Ultrastructure of cells from the liver-endocrine pancreas system in hibernating animals

In hibernated ground squirrel (Citellus erythrogenys) during various periods of hibernation morphological changes in organs participating in insurance of energoplastic homeostasis (liver, endocrine part of the pancreas) have been followed. At the beginning of hibernation certain signs of functional strain in the liver are observed-shortening and swelling of the endoplasmic reticulum (ER) canaliculi, decreases of glycogen resources and activation of the lysosomal apparatus, as well as certain features demonstrating its decreased activity--shortening cisterns in the Golgi complex, poorly manifested granular ER, tightening of mitochondrial matrix. At a deep hibernation, the signs of strain disappear, ultrastructure of hepatocytes corresponds to a new lower level of functioning in comparison to that in the active state and at the beginning of hibernation. During these periods the structure of endocrine cells in the pancreas demonstrates certain changes in the character of functioning of the cells. Ultrastructural transformations in the liver and endocrine part of the pancreas in the ground squirrel are mutually correlated.     

中华蟾蜍糖原含量的季节变化

于2005年11月至2006年10月,用硫酸-蒽酮比色法和比重法测定了中华蟾蜍各月份的肝糖原和肌糖原含量及肝比重。结果显示:冬眠期间(11月至次年的2月),糖原含量逐月下降;2月份时出现临时回升,然后继续下降;4月份时肝糖原含量最低;5月份起,肝糖原含量逐渐上升;5月份时肌糖原含量为最低;6月份起,肌糖原含量逐渐上升。虽然在7-8月间出现过下降,两种糖原的含量在10月份时达到一年中的最高值。这些结果表明,蟾蜍糖原含量在一年中呈现显著的季节性波动。越冬前所储备糖原的一部分可能用于越冬期间维持高水平的血糖,一部分用于基础代谢。2月份时糖原含量的临时上升,可能是血液中作为防冻保护剂的葡萄糖运回肝脏和肌肉中再合成糖原的结果。7-8月间糖原含量降低可能与蟾蜍夏蛰有关。雌性5月至10月期间的肝糖原总体水平显著低于雄性,可能与依赖可得到葡萄糖的卵母细胞中的糖原合成有关。糖原含量的季节变化与蟾蜍的生活状态(越冬、繁殖等)有关,并与血糖含量有联动关系。     

Liver glycogen,glucose mobilization and freezing survival in chorus frogs,Pseudacris triseriata

We compared liver glycogen stores and glucose mobilization during freezing among winters in chorus frogs, Pseudacris triseriata, where populations varied in freezing survival. We also characterized tissue glycogen levels across the annual cycle. Frogs with low liver glycogen stores mobilized low amounts of glucose during freezing, and these were correlated with population variation in freezing survival. Moreover, liver glycogen stores were significantly and positively related to body mass. These data suggest that chorus frogs store liver glycogen in preparation for hibernation and that body size and glycogen stores must reach threshold levels for successful survival of freezing bouts during the winter.     

Novel findings on ultrastructural protection of skeletal muscle fibers during hibernation of Daurian ground squirrels: Mitochondria,nuclei, cytoskeleton,glycogen

We examined ultrastructure protective phenomena and mechanisms of slow and fast muscles in hibernating Daurian ground squirrels (Spermophilus dauricus). Some degenerative changes such as slightly decreased sarcomere length and vacuolization occurred in hibernation, but periaxonal capsular borders in intrafusal fibers remained distinct and the arrangement of extrafusal fibers and Z-lines unscathed. In soleus samples, the number of glycogenosomes more than tripled during hibernation. The expression of phosphorylated glycogen synthase remained unaltered while that of glycogen phosphorylase decreased during hibernation. The number of extensor digitorum longus glycogenosomes decreased and the expression of phosphorylated glycogen synthase decreased, while glycogen phosphorylase expression remained unaltered. The nuclei number remained unchanged. Kinesin and desmin, preventors of nuclear loss and damage, were maintained or just slightly reduced in hibernation. The single-fiber mitochondrial concentration and sub-sarcolemmal mitochondrial number increased in both muscle types. The expression of vimentin, which anchors mitochondria and maintains Z-line integrity, was increased during and after hibernation. Also, dynamin-related protein 1, mitochondrial fission factor, and adenosine triphosphate synthase were elevated in both muscle types. These findings confirm a remarkable ultrastructure preservation and show an unexpected increase in mitochondrial capacity in hibernating squirrels.     

