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 optimal depot fat composition for hibernation by golden-mantled ground squirrels (Spermophilus lateralis)

Golden-mantled ground squirrels (Spermophilus lateralis) are herbivores that hibernate during winter. Although little is known about the nutritional/physiological constraints on hibernation, numerous studies have demonstrated that increasing the amount of linoleic acid (a polyunsaturated fatty acid) in the diet enhances hibernation. This is probably because high linoleic acid diets reduce the melting points of the depot fats produced for hibernation which makes them more metabolizable at low body temperatures. This suggests that a major limitation on hibernation may be obtaining enough linoleic acid in the diet for proper hibernation. In all previous studies, however, the amount of linoleic acid in the diets of free-ranging animals was either not considered, or the range of dietary linoleic acid contents in the experiments was less than that of natural diets. It is thus not known whether the amount of linoleic acid available to hibernators under natural conditions actually limits their torpor patterns. A series of laboratory feeding and hibernation experiments were conducted with S. lateralis and artificial diets with different linoleic acid contents that were either below or above the linoleic acid content of the natural diet. The results demonstrated that when dietary linoleic acid contents are either below or above natural levels, hibernation ability is greatly reduced. Hibernation ability was reduced when the squirrels were maintained on a high linoleic acid diet probably by the production of toxic lipid peroxides in brown adipose tissues. The results indicate that there is an optimal level of dietary linoleic acid for proper hibernation, and this is equal to that of the natural diet. The amount of linoleic acid available in the diet thus does not limit hibernation under normal natural conditions.Abbreviations BAT brown adipose tissue - bm body mass - FA fatty acid - PUFA polyunsaturated fatty acid - T _a ambient temperature - T _b body temperature - WAT white adipose tissue     

Prematurely induced arousal from hibernation alters key aspects of warming in golden-mantled ground squirrels, Callospermophilus lateralis

Hibernation researchers have long been interested in the transitions between the dissimilar states of torpor and euthermy. Natural arousal from torpor occurs spontaneously with highly predictable timing. However, animals can also be induced to arouse prematurely in response to various disturbances. While many investigations have used natural and induced arousals synonymously, direct comparisons of these two types of arousal have been limited. We address the question of whether natural and prematurely induced arousals generate the same patterns of warming at the level of the whole organism. We compare the effects of ambient temperature on the dynamics of natural versus induced arousals. Arousal duration, maximum rewarming rate, and the variance associated with increases in body temperature differed between natural and induced arousals. Prematurely inducing arousal also alters the duration of the interbout aroused (IBA) period. We recommend that careful consideration be given to experimental design and data interpretation related to the arousal phase of a torpor bout.     

Temperature-independence of circannual variations in circadian rhythms of golden-mantled ground squirrels

In golden-mantled ground squirrels, phase angles of entrainment of circadian locomotor activity to a fixed light-dark cycle differ markedly between subjective summer and winter. A change in ambient temperature affects entrainment only during subjective winter when it also produces pronounced effects on body temperature (Tb). It was previously proposed that variations in Tb are causally related to the circannual rhythm in circadian entrainment. To test this hypothesis, wheel-running activity and Tb were monitored for 12 to 14 months in castrated male ground squirrels housed in a 14:10 LD photocycle at 21 degrees C. Animals were treated with testosterone implants that eliminated hibernation and prevented the marked winter decline in Tb; these squirrels manifested circannual changes in circadian entrainment indistinguishable from those of untreated animals. Both groups exhibited pronounced changes in phase angle and alpha of circadian wheel-running and Tb rhythms. Seasonal variation in Tb is not necessary for circannual changes in circadian organization of golden-mantled ground squirrels.     

Cloning and sequencing of myosin heavy chain isoform cDNAs in golden-mantled ground squirrels: effects of hibernation on mRNA expression.

