Diversity and Evolution of Highly Repetitive DNA Sequences Constituting Chromosome Site-Specific Heterochromatin in Two Gerbillinae Species.

Constitutive heterochromatin, consisting of repetitive sequences, diverges very rapidly; therefore, its nucleotide sequences and chromosomal distributions are often largely different, even between closely related species. The chromosome C-banding patterns of two Gerbillinae species, Meriones unguiculatus and Gerbillus perpallidus, vary greatly, even though they belong to the same subfamily. To understand the evolution of C-positive heterochromatin in these species, we isolated highly repetitive sequences, determined their nucleotide sequences, and characterized them using chromosomal and filter hybridization. We obtained a centromeric repeat (MUN-HaeIII) and a chromosome 13-specific repeat (MUN-EcoRI) from M. unguiculatus. We also isolated a centromeric/pericentromeric repeat (GPE-MBD) and an interspersed-type repeat that was predominantly amplified in the X and Y chromosomes (GPE-EcoRI) from G. perpallidus. GPE-MBD was found to contain a 17-bp motif that is essential for binding to the centromere-associated protein CENP-B. This indicates that it may play a role in the formation of a specified structure and/or function of centromeres. The nucleotide sequences of the three sequence families, except GPE-EcoRI, were conserved only in Gerbillinae. GPE-EcoRI was derived from the long interspersed nuclear elements 1 retrotransposon and showed sequence homology throughout Muridae and Cricetidae species, indicating that the repeat sequence occurred at least in the common ancestor of Muridae and Cricetidae. Due to a lack of assembly data of highly repetitive sequences constituting heterochromatin in whole-genome sequences of vertebrate species published to date, the knowledge obtained in this study provides useful information for a deep understanding of the evolution of repetitive sequences in not only rodents but also in mammals.

Testosterone interrupts binding of Neurexin and Neuroligin that are expressed in a highly socialized rodent, Octodon degus.

Octodon degus is said to be one of the most human-like rodents because of its improved cognitive function. Focusing on its high sociality, we cloned and characterized some sociality-related genes of degus, in order to establish degus as a highly socialized animal model in molecular biology. We cloned degus Neurexin and Neuroligin as sociality-related genes, which are genetically related to autism spectrum disorder in human. According to our results, amino acid sequences of Neurexin and Neuroligin expressed in degus brain, are highly conserved to that of human sequences. Most notably, degus Neuroligin4 is highly similar to human Neuroligin4X, which is one of the most important autism-related genes, whereas mouse Neuroligin4 is known to be poorly similar to human Neuroligin4X. Furthermore, our work also indicated that testosterone directly binds to degus Neurexin and intercepts intercellular Neurexin-Neuroligin binding. Moreover, it is of high interest that testosterone is another key molecule of the higher incidence of autism in male. These results indicated that degus has the potential for animal model of sociality, and furthermore may promote understanding toward the pathogenic mechanism of autism.

Equilibrium vitrification of mouse embryos using low concentrations of cryoprotectants.

Previously, we developed a method for vitrification of mouse embryos in a near-equilibrium state using EFS35c, PB1 medium containing 35% (v/v) ethylene glycol, and 0.98 M sucrose. This method has advantages in both slow freezing and vitrification. However, since the vitrification solution in this method contains high concentrations of cryoprotectants and thus has high osmolality, the solution would injure oocytes and embryos with high sensitivity to chemical toxicity and high osmolality. In this study, we examined whether embryos could be vitrified in a near-equilibrium state using a solution containing low concentrations of cryoprotectants and thus with low osmolality. To investigate whether embryos were vitrified in a near-equilibrium state, 2-cell mouse embryos were vitrified with EDFS10/10a, PB1 medium containing 10% (v/v) ethylene glycol, 10% (v/v) DMSO, and 0.4 M sucrose, in liquid nitrogen, stored at -80 °C for 4-28 days, and warmed in water at 25 °C. The viability of the embryos was evaluated by the appearance of embryos after warming and developmental ability. When embryos were vitrified in liquid nitrogen using EDFS10/10a, the survival and developmental ability into blastocysts after storage at -80 °C for 7 days were high, indicating that embryos were vitrified in a near-equilibrium state. A high proportion of embryos vitrified with EDFS10/10a developed to term after transportation with dry ice, re-cooling in liquid nitrogen, and transfer to recipients. Therefore, new equilibrium vitrification developed in this study may be useful for oocytes and embryos that are highly sensitive to the toxicity of cryoprotectants and high osmolality.

