Long-term behavioral effects of social separation during early life in a social mammal, Octodon degus.

Social separation is thought to induce a strong stress response in social juvenile mammals, but little is known about how this response might vary throughout the development. The present study examines the long-term effects of early-life stress (ELS) induced by social separation on individual behaviors later in life using the social and precocious species Octodon degus. Four experimental groups were established a positive control group of mothers and siblings from six litters comprised the socially housed (SH) group, while pups from seven litters were randomly assigned to three treatments: pups experiencing no separation (NS) treatment while their siblings did; repeated bouts of consecutive separation (CS); intermittent separation (IS). We analyzed the effects of separation treatment on the frequency and duration of freezing, rearing and grooming behaviors. ELS was correlated with higher hyperactivity, and hyperactivity increased with more frequent separation. However, the behavioral trend of the NS group changed to hyperactive in long-term observation. The findings suggest that the NS group was indirectly affected by ELS. In addition, suggesting ELS acts to converge an individual's behavioral tendencies in a certain direction.

Individual variation of daily torpor and body mass change during winter in the large Japanese field mouse (Apodemus speciosus).

Daily torpor is a strategy used by some overwintering small endotherms to aid in energy conservation. However, the pattern of torpor varies among individuals within species and populations, even under the same environmental conditions, with significant implications for survival rate and reproductive success. Body mass is one factor that may influence this variation, especially in some small mammals that accumulate fat stores prior to overwintering. However, to our knowledge there has been no previous study examining the detailed relationships between torpor expression and body mass change in small mammals that hoard food as an energy resource during winter. The large Japanese field mouse, Apodemus speciosus, whose winter survival strategy depends on food caches instead of fat stores, displays daily torpor under artificial winter conditions (short-day photoperiod and cold). The present study clarifies the characteristics and patterns of daily torpor and body mass change in this species in the laboratory. Although expression of daily torpor was facilitated progressively as in other species, the observed patterns of torpor expression and body mass change showed considerable individual variation. Moreover, there was no obvious correlation between body mass and daily torpor expression. Therefore, it is suggested that in A. speciosus body mass may not contribute to individual variation of daily torpor during winter. Daily torpor during winter may be adjusted by not only mechanisms common to other small mammals, but also species-specific factors relating to the external or internal reserves of energy in small mammals.

The effects of extracellular ATP and its receptor antagonists on pig oocytes during in vitro maturation.

We measured the ATP concentrations in the porcine follicular fluid derived from three sizes of follicles (small: 6 mm in diameter). Then, the effects of pre-treatment (100 µM each for 30 min before maturation) with antagonists for extracellular ATP receptor P2X or P2Y on the nuclear maturation rate of cumulus-cell-enclosed (COs) or -denuded oocytes (DOs) up to the preovulatory stage in the presence or absence of 20 nM ATP (a similar concentration to that of medium-sized follicle fluid) were investigated. The antagonists used were pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) or reactive blue 2 (RB2), for extracellular ATP receptor P2X and P2Y, respectively. In addition, the embryonic development rates of COs pre-treated with RB2 were also evaluated. It was found that when the follicular sizes increased, the ATP concentrations significantly decreased (P < 0.05). No differences were observed in the nuclear maturation rates among all COs, regardless of pre-treatment with (+) or without (-) PPADS and in the presence (+) or absence (-) of ATP during maturation. In contrast, the nuclear maturation rate of the COs, but not DOs, in the ATP(-) RB2(+) group was significantly lower (P < 0.05) than that of the ATP(-) RB2(-) and ATP(+)RB2(-) groups. The pronuclear formation and blastocyst formation rates by parthenogenetic activation in the ATP(-) RB2(+) and ATP(+) RB2(+) groups were significantly lower (P < 0.05) than those in the ATP(-) RB2(-) group. In conclusion, it is suggested that the nuclear maturation of porcine oocytes may be influenced by the ATP receptor P2Y present in the cumulus cells.

Persistence and accumulation of micronucleated hepatocytes in liver of rats after repeated administration of diethylnitrosamine.

