The mammalian circadian oscillator is composed of interacting positive and negative transcription events. The clock proteins PER1 and PER2 play essential roles in a negative limb of the feedback loop that generates the circadian rhythm in mammals. In addition, the proteins CLOCK and BMAL1 (also known as ARNTL) form a heterodimer that drives the Per genes via the E-box consensus sequences within their promoter regions. In the present study, we demonstrate that Id2 is involved in stabilization of the amplitudes of the circadian oscillations by suppressing transcriptional activation of clock genes Clock and Bmal1. Id2 shows dynamic oscillation in the SCN, with a peak in the late subjective night. Under constant dark conditions (DD), Id2^-/- mice showed no apparent difference in locomotor activity, however, under constant light conditions (LL), Id2^-/- mice exhibit aberrant locomotor activity, with lower circadian oscillation amplitudes, although the free running periods in Id2^-/- mice show no differences from those in either wild type or heterozygous mice. Id2^-/- animals also exhibit upregulation of Per1 in constant light, during both the subjective night and day. In wild type mice, Id2 is upregulated by constant light exposure during the subjective night. We propose that Id2 expression in the SCN contributes to maintenance of dynamic circadian oscillations.

The incidence and prevalence of depression is higher in women than in men, but the cause of this sex discrepancy remains unknown. Brain-derived neurotrophic factor (BDNF) is a key protein for maintaining neuronal integrity. The purpose of this study was to investigate the female preponderance in behavioral responsivity to restraint stress focusing on the stress reactivity of BDNF in the hippocampus. Male and female ICR mice were exposed to a 3-h session of restraint stress. Plasma corticosterone was measured by high-performance liquid chromatography. BDNF mRNA expression in the whole hippocampus was measured by quantitative real-time reverse transcription-polymerase chain reaction. Wheel-running activity was monitored during the dark period. In response to restraint stress, the increase in levels of serum corticosterone was higher in female than in male mice. Restraint stress resulted in decreased voluntary wheel-running behavior that was greater in female than male animals. In addition to these sex differences in stress reactivity, we found a significant sex difference in BDNF levels in the hippocampus of restraint-stressed mice; total BDNF levels significantly decreased in female mice, but not in male mice in response to the stress. Furthermore, BDNF exon I and IV mRNA expression also showed the same tendency. These data indicate that the reduction in levels of voluntary wheel-running activity in response to stress can be significantly influenced by sex. Moreover, our findings suggest a link between the sex differences in this behavioral response to stress and differential stress reactivity in the production of BDNF in the hippocampus.

In sika deer Cervus nippon, rutting vocalizations play an important role in breeding behavior. This study investigated two types of rutting vocalizations, the moan and the howl, of the Formosan sika deer C. n. taiouanus, including the acoustic characteristics of the vocalizations, the diurnal and seasonal variations of vocal activity, and individual acoustic variation and identification. The results showed that the sound levels were approximately 81–88 dB(A) for the moan and 92–96 dB(A) for the howl, at a distance of 7 m from the sources. From October 2006 to January 2007, eight days of continuous observations were conducted to record the type and amount of vocalizations. Both moan and howl began to occur in the middle of October and reached peaks in the middle and end of November. Thereafter, few vocalizations were recorded until mid-January 2007. Moreover, we found that 74.5% of the first portion of moan, 65.3% of the second portion of moan, and 64.2% of howl could be identified on an individual basis by using discriminant analysis with 200 iterations of cross-validation test. These results revealed that the sounds differed among individuals, and also that they could be correctly identified. Our findings add to the scientific knowledge of sika deer behavior and provide the basis for a novel method of monitoring sika deer populations.

Autologous bone marrow-derived mesenchymal stem cell (BMSCs)-based therapies show great potential in regenerative medicine. However, long-term storage and preservation of BMSCs for clinical use is still a great clinical challenge. The present study aimed to analyze the effect of long-term cryopreservation on the regenerative ability of BMSCs. After cryopreservation of BMSCs from beagle dogs for three years, cell viability, and quantitative analysis of alkaline phosphatase (ALP) activity, surface adherence, and mineralized nodule formation were analyzed. BMSCs in cell-scaffold complex were then implanted into nude mice. There was no significant difference in cell viability and ALP activity between osteogenic differentiation and non-osteogenic differentiation of BMSCs, and BMSCs in cell-scaffold complex retained osteogenic differentiation ability in vivo. These results indicate that long-term cryopreserved BMSCs maintain their have capacity to contribute to regeneration.