Experimental ecology and hibernation of Onchidium struma (Gastropoda: Pulmonata: Systellommatophora)

This study investigated both laboratory-reared and pond-cultured subjects to explore the habit and hibernation patterns of the sea slug Onchidium struma (Mollusca, Gastropoda, Systellommatophora, Onchidioidea, Onchidiidae) from Shanghai and Zhejiang in China. Movement and feeding habits and the process of hibernation were observed in culturing tanks from June 2004 to March 2008. Our results showed that the conditions of movement are as follows: a minimum air temperature of 12 °C; a maximum light intensity of 25 lux and a minimum relative air humidity of 72%. Major movement is usually at dusk and during the night, and the average temperatures for fasting and beginning hibernation are 13.8 °C and 11.4 °C respectively. The analysis showed that the temperature is an essential factor affecting movement and feeding of O. struma and that RH and light intensity also play an important role, but are not necessarily required at the same time. In this study, the survival rate of O. struma through hibernation in high-biodiversity culturing tanks is 77.57% ± 2.86%.     

Caste specific differences in fat body glycogen metabolism of the bumblebee,Bombus terrestris

Bumblebee queens of Bombus terrestris store large amounts of glycogen in the fat body during the first days after eclosion. The accumulation of the reserves is complete prior to hibernation. Comparative studies on the glycogen metabolism in queens and workers show that the increased activity of UDP-glucose: glycogen 4-α-d-glucosyltransferase (EC 2.4.1.11) could account for glycogen accumulation in queens. The enzymatic activities are nearly the same in newly emerged bees, significant differences between castes are detected by day three after eclosion. The activity of glycogen phosphorylase (EC 2.4.1.1), in contrast, is not different between the castes. After injection of juvenile hormone into newly emerged queens the activity of UDP-glucose:glycogen 4-α-d-glucosyltransferase remains low and no glycogen is accumulated in the fat body. Since eggs are formed simultaneously, the lowered activity of the enzyme is thought to depend on the changed metabolism of the fat body related to induced oogenesis.     

Sugar accumulation in hibernating adult bees: An example of the unique energy reservoir for hibernation

• 1.1. Sugars, sugar-alcohols, glycogen, triacylglycerol contents in hibernating adult bees are compared in relation to the hibernating habits of the bees. Monosaccharide content is low in Andrena and Osmia which hibernate in natal blood cells, which very high in Lasioglossum spp. which feed before hibernation.
• 2.2. Distribution of sugars within the body of hibernating Lasioglossum reveals that trehalose is the major sugar accumulated in hemolymph, and that the crop contains a large amount of monosaccharides.
• 3.3. Seasonal changes of sugars and glycogen contents in L. duplex strongly suggest that trehalose accumulated is derived, not from the glycogen in the fat body, but from monosaccharides in the crop.

     

Relations Between Hibernation and Ovarian Functions in a Temperate Zone Frog,Rana temporaria

Adult female frogs were exposed to artificial hibernation in a refrigerator, at about 1–3°C, 1–2 months earlier than in nature, isolated or together with males. Ovarian functional state at the beginning of hibernation was assessed from ovarian biopsies taken in mid-September. All frogs ovulated during hibernation which was interrupted in mid-May, but in seven out of 19 frogs ovulation was incomplete. The number of non-ovulated oocytes varied from a minor fraction to about two thirds of the complement of vitellogenic oocytes, indicating that the follicle size reached at the time of induced hibernation was at the critical level for successful maturation. This critical size was about half the normal oocyte volume at the onset of natural hibernation. There was no vitellogenic growth during hibernation, which presumably constitutes the period of follicle maturation. In five of the frogs, oogenesis sensu stricto had occurred during the period of hibernation, some oogenic events being completely finished at autopsy in mid-May and others still progressing.     