A repeat-dose micronucleus assay in adult rat liver was recently developed [Mutat. Res. 747 (2012) 234-239]. This assay demonstrated a high detectability of hepatocarcinogens at relatively low doses, as indicated by dose-dependent micronucleus induction. Because the adult rat liver is known to have a long life-span, this desirable property of the assay will be an advantage in detecting micronucleated hepatocytes (MNHEPs) that have persisted for long periods in the liver following repeated dosing. However, no data directly supporting the underlying mechanisms have been published to date. In the present study, we verified the mechanisms by means of pulse-labeling of micronucleated hepatocytes with the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU). The rodent hepatocarcinogen diethylnitrosamine (DEN) was repeatedly administered orally to male Crl:CD (SD) rats (6 weeks old) for up to 2 weeks, and EdU was injected intraperitoneally on days 1, 7, or 14. Hepatocytes were isolated by use of a non-perfusion technique at 24h, 1 week, or 2 weeks after EdU injection and analyzed for EdU incorporation and micronucleus formation. The results of our study confirmed that MNHEPs labeled with EdU on the first day of DEN administration persisted until 2 weeks post-administration in the rat livers. However, the frequency of MHNEPs among EdU-labeled hepatocytes decreased over time. In addition, the number of terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL)-positive cells in the liver tissue increased, suggesting selective removal of micronucleated cells. Theoretical calculation of the cumulative MNHEP frequency on each of the days on which DEN was administered, taking into account the rate of loss, came out closer to the actual value observed in the liver micronucleus test. Taken together, these results indicate that although micronucleated cells induced in rat livers by administration of the genotoxic hepatocarcinogen DEN undergo selective removal, they persist for a long time in a certain proportion, and repeated administration results in their accumulation and increased frequency.

Regulation of fowl sperm motility: evidence for the indirect, but not direct, involvement of dynein-ATPase activity on the reversible temperature-dependent immobilization.

Potential mechanisms of the reversible temperature-dependent immobilization of fowl sperm were investigated. At 30 °C, motility of demembranated fowl sperm was inhibited by adding 2 mM ethylene glycol-bis (2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), but restored immediately after the subsequent addition of 2 mM CaCl(2), whereas at 40 °C, such additions did not appreciably affect motility (which remained almost negligible). With intact sperm, 10(-9) to 10(-3) M Ca(2+) had no effect on motility at 30 °C, which remained high. In contrast, intact sperm at 40 °C were almost immotile below 10(-5) M Ca(2+), and then gradually recovered motility at higher Ca(2+) concentrations. The negligible motility of demembranated sperm at 40 °C, and at 30 °C in the presence of EGTA, was stimulated by addition of 100 nM of the protein phosphatase inhibitor calyculin A. Dynein-ATPase activities of sperm at 40 °C in the presence of 2 mM EGTA, 2 µM CaCl(2), 2 mM CaCl(2,) or 100 nM calyculin A were higher than those at 30 °C. Therefore, stimulation of fowl sperm motility by temperature, Ca(2+), and phosphatase inhibition was not simply associated with an increase of flagellar dynein-ATPase activity. Furthermore, Ca(2+) was essential, at the axonemal level, for initiation of the 'intrinsic' motility of fowl sperm at 30 °C, but this Ca(2+)-dependent mechanism might be different from that involved in restoration of motility of intact sperm at 40 °C. In addition, perhaps inhibition of protein phosphatase activity was involved in initiation of sperm motility, but acting at a location different from Ca(2+) on the axoneme.

Development of a repeated-dose liver micronucleus assay using adult rats: an investigation of diethylnitrosamine and 2,4-diaminotoluene.

