Tsuneo Sekijima

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.

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.

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.

Phylogenetic Background of Hibernation and Hibernation-Specific Proteins in Sciuridae

In mammals, hibernation is expressed in only a limited number of species, and the molecular mechanisms underlying hibernation are not well understood. We have previously demonstrated a decrease in hibernation-specific protein (HP) in the blood of chipmunks during hibernation. Recently, we revealed that HP was regulated by an endogenous circannual rhythm and conveys a signal to the brain essential for hibernation control. In most mammalian hibernators other than some squirrel species, however, the relationship between HP and hibernation remains unknown. Here, we describe the phylogenetic relationships between HP and hibernation in Sciuridae. The expression of HP gene was observed only in hibernating species. Non-hibernating species did not express HP gene even though they possessed the HP-20, -25, and -27 genes. The hibernation-associated HP cycle found in the chipmunk also occurred in other hibernating species. The phylogenetic tree in Sciuridae demonstrated that hibernating species expressing HP like ground squirrel and chipmunk, which belong to Marmotini, diverged recently from non-hibernating species not expressing HP, such as Funambulini and Protoxerini. Thus, HP is closely related to the evolution of hibernation in Sciuridae.

Genetic Variation in AhR Gene Related to Dioxin Sensitivity in the Japanese Field Mouse, Apodemus speciosus

Human beings have developed many tools, technologies, and chemicals for their convenience and comfort. For example, herbicides and/or insecticides that are sprayed on crop lands prevent damage from pests and have resulted in remarkable increases in crop yield. Polychlorinated biphenyls (PCBs), which have insulating properties and are incombustible, were widely used in the past in electronic instruments and by the electric industry. However, some of these chemicals have harmful effects on organisms. In Seveso, Italy, a large amount of dioxins was emitted by explosion of an agrochemical factory. This accidental release of dioxins killed a lot of farm animals and many people living near the factory developed skin inflammation due to exposure to the high concentrations of dioxins. As seen above, many similar chemical spill disasters have occurred and new chemicals are still being produced. Dioxins are one of the most toxic groups of manmade chemicals known. Dioxins are not only highly toxic, but they also insidiously disrupt reproductive function by mimicking the actions of hormones in the body. Their effects on reproduction, such as reducing the number of sperm and affecting the sex ratio in offspring, may impair the fitness of individuals. Decreased reproductive success of individuals in a population may result in the extinction of local populations and eventually species extinction. In this chapter, we describe the effects of the most toxic chemical pollutant, dioxins, on the Japanese field mouse, Apodemus speciosus. We also discuss the diversity of dioxin sensitivity and attempted to identify dioxin sensitivity in mice using a molecular indicator. Our findings suggest that it is important to take into consideration the differences in dioxin response in each mouse for an accurate estimation of the impact of the pollution.

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.

Endogenous Changes in Hibernation-specific Protein in Chipmunk Cerebrospinal Fluid

Hibernation-specific protein (HP) which was found in the blood of chipmunks (Tamias sibiricus asiaticus), a rodent hibernator, has been demonstrated to be downregulated by differential gene expression in the liver in association with an endogenous circannual rhythm. In the present paper, we investigated the existence and the structural properties of HP in cerebrospinal fluid (CSF) from the lateral ventricle. A much less amount of HP was detected in CSF in nonhibernating state. HP in CSF was markedly increased in association with a circannual rhythm of hibernation and structurally differed from HP in plasma. A similar increase in HP in CSF was induced by the administration of thyroxine that has been shown to cause the change in HP contents in plasma as seen during hibernation. These results suggested that HP plays a role in the brain during hibernation through structural changes. Thyroxine may be involved in the regulation of HP during hibernation.