Equilibrium vitrification of mouse embryos at various developmental stages using low concentrations of cryoprotectants.

We previously developed a new vitrification method (equilibrium vitrification) by which two-cell mouse embryos can be vitrified in liquid nitrogen in a highly dehydrated/concentrated state using low concentrations of cryoprotectants. In the present study, we examined whether this method is effective for mouse embryos at multiple developmental stages. Four-cell embryos, eight-cell embryos, morulae, and blastocysts were vitrified with EDFS10/10a, 10% (v/v) ethylene glycol and 10% (v/v) DMSO in FSa solution. The FSa solution was PB1 medium containing 30% (w/v) Ficoll PM-70 plus 0.5 M sucrose. The state of dehydration/concentration was assessed by examining the survival of vitrified embryos after storage at -80°C. When four-cell embryos and eight-cell embryos were vitrified with EDFS10/10a in liquid nitrogen and then stored at -80°C, the survival rate was high, even after 28 days, with relatively high developmental ability. On the other hand, the survival of morulae and blastocysts vitrified in liquid nitrogen and stored at -80°C for four days was low. Therefore, morulae and blastocysts cannot be vitrified in a highly dehydrated/concentrated state using the same method as with two-cell embryos. However, when blastocysts were shrunken artificially before vitrification, survival was high after storage at -80°C for four days with high developmental ability. In conclusion, the equilibrium vitrification method using low concentrations of cryoprotectants, which is effective for two-cell mouse embryos, is also useful for embryos at multiple stages. This method enables the convenient transportation of vitrified embryos using dry ice.

Individual differences in torpor expression in adult mice are related to relative birth mass.

Daily torpor is a physiological adaptation in small mammals and birds, characterised by drastic reductions in metabolism and body temperature. Energy-constraining conditions, such as cold and starvation, are known to cause the expression of daily torpor. However, the reason for high degrees of inter- and intra-individual variation in torpor expression (TE) in similar situations is not clear. As littermates of altricial animals are exposed to an uneven allocation of maternal resources from conception to weaning, we tested whether early nutritional experiences have long-term effects on TE in adults. We used full-sibling littermates of laboratory mice that as adults were starved overnight to induce torpor. We measured body mass from birth until adulthood as an indicator of nutritional status, and calculated the relative body mass (RBM) as an indicator of the difference in nutritional status within a litter. After maturation, we subjected mice to five repeated torpor induction trials involving 24 h of fasting and 5 days of recovery. Half of the female mice displayed great individual variation in TE whereas male mice rarely exhibited daily torpor. In females, RBM at birth influenced TE, irrespective of body mass in adulthood; thus, female mice born with low RBMs displayed high TE in adulthood. In conclusion, we provide evidence that TE in mice differs among littermates, and that this variation is linked closely to heterogeneous nutritional experiences during the fetal period.

Susceptibility of muridae cell lines to ecotropic murine leukemia virus and the cationic amino acid transporter 1 viral receptor sequences: implications for evolution of the viral receptor.