Various liver micronucleus assay methods, such as those involving partial hepatectomy, treatment with mitogens, and the use of juvenile animals, have been developed. These assays have been proven to be of high sensitivity and specificity to predict hepatocarcinogenicity of compounds that cannot be detected by bone marrow micronucleus assays. On the contrary, the existing assays have only been evaluated for their use in detecting micronucleus induction in the settings of relatively short-term cell proliferation. However, the integration of in vivo genotoxicity endpoints into routine toxicity studies is increasingly desired from the viewpoint of animal welfare to reduce the number of animals used. In the present study, the rodent hepatocarcinogens diethylnitrosamine (DEN) and 2,4-diaminotoluene (2,4-DAT) were repeatedly administered orally to male Crl:CD (SD) rats (6 weeks old at the beginning of administration) for 5, 14, and 28 days, and changes in the frequency of hepatocytes with micronuclei in liver tissues that had undergone no artificial treatment to accelerate cell proliferation were evaluated. At the same time, a new method of hepatocyte isolation involving the treatment of a portion of the liver with collagenase in a centrifuge tube, without the use of in situ perfusion, was established. The induction of micronucleated hepatocytes was achieved after the repeated administration of DEN for 5 days or longer and of 2,4-DAT for 14 days or longer. Micronucleus frequencies were increased depending on the number of administrations, indicating that micronucleated hepatocytes had possibly remained for a long period of time and accumulated additively. It therefore appears that even in adult rat liver with low mitotic activity, a repeated-dose of a chemical substance for 14 days or longer enables the detection of micronucleus induction. In addition, the establishment of a method to isolate hepatocytes without perfusion using only a part of the liver enables the integration of liver micronucleus assays into general toxicity studies.

The effects of cysteine addition during in vitro maturation on the developmental competence, ROS, GSH and apoptosis level of bovine oocytes exposed to heat stress.

In the present study, we investigated the effects of various concentrations of cysteine (0.0, 0.6, 1.2 and 1.8 mM) added to the maturation medium on nuclear maturation and subsequent embryonic development of bovine oocytes exposed to heat stress (HS: set at 39.5 °C for 5 h, 40.0 °C for 5 h, 40.5 °C for 6 h, and 40.0 °C for 4 h versus 38.5 °C for 20 h as the control group). This regime mimicked the circadian rhythm of the vaginal temperature of lactating dairy cows during the summer season in southwestern Japan. Moreover, we also evaluated the oocyte's reactive oxygen species (ROS) and glutathione (GSH) levels and the apoptosis levels of the oocytes and cumulus cells in the presence or absence of 1.2 mM cysteine. As a result, HS in the without-cysteine group significantly suppressed (p < 0.05) both the nuclear maturation rate up to the metaphase (M)II stage and the blastocyst formation rate compared with that of the control group. In addition, this group showed significantly higher (p < 0.05) ROS levels and significantly lower (p < 0.05) GSH levels than those of the control group. Moreover, the level of TdT-mediated dUTP nick end labelling (TUNEL)-positive cumulus cells in the HS without-cysteine group was significantly higher (p < 0.05) than that of the control group. However, the addition of 1.2 mM cysteine to the maturation medium restored not only the nuclear maturation, blastocyst formation rates and GSH contents, but also increased the ROS and TUNEL-positive levels of the cumulus cells, but not oocytes, to that of the control group. These results indicate that the addition of 1.2 mM cysteine during in vitro maturation (IVM) may alleviate the influence of heat stress for oocyte developmental competence by increasing GSH content and inhibiting the production of oocyte ROS followed by apoptosis of cumulus cells.

The role of mitochondrial transition pores on bovine oocyte competence after heat stress, as determined by effects of cyclosporin A.

This study was undertaken to investigate the effects on the nuclear maturation and subsequent embryonic development of bovine oocytes exposed to heat stress (HS) when treating bovine oocytes before in vitro maturation (IVM) with 1 µM cyclosporin A (CsA), an inhibitor of mitochondrial permeability transition pore opening. Mitochondrial activity, reactive oxygen species (ROS), and apoptosis levels of the oocytes were also assessed. Nuclear maturation rates of both the HS-exposed oocytes treated with or without CsA groups (HS + CsA or HS group) were significantly lower (P<0.05) than that of the control group, while the rate of the HS + CsA group was significantly higher (P<0.05) than that of the HS group. Furthermore, although the cleavage and blastocyst formation rates of the HS group were significantly lower than those of the control groups (P<0.05), both rates of the HS + CsA group recovered to the same level as those of the control group. The HS group showed a significantly higher ROS level, lower mitochondrial activity in the oocytes, and TUNEL-positive cumulus cells, but not oocytes, compared with those of the control group (P<0.05), whereas the TUNEL-positive and mitochondrial activity levels of the HS + CsA group recovered to those of the control group. These results indicate that 1 µM CsA treatment before IVM may mitigate reduced mitochondrial activity, increase number of apoptotic cumulus cells under HS, and improve the nuclear maturation and developmental competence of bovine oocytes.