The hawksbill turtle is an ectotherm, whose sex is determined by temperature during embryonic development. This study aimed to determine whether embryonic hawksbill turtle cells respond differently to temperature than mammalian cells. Embryonic hawksbill turtle cells were established in culture, and thermal effects on these cells were investigated in vitro. Cells were maintained in Dulbecco's Modified Eagle Medium supplemented with non-essential amino acids, vitamin solution, sodium pyruvate, and 10% fetal bovine serum at 33°C and cell proliferation occurred at 25–33°C. When cells were incubated at 37°C (the temperature of mammalian cell culture) for 24 h, cell growth was completely inhibited. This growth inhibition was evidently recovered by changing the incubation temperature back to 33°C. Expression of heat shock protein was found to increase with elevating culture temperature from 25 to 33°C.

In eukaryotic cells that multiply by binary fission, the interaction of actin filaments with myosin II in the contractile ring is widely recognized to generate force for membrane ingression into the cleavage furrow; however, the expression of myosin II is restricted in animals, yeast, fungi, and amoeba (collectively, unikonts). No corresponding motor protein capable of forming mini-filaments that could exert sufficient tension to cleave the cell body is found in bikonts, consisting of planta, algae, and most protozoa; however, cells in some bikont lineages multiply by binary fission, as do animal cells. Of these, the ciliate Tetrahymena is known to form an actin ring beneath the division furrow in cytokinesis. Here, we investigated the role of filamentous actin in the cytokinesis of Tetrahymena pyriformis by treating synchronized dividing cells with an actin-inhibiting drug, Latrunculin-A. Video microscopic observation of live cells undergoing cytokinesis was performed, and contrary to expectation, we found that initiation of furrow ingression and its progress are not suppressed under the inhibitory condition of actin polymerization in Tetrahymena cells. We suggest that an actin filament-independent mechanism of binary fission may have been acquired during the evolution in this organism.

In our previous study, we clarified the toxicity of 2,2′-dipyridyldisulfide [(PS)2], one of photodegradation products of a metal pyrithione that is used as an alternative antifouling paint biocides to organotin compounds in Japan. In early life stage toxicity tests, we exposed the mummichog, (Fundulus heteroclitus) to (PS)2, and the hatched larvae subsequently displayed notochord undulations and skeletal deformities (Mochida et al., 2012). Runx2, a transcription factor of the runt family, is a key regulator in skeletal development in mammals. It is possible that (PS)2 inhibits Runx2 gene expression, inducing the skeletal deformities in mummichog. In the present study, we cloned two Runx2 cDNAs (type I and type II) from mummichog embryos. The deduced amino acid sequences of type I and type II contain an open reading frame encoding 450 and 464 amino acid residues, respectively. The derived amino acid sequence of Fundulus Runx2 type I showed the highest identity (93.8%) with Takifugu Runx2 type I, and Fundulus Runx2 type II showed 94.6% homology with medaka Runx2. The expression level of Runx2 mRNA in the early stage series was measured using a real-time quantitative PCR assay. Expression levels tended to increase in both the blastula-gastrula and the retinal pigmentation stage. To examine the effect of toxic compounds on skeletal formation, mummichog embryos in the late blastula to retinal pigmentation stage were exposed to (PS)2. After exposure to (PS)2 for one week, the expression level of Runx2 mRNA was unchanged. These results suggest that there is no inhibition of Runx2 gene expression due to (PS)2 exposure.

During precopulatory courtship, male Drosophila typically produce wing vibration to generate species-specific songs before mounting females. Three species in the lini clade of the montium species subgroup have been found to produce species-specific sine song only after mounting and during copulation. Here we investigated and analyzed the courtship behavior of 29 species in the montium subgroup from video and song recordings and measured the duration of wing vibration. We describe a great diversity of courtship behavior in the montium subgroup. The courtship patterns can be categorized into four types in the montium subgroup: 1) type P/C, species with both precopulatory and copulatory courtship, such as D. parvula and D. nikananu, 2) type P-/C, species with sporadic precopulatory and mainly copulatory courtship, such as D. auraria and D. triauraria. 3) type C, species with only copulatory courtship, such as D. tani and D. pectinifera, 4) type C-, species with only very brief copulatory courtship, such as D. rufa and D. asahinai. According to a phylogenetic tree based on sequences of mitochondrial COI and COII, and the nuclear Adh, both precopulatory courtship and copulatory courtship were present in the most basal species D. parvula. Each of two branches in the montium subgroup contains four types of courtship behavior. Type C is present in each sub-branch. These results suggest that the courtship behavior initially involved both precopulatory and copulatory courtship, but that subsequently precopulatory courtship has gradually been lost in the montium subgroup. We suggest reasons why precopulatory behavior might come to be lost in the montium subgroup.