Ecotropic murine leukemia viruses (Eco-MLVs) infect mouse and rat, but not other mammalian cells, and gain access for infection through binding the cationic amino acid transporter 1 (CAT1). Glycosylation of the rat and hamster CAT1s inhibits Eco-MLV infection, and treatment of rat and hamster cells with a glycosylation inhibitor, tunicamycin, enhances Eco-MLV infection. Although the mouse CAT1 is also glycosylated, it does not inhibit Eco-MLV infection. Comparison of amino acid sequences between the rat and mouse CAT1s shows amino acid insertions in the rat protein near the Eco-MLV-binding motif. In addition to the insertion present in the rat CAT1, the hamster CAT1 has additional amino acid insertions. In contrast, tunicamycin treatment of mink and human cells does not elevate the infection, because their CAT1s do not have the Eco-MLV-binding motif. To define the evolutionary pathway of the Eco-MLV receptor, we analyzed CAT1 sequences and susceptibility to Eco-MLV infection of other several murinae animals, including the southern vole (Microtus rossiaemeridionalis), large Japanese field mouse (Apodemus speciosus), and Eurasian harvest mouse (Micromys minutus). Eco-MLV infection was enhanced by tunicamycin in these cells, and their CAT1 sequences have the insertions like the hamster CAT1. Phylogenetic analysis of mammalian CAT1s suggested that the ancestral CAT1 does not have the Eco-MLV-binding motif, like the human CAT1, and the mouse CAT1 is thought to be generated by the amino acid deletions in the third extracellular loop of CAT1.

Effects of type III antifreeze protein on sperm and embryo cryopreservation in rabbit.

We investigated the effects of antifreeze protein (AFP) III supplementation on the cryopreservation of rabbit sperm cells and embryos. Ejaculated semen was collected from male Japanese white (JW) rabbits and divided into four AFP-supplemented groups (0.1 µg/ml, 1 µg/ml, 10 µg/ml, 100 µg/ml) and one control group with no AFP-supplementation. The semen samples were treated with egg-yolk HEPES extender containing 6% acetamide before the sperm was cooled from room temperature to 5 °C, then packed into sperm straws. The straws were frozen in steam of liquid nitrogen (LN2) and then preserved in the LN2. The motility of the sperm after thawing in 37 °C water was analyzed. The percentage of rapidly motile sperm in the 1 µg/ml AFP group was significantly higher than in the control group. Morulae were collected from female JW rabbits and divided into three AFP-supplemented groups (100 ng/ml, 500 ng/ml, 1000 ng/ml) and one control group. The morulae, immersed in an embryo-freezing solution (M199-HEPES containing 20% ethylene glycol, 20% dimethylsulfoxide, 10% fetal bovine serum and 0.25 M sucrose), were packed into open pulled embryo straws and vitrified in LN2. The frozen embryos were thawed in the embryo-freezing solution, and the rates of embryo survival and development to blastocyte stage were analyzed after incubation for 72 h. The development rate of the embryos in the 500 ng/ml AFP group was significantly higher than in the control group, but that in the 1000 ng/ml AFP group was significantly lower. In conclusion, the appropriate dose of AFP III increased the number of rapidly motile sperm and embryo survival following freezing and thawing. The results suggest that supplementation with AFP III can increase the efficiency of cryopreservation of rabbit sperm cells and embryos.

Rapid movement of water and cryoprotectants in pig expanded blastocysts via channel processes: its relevance to their higher tolerance to cryopreservation.

Pig oocytes and embryos are highly sensitive to cryopreservation; however, tolerance to cryopreservation increases in embryos at the expanded blastocyst stage. This increased tolerance may be attributed to a decrease in cytoplasmic lipid droplets at this stage. We previously showed that an increase in the permeability of the plasma membrane in mouse oocytes to water and cryoprotectants, caused by the artificial expression of aquaporin 3, an aquaglyceroporin, enhanced tolerance to cryopreservation. In the present study, we investigated whether membrane permeability was also involved in the tolerance of pig embryos to cryopreservation. The permeability of oocytes and morulae to water and glycerol was low, whereas that of expanded blastocysts was high. Activation energy for permeability to water, glycerol, ethylene glycol, and dimethyl sulfoxide was markedly lower for expanded blastocysts than for oocytes. This suggests that water and these cryoprotectants move through expanded blastocysts predominantly by facilitated diffusion and through oocytes predominantly by simple diffusion. Aquaporin 3 mRNA was expressed in expanded blastocysts abundantly, but less so in oocytes. On the other hand, the permeability of expanded blastocysts to propylene glycol was as low as that of oocytes, and activation energy for its permeability was similar to that of oocytes, which suggests that propylene glycol moves through oocytes and embryos predominantly by simple diffusion. These results suggest that the higher tolerance of pig expanded blastocysts to cryopreservation is also related to high membrane permeability due to the expression of water/cryoprotectant channels, in addition to the decrease in cytoplasmic lipid droplets.