The golden-mantled ground squirrel is a small rodent hibernator that demonstrates unusual myosin heavy chain (MHC) isoform plasticity during several months of torpor, punctuated by bouts of rewarming and shivering thermogenesis. We measured MHC mRNA levels to determine whether pretranslational control mechanisms were responsible for differences in MHC2x protein expression, as we previously observed between active and hibernating ground squirrels. We first cloned cDNA using the 3' rapid amplification of cDNA ends (3' RACE) technique and identified three sequences corresponding to MHC1, MHC2x, and MHC2b. A DNA control fragment was developed to be used in conjunction with a coupled RT-PCR reaction to simultaneously measure MHC mRNA levels for each isoform in the skeletal muscle of ground squirrels. MHC mRNA and protein expression were strongly correlated, and type IIx and IIb mRNA levels were significantly different between active and hibernating ground squirrels. Pretranslational control of MHC protein is apparently an important process during hibernation, although the exact stimulus is not known. The techniques presented can be used to obtain MHC cDNA sequences and to measure mRNA expression in many vertebrate groups.     

Circannual cycles in golden-mantled ground squirrels: fall and spring cold pulses

Summary Endogenous circannual rhythms of male golden-mantled ground squirrels (Spermophilus lateralis) exposed to low ambient temperature (6 °C) at various times were compared to the rhythms of a control group kept in a warm room (21 °C) throughout. A 20-week pulse of cold in the spring delayed the subsequent peak body weight and molt offset. However, in the second year after the spring cold pulse, a delay in the rhythm was evident only using peak weight as a phase marker. A 20-week cold pulse in the fall resulted in an earlier peak body weight and earlier onset of pigmented scrotum and descended testes, but there was no evidence from subsequent years that rhythms had been phase advanced. A third of the animals kept continuously at 6 °C remained with pigmented scrotum, descended testes and low body weight for more than a year. Circannual periodicities of animals that continued to display rhythms in the cold room were not significantly longer than those of controls in the warm room. The results suggest that low temperature in the fall can alter the expression of circannual rhythms without much affecting their phasing, while low temperature in the spring produces phase delays which last longer but have not been proved to be persistent.Abbreviation PRC phase response curve     

Regulation of Akt during hibernation in Richardson's ground squirrels

Akt (or protein kinase B) plays a central role in coordinating growth, survival and anti-apoptotic responses in cells and we hypothesized that changes in Akt activity and properties would aid the reprioritization of metabolic functions that occurs during mammalian hibernation. Akt was analyzed in skeletal muscle and liver of Richardson's ground squirrels, Spermophilus richardsonii, comparing the enzyme from euthermic and hibernating states. Akt activity, measured with a synthetic peptide substrate, decreased by 60–65% in both organs during hibernation. Western blotting showed that total Akt protein did not change in hibernation but active, phosphorylated Akt (Ser 473) was reduced by 40% in muscle compared with euthermic controls and was almost undetectable in liver. Kinetic analysis of muscle Akt showed that S_0.5 values for Akt peptide were 28% lower during hibernation, compared with the euthermic enzyme, whereas S_0.5 ATP increased by 330%. Assay at 10 °C also elevated S_0.5 ATP of euthermic Akt by 350%. Changes in ATP affinity would limit Akt function in the hibernator since the muscle adenylate pool size is also strongly suppressed during cold torpor. Other parameters of euthermic and hibernator Akt were the same including activation energy calculated from Arrhenius plots and sensitivity to urea denaturation. DEAE Sephadex chromatography of muscle extracts revealed three peaks of Akt activity in euthermia but only two during hibernation suggesting isozymes are differentially dephosphorylated during torpor. Altered enzyme properties and suppression of Akt activity would contribute to the coordinated suppression of energy-expensive anabolic and growth processes that is needed to maintain viability during over weeks of winter torpor.     

Auditory brainstem responses in ground squirrels arousing from hibernation

Summary Auditory brainstem responses (ABRs) were recorded in ground squirrels (Citellus lateralis) arousing from hibernation. Squirrels implanted with recording screws to record ABRs, and a thermistor to record brain temperature, were placed in a cold room at 9 °C on a 2L:22D light-dark cycle. Hibernating animals were moved from the cold room and ABRs recorded during arousal. The responses showed a gradual development of all brainstem peaks.At low temperatures there were very long latencies to the peaks. The amplitudes of the peaks increased (with fluctuations) as brain temperature increased. The data indicate that neural generators on the brainstem auditory pathway were all activated early in arousal.These results do not support the hypothesis that successive peaks appear and grow in amplitude only after previous peaks are fully developed.     