Effect of the temperature-humidity index on body temperature and conception rate of lactating dairy cows in southwestern Japan.

In the present study, we investigated the relationship between the temperature-humidity index (THI) and the conception rate of lactating dairy cows in southwestern Japan, one of the hottest areas of the country. We also investigated the relationship between measurement of the vaginal temperature of lactating dairy cows as their core body temperature at one-hour intervals for 25 consecutive days in hot (August-September, n=6) and cool (January-February, n=5) periods and their THI. Furthermore, we discussed the above relationship using these vaginal temperatures, the conception rates and the THI. As a result, when the conception rates from day 2 to 0 before AI were classified into day 2, 1 and 0 groups by the six maximum THI values in each group (mTHI; <61, 61-65, 66-70, 71-75, 76-80, >80), only the conception rate for the mTHI over 80 at 1 day before AI group was significantly lower (P<0.05) than the other groups. The conception rate for days 15 to 17, but not days 19 to 22 and 30 to 35, after AI in the cows that experienced average mTHI over 80 (amTHI>80) was significantly lower (P<0.05) than that of the cows that did not experience amTHI>80. There was a significant positive correlation (P<0.01) between the mTHI and the mean daily vaginal temperature, but not during the cool period. When the mTHI reached 69, the vaginal temperature started to increase. As for the relationship between the conception rates and vaginal temperatures for all mTHI classes, in the mTHI>80 at 1 day before AI group, the vaginal temperature increased by 0.6 C from 38.7 C, resulting in a reduction of 11.6% in the conception rate from 40.5%. In conclusion, these results suggest that one of the causes of the fall in conception rate of lactating dairy cows during the summer season in southwestern Japan may be an increase in their core body temperature with a higher mTHI than the critical mTHI of 69 at 1 day before AI.

Novel glycosaminoglycan biosynthetic inhibitors affect tumor-associated angiogenesis.

Heparan sulfate proteoglycans (HSPGs) are essential players in several steps of tumor-associated angiogenesis. As co-receptors for several pro-angiogenic factors such as VEGF and FGF, HSPGs regulate receptor-ligand interactions and play a vital role in signal transduction. Previously, we have employed an enzymatic strategy to show the importance of cell surface HSPGs in endothelial tube formation in vitro. We have recently found several fluoro-xylosides that can selectively inhibit proteoglycan synthesis in endothelial cells. The current study demonstrates that these fluoro-xylosides are effective inhibitors of endothelial tube formation in vitro using a matrigel based assay to simulate tumor-associated angiogenesis. These first generation scaffolds offer a promising stepping-stone to the discovery of more potent fluoro-xylosides that can effectively neutralize tumor growth.

4-deoxy-4-fluoro-xyloside derivatives as inhibitors of glycosaminoglycan biosynthesis.

Various 4-deoxy-4-fluoro-xylosides were prepared using click chemistry for evaluating their potential utility as inhibitors of glycosaminoglycan biosynthesis. 2,3-Di-O-benzoyl-4-deoxy-4-fluoro-ß-D-xylopyranosylazide, obtained from L-arabinopyranose by six steps, was treated with a wide variety of azide-reactive triple bond-containing hydrophobic agents in the presence of Cu(2+) salt/ascorbic acid, a step known as click chemistry. After click chemistry, benzoylated derivatives were deprotected under Zemplén conditions to obtain 4-deoxy-4-fluoro-xyloside derivatives. A mixture of α:ß-isomers of twelve derivatives were then separated on a reverse phase C18 column using HPLC and the resulting twenty four 4-deoxy-4-fluoro-xylosides were evaluated for their ability to inhibit glycosaminoglycan biosynthesis in endothelial cells. We identified two xyloside derivatives that selectively inhibit heparan sulfate and chondroitin sulfate/derman sulfate biosynthesis without affecting cell viability. These novel derivatives can potentially be used to define the biological actions of proteoglycans in model organisms and also as therapeutic agents to combat various human diseases in which glycosaminoglycans participate.