Glycemia and free sugar levels of the goby <Emphasis Type="Italic">Perccottus glehni</Emphasis> depending on period before the beginning and after the end of hibernation

The goby Perccottus glehni is one of the most winterhardy species of fresh-water eurythermal fish. Study of grounds of biochemical adaptation of these animals to hibernation under conditions of ice, which are currently absent, are of undoubted interest for understanding of the nature of hypometabolic states. This work deals with a study of changes in the content of glucose and other free sugars in goby blood and muscle tissue under different physiological states: active, prehibernation, and arousal after experimental cooling to negative near-zero temperature. A relatively high glycemia level with fluctuation amplitude from 9.8 ± 2.1 to 24.4 ± 2.4 mmol/ml is revealed. The minimal value of these fluctuations is recorded at the period almost coinciding with the beginning of hibernation, while the maximal value-at the period of artificial termination of the three-month hibernation in ice at ?5°C. The high blood glycemia level correlating with that in the muscle tissue might probably be due to a peculiar, probably, protector role of this sugar in adaptive mechanisms of the studied fish, not much due to winter hypothermia, but rather to winter hibernation on ice. The level of disaccharide maltose in muscle tissue that is maximal in April is predominantly in reciprocal dependence on the fructose content that is maximal in July. Dynamics of changes in the mono- and disaccharide content depending on the stage of preparation for hibernation basically coincided with the previously revealed dynamics for the fresh-water mollusc Lymnaea stagnalis, which indicates homology of mechanisms of the low-temperature adaptation for animals of different phylogenetic levels.     

Free amino acid composition in blood and muscle of the gobi <Emphasis Type="Italic">Precottus glehni</Emphasis> at the period of preparation and completion of hibernation

The freshwater fish gobi Preccottus glehni survives after wintering in ponds frozen in winter till the very bottom. In adaptation of poikilothermal animals to wintering at near-zero temperatures, an essential role is played by free amino acids; accumulation of a large amount of some particular acid at the period of preparation to the state of hibernation can indicate the protective role of this acid in the low-temperature adaptation. In the present work it has been shown that as soon as by the end of August, in the gobi muscle, the taurine concentration increases three times as compared with that in July, the taurine pool after this reaching 50% of the total pool of free amino acids in the muscle tissue. During December and after the 3-month hibernation in ice, it exceeds the April and July levels 8 and 4 times, respectively, and amounts to 50% of the total free amino acid pool for muscle and to 40% for blood. Several days prior to the beginning of winter hibernation under natural conditions, both in blood and in muscle, there is revealed disappearance or a sharp fall of concentration of essential amino acids. An essential peculiarity of the change in the free amino acid composition after hibernation was a significant rise of alanine concentration in muscle—3.5 times as compared with July and 1.4–1.8 times as compared with changes in December. The total free amino acid pool in muscle in December as compared with that in July increased almost 1.5 times (34.76 ± 1.12 μmol/g wet weight), while after hibernation—2 times. Peculiarities of taurine accumulation long before the beginning of winter cold allow suggesting that role of taurine consists in providing a possibility of existence of eurythermal fish at near-zero temperature.     

