyte tolerance to subsequent stress imposed by extended torpor or the arousal process.     

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.     

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.     

Prediction of the changes in thermodynamic stability of proteins caused by single amino acid substitutions

The functional (synthetic) activity of blood lymphocytes and bone marrow hematopoietic cells in ground squirrels was studied in different seasons and at different stages of the torpor-arousal cycle. The effect of γ-irradiation on animals in different physiological states was also studied. The synthetic activity of cells was estimated from the amount of active RNA per unit DNA in the cell (parameter α). The α values in lymphocytes were minimal in hibernating animals (January–March), reached a peak upon their complete awakening (April), slightly decreased in the summer activity period, and decreased further in the prehibernation autumn period (November). During winter arousals between torpor bouts, this parameter reached the same values as in summer. The dynamics of parameter α in bone marrow hematopoietic cells were generally similar: minimal values in November and higher between torpor bouts than in summer. The peak of synthetic activity of proliferating hematopoietic cells recorded upon awakening from hibernation in April was mainly due to the accumulation of cells in the G₁ and G₂ phases of the cell cycle, and its decrease in summer reflected prevalent transition from G₂ to mitosis and then partly to G₀. In the torpor-arousal-euthermia cycle, two stages of awakening were distinguished, differing considerably in most of the test parameters. The synthetic activity and the total number of blood and bone marrow cells in ground squirrels irradiated in the state of torpor did not differ significantly from those in nonirradiated torpid animals. The adverse effect of radiation in animals irradiated at the initial stage of awakening was lesser than in animals irradiated in the active state, whereas animals at the second stage of awakening proved more vulnerable to acute irradiation. The physiological state of ground squirrels exposed to ionizing radiation at different phases of the torpor-arousal-euthermia cycle plays a key role in the dynamics of qualitative and quantitative characteristics of blood system cells. The results of this study indicate that the hypometabolic state of ground squirrels during hibernation is a factor of protection from the impact of ionizing radiation on the whole body and on the immune system in particular.     

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.     

Ubiquitin conjugate dynamics in the gut and liver of hibernating ground squirrels

Protein synthesis is severely depressed in hibernating mammals. In the absence of significant protein synthesis, the continued turnover of proteins as a function of normal cellular activity would result in the net depletion of protein pools. We measured levels of ubiquitylated proteins in the gut of thirteen-lined ground squirrels ( Spermophilus tridecemlineatus) and liver of golden-mantled ground squirrels ( Spermophilus lateralis). In both tissues, ubiquitin conjugate concentrations increased during entrance into torpor and were elevated 2-3 fold by late torpor compared with levels in active animals. The data are consistent with a depression of proteolysis with a resultant high level of ubiquitylated proteins during the natural hypothermia of torpor. The periodic returns to euthermy during the hibernation season allow for degradation of these conjugated proteins and may serve to restore protein pools.     

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.     

The influence of cholecystokinin octapeptide and angiotensin II on in vitro duodenal motility in hibernating and aroused 13-lined ground squirrels

Because cholecystokinin octapeptide and angiotensin II are directly involved in intestinal food and water absorption, the effect of these two compounds on intestinal motor responses of hibernating and alert 13-lined ground squirrels was investigated. Both cholecystokinin octapeptide and angiotensin II caused a greater increase in the composite motility of intestinal segments of normothermic in contrast to hypothermic hibernating ground squirrels. Additionally, cholecystokinin caused an increase in the contraction frequency of the intestine from normothermic as compared to hypothermic squirrels. This differential response may provide an adaptive advantage by decreasing food and water consumption; depressing motility and inhibiting digestive enzyme release; reducing the release of bile which also may have an irritant effect on the intestinal mucosa under prolonged exposure. As a result, the GI tract is devoid of food and digestive enzyme irritants during torporous periods.