Temperature-dependent regulation of sperm motility of Ijima's copper pheasants (Syrmaticus soemmerringii ijimae), one of 'near threatened' species.

In order to conserve the copper pheasants, one of the Japanese 'near threatened' species, the knowledge of the sperm characteristics is the inevitable issue. Therefore, temperature-dependent regulation of copper pheasant sperm motility was investigated in comparison with that of domestic fowl spermatozoa. Motility of intact spermatozoa from both species was markedly affected by temperature. During incubation at 30 degrees C, copper pheasant spermatozoa showed around 60-70% motility, but became almost immotile when the temperature was raised to 40 degrees C. Then, when the temperature of the sperm suspension was subsequently cooled to 30 degrees C, the spermatozoa regained their motility. The motility of domestic fowl spermatozoa showed a similar pattern. Temperature also affected the motility of both demembranated copper pheasant and domestic fowl spermatozoa in the same way. The motility of intact copper pheasant and domestic fowl spermatozoa at 30 degrees C was unaffected following the addition of 2 mM CaCl(2), 100 nM calyculin A, an inhibitor of protein phosphatase-type 1 (PP1), or 4 mM diB-cAMP, respectively, compared with those with no effectors. However, the presence of 10 microM ML-7, a selective inhibitor of myosin light chain kinase (MLCK), inhibited motility of spermatozoa from both species. At 40 degrees C, the presence of CaCl(2) or calyculin A restored the motility of spermatozoa from both species, but the addition of diB-cAMP or ML-7 could not prevent the immobilization of spermatozoa. At 30 degrees C in the presence of ATP, the motility of demembranated copper pheasant spermatozoa was over 60% but was inhibited following the addition of 10 microM ML-7; a similar pattern was found with demembranated domestic fowl sperm motility. The motility of demembranated spermatozoa from both species was inhibited following the addition of 2mM EGTA to the reactivation medium at 30 degrees C, but restored by the subsequent addition of 4 mM CaCl(2). These results suggest that copper pheasant sperm motility might be regulated by similar mechanisms to that of domestic fowl spermatozoa: i.e., the balance of Ca(2+)/MLCK or an MLCK-like protein-dependent phosphorylation and PP1-dependent dephosphorylation. The similarity in physiological regulation of spermatozoa from both species shows that extensive technology developed for artificial breeding of the domestic fowl might be applicable to captive breeding of copper pheasants.

Intracellular signal transduction pathways in the regulation of fowl sperm motility: evidence for the involvement of phosphatidylinositol 3-kinase (PI3-K) cascade.

The possible role of PI3-K in the reversible temperature-dependent immobilization of fowl sperm motility was investigated by using PI3-K inhibitor (LY294002) and its inactive analogue (LY303511). The existence of the PI3-K in fowl spermatozoa was also confirmed by Western blotting analysis. Fowl sperm motility in TES/NaCl buffer remained negligible at the avian body temperature of 40 degrees C but was maintained vigorously when the temperature was decreased to 30 degrees C. At 30 degrees C, no stimulation or inhibition of motility was observed after the addition of 2 mM CaCl2 and 10 microM LY294002 or LY303511: around 70-80% of spermatozoa remained motile. In contrast, at 40 degrees C, the motility of spermatozoa was activated immediately after the addition of Ca(2+), but the subsequent addition of LY294002 inhibited the motility again. The addition of LY303511 did not appreciably affect the Ca(2+)-supplemented sperm motility, which was maintained for at least 15 min. The ATP concentrations of spermatozoa after the addition of LY294002 + Ca(2+) or LY303511 + Ca(2+) were almost the same values compared with those of Ca(2+) alone at 40 degrees C, suggesting that the addition of LY294002 was not simply affecting membrane damage or inhibiting energy production in the spermatozoa, but may be acting on some part of the motility-regulating cascade. Immunoblotting of sperm extract using an antibody to PI3-K revealed a major cross-reacting protein of 85 kDa, which corresponds to the molecular weight of the subunit of PI3-K. These results suggest that PI3-K may be positively involved in the calcium-regulated maintenance of flagellar movement of fowl spermatozoa at 40 degrees C.