Physiological regulation of tau phosphorylation during hibernation

The microtubule-associated protein tau is abnormally hyperphosphorylated in the brains of individuals with Alzheimer disease and other tauopathies, and is believed to play a critical role in the pathogenesis of these diseases. While the mechanisms leading to abnormal tau phosphorylation remain elusive, the recent demonstration of reversible tau phosphorylation during hibernation provides an ideal physiological model to study this critical process in vivo . In this study, arctic ground squirrels (AGS) during hibernation were used to study mechanisms related to tau hyperphosphorylation. Our data demonstrate that tau is hyperphosphorylated at all six sites (S199, T205, S214, S262, S396, and S404) examined in hibernating AGS. Interestingly, only three of these sites (S199, S262, and S404) are dephosphorylated in aroused animals, suggesting a reversible phosphorylation at selective sites. Summer-active AGS demonstrated the lowest tau phosphorylation at all these sites. To explore the mechanisms underlying increased tau phosphorylation during hibernation, the expression level and enzyme activity of various potential tau kinases and protein phosphatases were examined. The kinetic analysis of enzyme activity at different temperatures revealed differential changes in enzyme activity with temperature decline. Specifically, increased protein kinase A activity, decreased protein phosphatase 2A activity, as well as substantial contribution from glycogen synthase kinase-3β, likely play a key role in increased tau phosphorylation during hibernation in AGS.     

Emergence activity at hibernacula differs among four bat species affected by white‐nose syndrome

Prior to the introduction of white‐nose syndrome (WNS) to North America, temperate bats were thought to remain within hibernacula throughout most of the winter. However, recent research has shown that bats in the southeastern United States emerge regularly from hibernation and are active on the landscape, regardless of their WNS status. The relationship between winter activity and susceptibility to WNS has yet to be explored but warrants attention, as it may enable managers to implement targeted management for WNS‐affected species. We investigated this relationship by implanting 1346 passive integrated transponder (PIT) tags in four species that vary in their susceptibility to WNS. Based on PIT‐tag detections, three species entered hibernation from late October to early November. Bats were active at hibernacula entrances on days when midpoint temperatures ranged from −1.94 to 22.78°C (mean midpoint temperature = 8.70 ± 0.33°C). Eastern small‐footed bats (Myotis leibii), a species with low susceptibility to WNS, were active throughout winter, with a significant decrease in activity in mid‐hibernation (December 16 to February 15). Tricolored bats (Perimyotis subflavus), a species that is highly susceptible to WNS, exhibited an increase in activity beginning in mid‐hibernation and extending through late hibernation (February 16 to March 31). Indiana bats (M. sodalis), a species determined to have a medium–high susceptibility to WNS, remained on the landscape into early hibernation (November 1 to December 15), after which we did not record any again until the latter portion of mid‐hibernation. Finally, gray bats (M. grisescens), another species with low susceptibility to WNS, maintained low but regular levels of activity throughout winter. Given these results, we determined that emergence activity from hibernacula during winter is highly variable among bat species and our data will assist wildlife managers to make informed decisions regarding the timing of implementation of species‐specific conservation actions.     

Phosphatases in the rice stem borer, Chilo suppressalis Walker (Lepidoptera; Pyralidae): Some properties and changes of the activities during hibernation

To elucidate the regulation of glycerol synthesis from α-glycerophosphate (α-GP) in larvae of the rice stem borer, Chilo suppressalis Walker, phosphatase activities were examined during hibernation. The presence of alkaline and acid phosphatase was demonstrated in hibernating larvae. The activity of the alkaline type decreased during diapause entry while the activity of the acid type increased at the initiation of diapause and then decreased during postdiapause stage. This pattern of acid type activity change coincided with that of glycerol accumulation in hemolymph during hibernation. Thus, the acid type phosphatase may largely be involved in glycerol synthesis.     

Evidence of prolonged torpor in Goodman’s mouse lemurs at Ankafobe forest,central Madagascar

The small-bodied mouse lemurs of Madagascar (Microcebus) are capable of heterothermy (i.e., torpor or hibernation). The expression of these energy-saving strategies has been physiologically demonstrated in three species: M. berthae, the pygmy mouse lemur (daily torpor), M. murinus, the gray mouse lemur (daily torpor and hibernation), and M. griseorufus, the reddish-gray mouse lemur (daily, prolonged torpor and hibernation). Additional evidence, based on radiotracking and seasonal body mass changes, indicated that mouse lemur capabilities for heterothermy extended to M. lehilahytsara, the Goodman’s mouse lemur. In this study, we confirm the use of hibernation in Goodman’s mouse lemurs at a new location, a high-plateau forest fragment in Ankafobe, central Madagascar. Our evidence is based on sleeping site monitoring of radiocollared individuals and the retrieval of three mouse lemurs from inside a tree hole, all of which displayed a lethargic state. Though our data are preliminary and scant, we show that hibernation occurs in high-plateau mouse lemurs, and suggest that a buffered environment (i.e., tree holes instead of nests) may be crucial to avoiding potentially extreme ambient temperatures.     

