Mating in the cold. Prolonged sperm storage provides opportunities for forced copulation by male bats during winter.

In a wide range of heterothermic mammals, hibernation interrupts the reproductive cycle by forcing reproductive delays. In hibernating bats with delayed fertilization, an opportunity for sperm competition is enhanced by extending a time-window between copulations and fertilization. In order to achieve greater fertilization success, males are expected to show adaptations for sperm competition by increasing their opportunities for mating over an extended period. We aimed to clarify the physiological and behavioral characteristics of male bats experiencing increased risks of sperm competition. We investigated the characteristics of the reproductive cycle of the little horseshoe bat (Rhinolophus cornutus), and examined whether males retain reproductive physiology related to sexual behavior, and attempt to copulate with females even during the hibernation period. Field observations and histological examinations of the reproductive cycle confirmed that females, having mated in the autumn, store spermatozoa in the uterus during hibernation and give birth in the early summer to just one offspring per year, thus males face a low certainty of successful fertilization. Although their testes regressed rapidly and their testosterone levels were lower during winter than in autumn, males stored motile spermatozoa in their cauda epididymides from autumn throughout the winter. During hibernation, we found that males occasionally aroused from torpor and attempted to mate forcibly with torpid females. Forced copulations appear to increase a male's chances of obtaining a mate while avoiding pre-copulatory female choice. Epididymal sperm storage could be advantageous for males in allowing them to extend their potential mating period even though their testes have regressed. We also found that some hibernating nulliparous females were ready for fertilization in spring after hibernation, whereas few parous females appeared in the same roost. In contrast to males, forced copulations would be maladaptive for females because they cannot opt for higher-quality males while in torpor. Females that have experienced sexual coercion when young may subsequently avoid hibernacula where adult males are present.

Differential AMPK-mediated metabolic regulation observed in hibernation-style polymorphisms in Siberian chipmunks.

Hibernation is a unique physiological phenomenon allowing extreme hypothermia in endothermic mammals. Hypometabolism and hypothermia tolerance in hibernating animals have been investigated with particular interest; recently, studies of cultured cells and manipulation of the nervous system have made it possible to reproduce physiological states related to hypothermia induction. However, much remains unknown about the periodic regulation of hibernation. In particular, the physiological mechanisms facilitating the switch from an active state to a hibernation period, including behavioral changes and the acquisition of hypothermia tolerance remain to be elucidated. AMPK is a protein known to play a central role not only in feeding behavior but also in metabolic regulation in response to starvation. Our previous research has revealed that chipmunks activate AMPK in the brain during hibernation. However, whether AMPK is activated during winter in non-hibernating animals is unknown. Previous comparative studies between hibernating and non-hibernating animals have often been conducted between different species, consequently it has been impossible to account for the effects of phylogenetic differences. Our long-term monitoring of siberian chipmunks, has revealed intraspecific variation between those individuals that hibernate annually and those that never become hypothermic. Apparent differences were found between hibernating and non-hibernating types with seasonal changes in lifespan and blood HP levels. By comparing seasonal changes in AMPK activity between these polymorphisms, we clarified the relationship between hibernation and AMPK regulation. In hibernating types, phosphorylation of p-AMPK and p-ACC was enhanced throughout the brain during hibernation, indicating that AMPK-mediated metabolic regulation is activated. In non-hibernating types, AMPK and ACC were not seasonally activated. In addition, AMPK activation in the hypothalamus had already begun during high Tb before hibernation. Changes in AMPK activity in the brain during hibernation may be driven by circannual rhythms, suggesting a hibernation-regulatory mechanism involving AMPK activation independent of Tb. The differences in brain AMPK regulation between hibernators and non-hibernators revealed in this study were based on a single species thus did not involve phylogenetic differences, thereby supporting the importance of brain temperature-independent AMPK activation in regulating seasonal metabolism in hibernating animals.

AMPK activation, eEF2 inactivation, and reduced protein synthesis in the cerebral cortex of hibernating chipmunks.

During hibernation, mammalian cells are exposed to severe environmental stressors such as low temperature, lowered O2 supply, and glucose deficiency. The cellular metabolic rate is markedly reduced for adapting to these conditions. AMP-activated protein kinase (AMPK) senses the cellular energy status and regulates metabolism. Therefore, we examined AMPK signaling in several brain regions and peripheral tissues in hibernating chipmunk. Eukaryotic elongation factor 2 (eEF2) is a downstream target of AMPK. Phosphorylation of eEF2, indicating its inactivation, is enhanced in the cerebral cortex of hibernating chipmunks. The study indicated that the sequential regulation of AMPK-mammalian target of rapamycin complex 1-eEF2 signaling was altered and protein synthesis ability was reduced in the cerebral cortex of hibernating chipmunks.

Dioxin pollution disrupts reproduction in male Japanese field mice.

Dioxins cause various adverse effects in animals including teratogenesis, induction of drug metabolizing enzymes, tumor promotion, and endocrine disruption. Above all, endocrine disruption is known to disturb reproduction in adult animals and may, also seriously impact their offspring. However, most previous studies have quantified the species-specific accumulation of dioxins, whereas few studies have addressed the physiological impacts of dioxins on wildlife, such as reduced reproductive function. Here we clarify an effect of endocrine disruption caused by dioxins on the Japanese field mouse, Apodemus speciosus. Japanese field mice collected from various sites polluted with dioxins accumulated high concentrations of dioxins in their livers. Some dioxin congeners, especially, 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin, 3,3',4,4',5-pentachloro biphenyl, 1,2,3,4,6,7,8-heptachlorodibenzofuran, and octachlorodibenzo-p-dioxin, which showed high biota-soil accumulation factors, contributed to concentration of dioxins in mouse livers with an increase of accumulation of total dioxins. As for physiological effects on the Japanese field mouse, high levels of cytochrome P450 1A1 (CYP1A1) mRNA, a drug metabolizing enzyme induced by dioxins, were found in the livers of mice captured at polluted sites. Furthermore, at such sites polluted with dioxins, increased CYP1A1 expression coincided with reduced numbers of active spermatozoa in mice. Thus, disruption in gametogenesis observed in these mice suggests that dioxins not only negatively impact reproduction among Japanese field mice, but might also act as a kind of selection pressure in a chemically polluted environment.