The expression of Hedgehog genes (Ihh, Dhh) and Hedgehog target genes (Ptc1, Gli1, Coup-TfII) is affected by estrogenic stimuli in the uterus of immature female rats.

The objective of this study was to investigate the effects of estrogen receptor (ER) agonists and an ER antagonist on the expression of Hedgehog genes (Indian hedgehog: Ihh; Desert hedgehog: Dhh) and Hedgehog target genes (Patched 1: Ptc1; glioma-associated oncogene homolog 1: Gli1; chicken ovalbumin upstream promoter transcription factor II: Coup-TfII) in the rat uterus. Immature female rats were administered once with 17alpha-ethynyl estradiol (EE, an ER agonist), propyl pyrazole triole (PPT, an ERalpha-selective agonist), diarylpropionitrile (DPN, an ERbeta-selective agonist), or ICI 182,780 (an ER antagonist). Expression of mRNA for Ihh, Dhh, and Ptc1 was dose-dependently downregulated by EE in the uterus of immature rats, mediated by ER as confirmed by coadministration of ICI 182,780. The mRNA expression levels of Ptc1, Gli1, and Coup-TfII were simultaneously downregulated during the period in which the mRNA expression levels of Ihh and Dhh were downregulated in the uterus after administration of EE. PPT downregulated the transcription of Ihh, Dhh, Ptc1, Gli1, and Coup-TfII, indicating that expression of these genes was regulated by the ERalpha-dependent pathway. DPN also downregulated the transcription of Ihh and Dhh, although the effect was weaker than that of PPT, indicating that the regulation of uterine Ihh and Dhh transcription was also affected by the ERbeta-dependent pathway. These results suggest that the expression of Hedgehog genes (Ihh, Dhh) and Hedgehog target genes (Ptc1, Gli1, Coup-TfII) is affected by estrogenic stimuli in the uterus of immature female rats.

Differential expression patterns of Wnt and beta-catenin/TCF target genes in the uterus of immature female rats exposed to 17alpha-ethynyl estradiol.

To characterize the effects of an estrogen receptor (ER) agonist on the gene expressions in the uterus, immature female rats were administered once orally with 17alpha-ethynyl estradiol (EE, 3 mug/kg), a potent ER agonist. We focused on four categories of sex steroid hormone receptor genes: well-known estrogen target genes, Wnt genes, and beta-catenin/T-cell factor (TCF) target genes. ERalpha, ERbeta, progesterone receptor, and androgen receptor mRNAs were all downregulated at 24 and/or 48 h after EE administration. Complement C3 and insulin-like growth factor 1 mRNAs were markedly induced after EE administration. Although the time courses of Wnt4, Wnt5a, and Wnt7a mRNA status varied until 12 h after EE administration, all of them were simultaneously downregulated at 24 and 48 h. The remarkable downregulation of Wnt7a mRNA in response to EE was considered to be important to understand the various uterine phenomena affected by ER agonists. In the beta-catenin/TCF target genes, the downregulation of anti-Mullerian hormone type 2 receptor and bone morphogenetic protein 4 mRNA after EE administration appeared to be closely related to the downregulation of Wnt7a. The upregulation of cyclin D1 and follistatin mRNA at the early phase after EE administration was considered to have been affected by the upregulation of Wnt4. These results indicate that an ER agonist influences not only the mRNA expression of sex steroid hormone receptor genes and well-known estrogen target genes but also Wnt genes (Wnt4, Wnt5a, Wnt7a) and beta-catenin/TCF target genes in the uterus of immature rats, indicating that their molecules are the potential players affected by estrogenic stimuli.