Analysis of changes in heart rate and temperature of the ground squirrel Citellus undulatus in various physiological states

Heart rate (HR) of ground squirrel C. undulatus was studied in dependence of season, level of activity, physiological state and air temperature (T). In summer HR varies from 110-130 beat/min in sleep up to 420 beat/min at flight from danger. During winter hibernation HP was minimal (3-5 beat/min) at T 1-4 degrees C, the increase in T induced the growth of HP in correspondence to the Arrenius van't Hoff law. The temperature of the body in hibernation exceeded T on 1.5-3 degrees C. The time of getting off the hibernation increased with the decrease in T (6-7 hours at -1 degree C and 2.5-3 hours at +18 degrees C). At phase of increased thermogenesis during arousal heart temperature exceeded rectal one on 10-12 degrees C and heart rate run up to 360-420 beat/min i.e. 2-3 time higher than in active state. The decrease in T stimulated the increase in HP up to 3.8 in winter and 5.3 beat/min/degree C in summer. The highest values of Q10 for HP were revealed at the beginning of hibernation (15-20) and at the beginning of arousal (6-7), in other periods Q10 was similar to the normal biological values (2-2.5). Thus, at the beginning of transitional periods changes of HP were determined mainly by endogenic mechanisms that inhibited myocardium at the beginning of hibernation and activated in arousal. Some mechanisms of coordination between activities of heart and other systems of organism are considered. The features of hit exchange providing the hibernation in wide range of T are discussed.     

Hexose-, inositol-, and nucleoside phosphate esters in germinating seeds of crested wheatgrass

Net synthesis of phosphate esters was measured in crested wheatgrass seeds [Agropyron desertorum (Fisch.) Schult.] to learn what phosphorylating reactions occur in early stages of germination. Phosphate esters were separated on ion exchange columns and identified by cochromatography and chemical analyses. Synthesis of adenosine triphosphate and UDP-hexose was detected 6 hours after the beginning of water absorption. This synthesis occurred during the period of rapid increase in water potential. Synthesis of hexose phosphate and uridine triphosphate was detected 12 hours after the beginning of water absorption. The concentrations of these esters increased during 48 hours. Increases in concentration of inositol di-, tri-, and tetraphosphate suggested that hydrolysis of inositol hexaphosphate began after 12 hours.     

Quantitative ultrastructural changes of hepatocyte constituents in euthermic,hibernating and arousing dormice (Muscardinus avellanarius)

Hibernating animals represent a suitable model for investigating the structural effects of drastic changes in cell activity under physiological conditions. In this study we investigated by means of electron microscopy and morphometrical analysis the fine structural counterpart of functional rest in hepatocytes of the hibernating dormouse, Muscardinus avellanarius, in comparison with arousing and euthermic dormice. Our observations demonstrate that during hibernation several structural constituents of the hepatocyte undergo modifications. In particular, during deep hibernation, the total cell and cytoplasm area significantly reduced, as well as the total and percent glycogen and residual body area, and the Golgi apparatus almost disappeared. Upon arousal, the amount of glycogen was minimal, whereas total cell and cytoplasm area significantly increased towards the euthermic value as well as total and percent residual body area. In comparison with the euthermic condition, the total and percent cell lipid area significantly increased in early hibernation, reduced in deep hibernation and almost disappeared during arousal. Taken together, our findings give quantitative ultrastructural support to the marked reduction found in hepatocyte functional activities during hibernation. Such a reduced activity involves profound rearrangement of the euthermic cell structure, which is rapidly resumed upon arousal.     