Terrestrial environments surrounding aquatic resources are important and intensively used by semi-aquatic species. In the present work, terrestrial dispersal and nesting sites of the freshwater turtle Phrynops hilarii were analyzed in the floodplain of the Paraná River, using field data and variables obtained from remote sensing. A total of 112 turtles and 44 nests were recorded during road sampling for one year (covered a total of 786 km in 30 surveys). Individuals were at a mean distance of 171.45 m from water, with a negative correlation between number of turtles and distance from water bodies. No significant differences in distance of turtles from water were observed among seasons. Phrynops hilarii nested at a mean distance of 136.51 m from water, showing a negative correlation between number of nests and distance from water bodies. Mean elevation of nests relative to maximum level of water body nearest each record was 1.13 m. The correlation between number of nests and elevation of the nearest water body was positive and significant. The landscape surrounding wetlands is important for P. hilarii to complete the life cycle, as nesting is done in this environment. Our results show that the habitat selected for nesting and terrestrial dispersal was proportionally different from that available in the entire study area, with a higher proportion of wetlands, grasslands and forests.

We monitored annual fecal sex hormones and reproductive displays of five individuals of males and females Thai sarus crane (Grus antigone sharpii), a flock of five males and females black-headed Ibis (Threskiornis melanocephalus), and five pair bonded lesser adjutant stork (Leptoptilos javanicus), all maintained in captivity at Bangprha Waterbird Breeding Research Center. Reproductive behaviors were observed during 0600–1800 h, for four days during the second week of each month and feces were collected monthly to determine annual male total testosterone (mTT) and female estradiol (fE₂) levels by radioimmunoassay. Thai sarus crane exhibited a peak mTT in August following a fE₂, with a surge in July. Black-headed ibis demonstrated a peak mTT in January prior to a fE₂ with a surge in March. Lesser adjutant stork showed a maximal mTT coincidently with fE₂ with a surge in October. Thai sarus crane frequently displayed courtship in May-October, corresponding well with higher mTT rather than fE₂ levels. Black-headed ibis showed courtship-copulation displays in January, simultaneously with mTT, but not with fE₂ surge. Lesser adjutant stork often displayed courtship-copulation in October-January, seemingly corresponded with higher mTT and fE₂ levels during October-December and October—November, respectively. Male and female lesser adjutant stork displayed egg-incubation and chick-rearing behaviors in November—January and December—June, respectively. We suggest that mTT and/or fE₂ apparently played an important role in regulation of courtship-copulation displays but did not relate to both egg-incubation and chickrearing behaviors.

To gain a better understanding of the reproductive endocrinology of a primitive order clupeiform fish (Japanese anchovy, Engraulis japonicus), cDNAs encoding three gonadotropin-releasing hormone (GnRH) isoforms were isolated from the brain, and their distribution was analyzed using insitu hybridization (ISH). The three GnRH isoforms include GnRH1 (herring GnRH), GnRH2 (chicken GnRH-ll) and GnRH3 (salmon GnRH), and their full-length cDNAs encode 88, 86, and 89 deduced amino acids (aa), respectively. Alignment analysis of Japanese anchovy GnRH isoforms showed lower identities with other teleost fish. The major population of GnRH1 neurons was localized in the ventral telencephalon (VT) and nucleus preopticus (NPO) of the preoptic area (POA) with minor population in the anterior olfactory bulb (OB). GnRH2 neurons were restricted to the midbrain tegmentum (MT), specific to the nucleus of the medial longitudinal fasciculus (nMLF). GnRH3 neurons were localized in the olfactory nerve (ON), ventral OB, and transitional area between OB and ON. Interestingly, GnRH1 neurons were also localized in the olfactory bulb, in addition to its major population in the preoptic area. These results indicate the differential distribution of three GnRH isoforms expressed in the brain of the Japanese anchovy.