Effects of exposure to clothianidin on the reproductive system of male quails.

Clothianidin (CTD) is a neonicotinoid developed in the 1990s as an insecticide having selective toxicity, but it was later found to cause reproductive abnormalities in rats through oxidative stress. There is an attempt to preserve endangered animals, including the Japanese crested ibis, in Japan. However, there is a concern that neonicotinoid affects the reproduction of this bird, since it is used in its habitat. CTD toxicity in the birds is poorly understood, so we investigated whether or not the daily oral administration of CTD has any deleterious effects on the reproductive functions of mature male quails as experimental animals. The animals were randomly divided into four groups of 6 or 7 quails each, treated orally with 0, 0.02, 1 or 50 mg CTD/kg body weight (Control, CTD0.02, CTD1 and CTD50). After that the males bred with untreated females to estimate the egg weights, and rates of fertilization and normal development, the testes, liver and spleen were examined histologically. Vacuolization and the number of germ cells having fragmented DNA in seminiferous tubules, and the number and size of vacuoles in hepatocytes increased dose-dependently. There were no significant differences in egg weights and fertilization rates between the groups, but some eggs of the CTD1 and CTD50 groups failed to develop, and embryonic length decreased dose-dependently. Thus, it was found that CTD affected the reproduction of the male quail through the fragmentation of germ cells and the inhibition or delay of embryonic development.

Hypothermia-dependent and -independent effects of forced swim on the phosphorylation states of signaling molecules in mouse hippocampus.

Forced swim (FS) stress induces diverse biochemical responses in the brain of rodents. Here, we examined the effect of hypothermia induced by FS in cold water on the phosphorylation of FS-sensitive signaling molecules in the mouse brain. As we have shown previously, FS in cold water induced a significant increase in the level of tyrosine phosphorylation of SIRPα, a neuronal membrane protein, in mouse hippocampus, while such effect of FS was markedly reduced in mice subjected to FS in warm water. FS in cold water also induced phosphorylation of mitogen-activated protein kinase kinase (MEK) as well as of cAMP response element-binding protein (CREB), or dephosphorylation of α isoform of Ca(2+)/calmodulin-dependent protein kinase II (αCaMKII) in the hippocampus. These effects of FS on the phosphorylation of these molecules were also lost in mice subjected to FS in warm water. Genetic ablation of SIRPα did not change the phosphorylation states of these molecules in the brain. Forced cooling of anesthetized mice, which induced a marked increase in the phosphorylation of SIRPα, induced dephosphorylation of αCaMKII in the brain, while the same treatment did not affect the phosphorylation level of MEK and CREB. Hibernation also induced an increase and a decrease of the phosphorylation of SIRPα and αCaMKII, respectively, in the brain of chipmunk. These results suggest that hypothermia is a major element that determines the levels of phosphorylation of αCaMKII and SIRPα during the FS in cold water, while it is not for the phosphorylation levels of MEK and CREB.

Polymorphisms and functional differences in aryl hydrocarbon receptors (AhR) in Japanese field mice, Apodemus speciosus.

Dioxins, which are unintentionally generated toxic pollutants, exert a variety of adverse effects on organisms. The majority of these effects, which include teratogenesis, immunosuppression, tumor promotion, and endocrine disruption, are mediated through aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor. Genetic variations in AhR result in different survivability under exposure to dioxin contamination, which might affect the genetic structure of wildlife populations through differential susceptibility to dioxin exposure. The aim of this study was to clarify the polymorphisms of AhR in Japanese field mice, Apodemus speciosus, and their functional differences in order to develop a molecular indicator for dioxin sensitivity. Wild Japanese field mice had abundant polymorphisms in AhR coding region. Seventy-one single nucleotide polymorphisms, 27 of which occur amino acid substitutions, and consequently 49 alleles were identified in 63 individuals. In the functional analysis of AhR variants using transient reporter assays, a Gln to Arg mutation at amino acid 799 exhibited a significant decrease in the level of transactivational properties (p=0.015) which might modify the dioxin susceptibility of an individual.

Circannual control of hibernation by HP complex in the brain.

Seasonal hibernation in mammals is under a unique adaptation system that protects organisms from various harmful events, such as lowering of body temperature (Tb), during hibernation. However, the precise factors controlling hibernation remain unknown. We have previously demonstrated a decrease in hibernation-specific protein (HP) complex in the blood of chipmunks during hibernation. Here, HP is identified as a candidate hormone for hibernation. In chipmunks kept in constant cold and darkness, HP is regulated by an individual free-running circannual rhythm that correlates with hibernation. The level of HP complex in the brain increases coincident with the onset of hibernation. Such HP regulation proceeds independently of Tb changes in constant warmth, and Tb decreases only when brain HP is increased in the cold. Blocking brain HP activity using an antibody decreases the duration of hibernation. We suggest that HP, a target of endogenously generated circannual rhythm, carries hormonal signals essential for hibernation to the brain.