Body temperature patterns and use of torpor in an alpine glirid species,woolly dormouse

Woolly dormice, Dryomys laniger Felten and Storch (Senckenbergiana Biol 49(6):429–435, 1968), are a small (20–30 g), omnivorous (mainly insectivorous), nocturnal glirid species endemic to Turkey. Although woolly dormice have been assumed to hibernate during winter, no information exists on body temperature patterns and use of torpor in the species. In the present study, we aimed to determine body temperature patterns and use of torpor in woolly dormice under controlled laboratory conditions. Accordingly, body temperature (Tb) of woolly dormice was recorded using surgically implanted Thermochron iButtons, small and inexpensive temperature-sensitive data loggers. Woolly dormice exhibited robust, unimodal daily Tb rhythmicity during the euthermic stage before the beginning of hibernation. They displayed short torpor before they began hibernation, although the tendency to enter short torpor was different among individuals. Woolly dormice began hibernation within 1–3 days after exposure to cold and darkness, i.e., on October 22–24, and ended hibernation in the first half of April. Hibernation consisted of a sequence of multiday torpor bouts, interrupted by euthermic intervals. Thus, the patterns of hibernation in woolly dormice were similar to those observed in classical hibernating mammals.     

Development of glycogen storage ability under cortisol control in primary cultures of rat fetal hepatocytes

Cortisol has been shown to induce glycogen storage function in primary cultures of fetal hepatocytes. The method we describe provides a homogeneous population of hepatocytes by elimination of hematopoietic cells. Hepatocytes transplanted from 15-day-old fetuses were grown in the absence or presence of cortisol (10^?5M) for periods of up to 4 days. In the presence of cortisol, after a lag period (24 hr), the glycogen content increased sharply, regardless of whether the medium was replaced or not. Incorporation of radioactivity from (U) ¹⁴C-glucose into glycogen paralleled glycogen accumulation, but the specific activity of the stored glycogen was lower than the final specific activity of the glucose in the medium. This result shows that free glucose is a good precursor of glycogen but not the only one. Data from chase and labeling experiments prove that the hormone acts on the synthetic pathway. If cortisol was removed the glycogen content dropped, suggesting that glycogen synthesis depends on the continuous presence of the hormone. The in vitro maturation of hepatocyte can be provoked by the hormone before the normal in vivo maturation stage of the onset of glycogen accumulation. Other studies of the same in vivo phenomenon have demonstrated that accumulation of glycogen in the liver prior to birth is corticosteroid dependent, but only an in vitro study could clearly show that the hormone acts at the cellular level.     

Factors Affecting Date of Implantation,Parturition, and Den Entry Estimated from Activity and Body Temperature in Free-Ranging Brown Bears

Knowledge of factors influencing the timing of reproduction is important for animal conservation and management. Brown bears (Ursus arctos) are able to vary the birth date of their cubs in response to their fat stores, but little information is available about the timing of implantation and parturition in free-ranging brown bears. Body temperature and activity of pregnant brown bears is higher during the gestation period than during the rest of hibernation and drops at parturition. We compared mean daily body temperature and activity levels of pregnant and nonpregnant females during preimplantation, gestation, and lactation. Additionally we tested whether age, litter size, primiparity, environmental conditions, and the start of hibernation influence the timing of parturition. The mean date of implantation was 1 December (SD = 12), the mean date of parturition was 26 January (SD = 12), and the mean duration of the gestation period was 56 days (SD = 2). The body temperature of pregnant females was higher during the gestation and lactation periods than that of nonpregnant bears. The body temperature of pregnant females decreased during the gestation period. Activity recordings were also used to determine the date of parturition. The parturition dates calculated with activity and body temperature data did not differ significantly and were the same in 50% of the females. Older females started hibernation earlier. The start of hibernation was earlier during years with favorable environmental conditions. Dates of parturition were later during years with good environmental conditions which was unexpected. We suggest that free-ranging pregnant brown bears in areas with high levels of human activities at the beginning of the denning period, as in our study area, might prioritize investing energy in early denning than in early parturition during years with favorable environmental conditions, as a strategy to prevent disturbances caused by human.