Genetic diversity at a major histocompatibility complex (MHC) class II B gene was examined for two wild and three captive populations of the endangered Tokyo bitterling Tanakia tanago. A specific primer set was first developed to amplify the MHC II B exon 2 locus. Using single strand conformation polymorphism (SSCP) and sequencing analysis, 16 DAB3 alleles were detected with 56 nucleotide substitutions in the 276-bp region. In the putative antigen-binding sites of exon 2, the rate of nonsynonymous substitutions was significantly higher than that of synonymous substitutions (dN/dS = 2.79), indicating positive selection on the retention of polymorphism. The population from the Handa Natural Habitat Conservation Area and that from the Tone River system exhibited low variation (one and three alleles, respectively), whereas the captive population that originated from a mix of three distinct populations had the highest amounts of variation (14 alleles). The levels of heterozygosity at the MHC varied considerably among populations and showed significant correlations with those at putative neutral microsatellite markers, suggesting that genetic drift following a bottleneck has affected MHC variability in some populations. Comparisons between endangered and non-endangered fish species in previous reports and the present results indicate that the number of MHC alleles per population is on average 70% lower in endangered species than non-endangered species. Considering the functional consequence of this locus, attention should be paid to captive and wild endangered fish populations in terms of further loss of MHC alleles.

Cortisol level changes in response to stocking density in the early stages of rainbow trout were measured. Eggs were exposed to low, normal, and high (2.55, 5.10 and 7.65 eggs cm^-2) densities during the incubation period. Cortisol of maternal origin was found in pre-fertilized eggs (5.09 ± 0.12 ng g^-1) of rainbow trout. In newly fertilized eggs, resting Cortisol levels (3.68 ± 0.14 ng g^-1) decreased to 0.58 ± 0.08, 0.60 ± 0.12, and 0.57 ± 0.16 ng g^-1 at low, normal and high densities by day 10 (organo-genesis), respectively. Resting Cortisol levels remained constant until the eyed stage (day 18). Then, Cortisol showed an increase at hatching to 1.16 ± 0.11, 1.20 ± 0.12, and 1.21 ± 0.14 ng g^-1 at low, normal, and high densities, respectively. The pattern of change in Cortisol level was similar in all three densities. Interrenal cells were observed in 1-day old alevins at all three densities. Hematopoietic tissue, renal tubules and nucleated red blood cells were clarified through the head part of kidney. Higher numbers and larger interrenal cells were observed at high-density groups. Chronic density stress test conducted on embryonic stages of rainbow trout revealed no differences in Cortisol levels, but had an effect on the abundance and size of the interrenal cells. Densities were equaled after hatching (200 alevins per replicate) to investigate the different densities of eggs on stress indices in rainbow trout alevins. An acute stress (air exposure of eggs for five minutes) was applied in three treatments two weeks after hatching, and samples were taken at 0, 1, 3, 6, and 24 hps (hours post stress). Cortisol content increased under low density in 1 hps and reached from 5.21 ± 0.13 ng g^-1 to 6.01 ± 0.18 ng g^-1 (P < 0.05). Cortisol levels increased under normal density in 1 hps from 6.03 ± 0.28 ng g^-1 to 10.84 ± 0.18 ng g^-1 (P < 0.05). In high density also Cortisol increased from 6.83 ± 0.23 ng g^-1 to 8.86 ± 0.26 ng g^-1 (P < 0.05). At 3 hps;, Cortisol level was returned to basal level under low (P > 0.05) and normal (P > 0.05) densities, but significantly decreased (P < 0.05) under high density. Results revealed that the Cortisol biosynthesis was observed in rainbow trout between eyeing to the hatching stage. An increase in the density of eggs until 7.65 egg cm^-2 impaired Cortisol secretion and feedback system in alevins. However, more studies are needed to identify the exact time for Cortisol synthesis ability from eyeing to hatching in this species.

The aim of this research was to evaluate two different diluents for sperm cryopreservation and to study functional parameters in relation to the response to heparin, lysophosphatidylcholine and progesterone, in frozen-thawed semen of fallow deer (Dama dama) during the reproductive season (brama). In this way, fallow deer can be used as a biological model of endangered cervids. Semen was obtained by electroejaculation. Heparin, progesterone and lysophosphatidylcholine were used as capacitation and acrosome reaction inducers, respectively. Capacitation and acrosome reaction were evaluated by chlorotetracycline epifluorescence technique (CTC), membrane integrity by Hypo-osmotic swelling test (HOS) and viability and acrosome integrity by trypan blue stain/DIC. Data was analyzed by ANOVA and Tukey Test (P < 0.05). Semen was cryopreserved in different diluents and Fructose-Tris-Glycine extender was selected. Capacitation with heparin at different incubation times determined that the highest capacitation percentage was obtained at 45 minutes incubation. Progesterone (1 ′M) and lysophosphatidylcholine in heparin capacitated sperm induced acrosome reaction (P < 0.05). This study contributes to improve cryopreservation methods and to increase the knowledge about capacitation and acrosome reaction in vitro in deer spermatozoa, allowing an advance in the development of reproductive biotechnologies.