Regulation of Akt during hibernation in Richardson's ground squirrels

Akt (or protein kinase B) plays a central role in coordinating growth, survival and anti-apoptotic responses in cells and we hypothesized that changes in Akt activity and properties would aid the reprioritization of metabolic functions that occurs during mammalian hibernation. Akt was analyzed in skeletal muscle and liver of Richardson's ground squirrels, Spermophilus richardsonii, comparing the enzyme from euthermic and hibernating states. Akt activity, measured with a synthetic peptide substrate, decreased by 60-65% in both organs during hibernation. Western blotting showed that total Akt protein did not change in hibernation but active, phosphorylated Akt (Ser 473) was reduced by 40% in muscle compared with euthermic controls and was almost undetectable in liver. Kinetic analysis of muscle Akt showed that S(0.5) values for Akt peptide were 28% lower during hibernation, compared with the euthermic enzyme, whereas S(0.5) ATP increased by 330%. Assay at 10 degrees C also elevated S(0.5) ATP of euthermic Akt by 350%. Changes in ATP affinity would limit Akt function in the hibernator since the muscle adenylate pool size is also strongly suppressed during cold torpor. Other parameters of euthermic and hibernator Akt were the same including activation energy calculated from Arrhenius plots and sensitivity to urea denaturation. DEAE Sephadex chromatography of muscle extracts revealed three peaks of Akt activity in euthermia but only two during hibernation suggesting isozymes are differentially dephosphorylated during torpor. Altered enzyme properties and suppression of Akt activity would contribute to the coordinated suppression of energy-expensive anabolic and growth processes that is needed to maintain viability during over weeks of winter torpor.     

Delayed effect of pinealectomy on hibernation of the golden-mantled ground squirrel

Pinealectomy or radical sham pinealectomy were performed on adult golden-mantled ground squirrels,Spermophilus (=Citellus) lateralis, approximately 1 month prior to the date of normal winter emergence. The first hibernatory period and subsequent active season were not different in either of the operated groups from intact animals. However, although the initiation of the second hibernatory period was not affected in the pinealectomized animals, this group failed to show the progressive increase in the length of heterothermic bouts that is characteristic of normal hibernation. Also, terminal arousal occurred approximately 6 weeks earlier in the second year after pinealectomy. Male squirrels showed a corresponding time compression in their annual gonadal cycle, as was assessed by testicular state.These results suggest that the pineal gland of the golden-mantled ground squirrel is involved in the expression of the annual hibernatory cycle. In the absence of the pineal gland the adult of this species is unable to sustain the normal depth and duration of hibernation in the second over-wintering period following pinealectomy.We have carried out additional experiments with young, laboratory-bornS. lateralis and with field-caught, adultS. richardsonii. The results of these studies also are described in this paper.Presented at the Ninth International Congress of Biometeorology, 23 Sept – 1 Oct 1981, Osnabrück and Hohenheim, FRG.     

Ubiquitylation of proteins in livers of hibernating golden-mantled ground squirrels, Spermophilus lateralis

Rodent hibernators experience low core body temperature (as low as −2 °C) and reduced metabolic rates during hibernation. Concordant with energetic constraints, protein synthesis is negligible during torpor. To maintain pools of key regulatory proteins, proteolysis must be depressed as well. Ubiquitin-dependent proteolysis consists of two major steps: (1) ubiquitylation or tagging of a protein substrate by ubiquitin and (2) the protein substrate’s subsequent degradation by the 26S proteasome. Earlier, we demonstrated that the low temperatures typical of torpor virtually arrest proteolytic processing. Here, we demonstrate that in vitro ubiquitylation still continues at greater than 30% of maximal rates at temperatures as low as 0 °C. Continued ubiquitylation in the presence of severely depressed proteolysis may explain the previously observed 2- to 3-fold increase of ubiquitin conjugates during torpor. We determined if there is a qualitative change in the type of ubiquitylation e.g., monoubiquitylation vs polyubiquitylation that occurs during torpor. We found no bias for monoubiquitylation in any state of the torpor cycle. We further determined that substrate limitation of free ubiquitin is not limiting ubiquitylation during torpor. We conclude that while the cold temperatures of torpor may limit proteolysis in accordance with metabolic demands, continued ubiquitylation may result in increased ubiquitin conjugate concentrations that must be processed upon arousal.     

Retention of social recognition after hibernation in Belding's ground squirrels

The retention of social memory during long periods of separation, such as hibernation or migration, has not been well documented, despite evidence for long-term social relationships in migrating species or in long-lived sedentary species. We investigated the ability of captive Belding's ground squirrels, Spermophilus beldingi, to remember previously familiar individuals as well as littermates after 9 months of isolation. Before hibernation, young ground squirrels discriminated between odours of familiar and unfamiliar individuals, as shown by greater investigation of a novel individual's odour. The following spring, these yearlings did not respond differentially to odours of previously familiar and unfamiliar individuals, suggesting that memory for familiar conspecifics was lost during hibernation. In contrast, both female and male yearlings continued to discriminate between odours of littermates and previously familiar nonlittermates. Thus, recognition of close kin was maintained during prolonged social isolation, but recognition of familiar, unrelated individuals was not. If re-establishment of familiarity is not costly or if adults rarely interact with the same individuals in successive years, then selection may not favour retention of individual memories of particular conspecifics over the winter. Even though males rarely encounter kin after dispersal, yearling males did recognize their siblings, suggesting that the relative costs of maintaining kin-recognition abilities year-round may be low. Possible mechanisms underlying the formation and maintenance of individual and kin recognition are discussed. Copyright 2000 The Association for the Study of Animal Behaviour.     

Spontaneous and induced summer hibernation in 13-lined ground squirrels

Among several mammalian hibernators, an endogenous circannual sequence of physiological events is believed to mediate the timing of torpor. Dawe and Spurrier (3) reported that a bloodborne substance (hibernation induction trigger) is important in initiating the torpor phase of those events in the 13-lined ground squirrel. We have reported the induction of summer hibernation among 13-lined ground squirrels using dialysates of serum from hibernating golden hamsters (a nonseasonal hibernator). While those animals receiving saline injections hibernated in 36.3 ± 2.9 days, an earlier induction (22 ± 8.8 days) occurred among those receiving the hibernation serum dialysate (P = 0.05). It was also observed that naive animals departed from a strict circannual rhythm and displayed a high incidence of hibernation, although not significant when compared to the experimental saline controls. The spontaneity of torpor in summer among the naive sample may in part be a characteristic of wild-caught animals employed in the bioassay. Nevertheless, the induction of hibernation among those animals receiving the hibernation serum preparations is supportive of the studies of Dawe and Spurrier (2, 3). That a “trigger” material apparently is present in the hamster, a phylogenetically distinct nonseasonal hibernator, suggests that a characteristic of rodent hibernators is the presence of a material which is associated with the initiation of torpor.     

Quantitative analysis of liver protein expression during hibernation in the golden-mantled ground squirrel

Mammals that enter deep hibernation experience extreme reductions in body temperature and in metabolic, respiratory, and heart rates for several weeks at a time. Survival of these extremes likely entails a highly regulated network of tissue- and time-specific gene expression patterns that remain largely unknown. To date, studies to identify differentially-expressed genes have employed a candidate gene approach or in a few cases broader unbiased screens at the RNA level. Here we use a proteomic approach to compare and identify differentially expressed liver proteins from two seasonal stages in the golden-mantled ground squirrel (summer and entrance into torpor) using two-dimensional gels followed by MS/MS. Eighty-four two-dimensional gel spots were found that quantitatively alter with the hibernation season, 68 of which gave unambiguous identifications based on similarity to sequences in the available mammalian database. Based on what is known of these proteins from prior research, they are involved in a variety of cellular processes including protein turnover, detoxification, purine biosynthesis, gluconeogenesis, lipid metabolism and mobility, ketone body formation, cell structure, and redox balance. A number of the enzymes found to change seasonally are known to be either rate-limiting or first enzymes in a metabolic pathway, indicating key roles in metabolic control. Functional roles are proposed to explain the changes seen in protein levels and their potential influence on the phenotype of hibernation.