Arterial (18)F-fluorodeoxyglucose uptake reflects balloon catheter-induced thrombus formation and tissue factor expression via nuclear factor-κB in rabbit atherosclerotic lesions.

BACKGROUND: Imaging modalities to assess atherosclerotic plaque thrombogenicity have not been established, so in this study the relationship between [(18)F]-fluorodeoxyglucose ((18)F-FDG) uptake and thrombus formation was investigated in rabbit atherosclerotic arteries. METHODS AND RESULTS: Atherosclerotic plaque was induced in the iliacofemoral artery by balloon injury and a 0.5% cholesterol diet. At 3 weeks after the first balloon injury, the arteries were visualized by (18)F-FDG positron emission tomography (PET) imaging 2h after an (18)F-FDG infusion, and then arterial thrombus was induced by a second balloon injury of both iliacofemoral arteries. Imaging with (18)F-FDG-PET revealed significantly more radioactivity along the injured (0.63 ± 0.12 SUVmax), than the contralateral non-injured artery (0.34 ± 0.08 SUV max, n=17, P<0.0001). Arterial radioactivity measured by autoradiography positively correlated with macrophage area, the number of nuclei that were immunopositive for nuclear factor κ B (NF-κB), and tissue factor (TF) expression. The immunopositive areas for glycoprotein IIb/IIIa and fibrin in thrombi were significantly larger in the atherosclerotic than in the contralateral arteries, and significantly correlated with radioactivity in PET (r=0.92, P<0.001, n=10) and autoradiography (r=0.73, P<0.0001, n=50) in the arteries. Inhibition of NF-κB significantly reduced TF expression in cultured atherosclerotic plaque. CONCLUSIONS: Arterial (18)F-FDG uptake reflects the thrombogenicity of atherosclerotic plaque following balloon injury.

A trial to cryopreserve immature medaka (Oryzias latipes) oocytes after enhancing their permeability by exogenous expression of aquaporin 3.

Fish oocytes have not been cryopreserved successfully, probably because it is difficult to prevent intracellular ice from forming. Previously, we have shown in medaka that immature oocytes are more suitable for cryopreservation than mature oocytes or embryos, in terms of permeability. We have also shown in immature medaka oocytes that the exogenous expression of aquaporin 3 (AQP3), a water/cryoprotectant channel, promotes the movement of water and cryoprotectants through the plasma membrane. In the present study, we attempted to cryopreserve immature medaka oocytes expressing AQP3. We first examined effects of hypertonic stress and the chemical toxicity of cryoprotectants on the survival of the AQP3-expressing oocytes. Exposure to hypertonic solutions containing sucrose decreased the survival of oocytes, but the expression of AQP3 did not affect sensitivity to hypertonic stress. Also, AQP3 expression did not markedly increase sensitivity to the toxicity of cryoprotectants. Of the four cryoprotectants tested, propylene glycol was the least toxic. Using a propylene glycol-based solution, therefore, we tried to cryopreserve immature oocytes by vitrification. During cooling with liquid nitrogen, all intact oocytes became opaque, but many AQP3-expressing oocytes remained transparent. This indicates that the expression of AQP3 is effective in preventing intracellular ice from forming during cooling. During warming, however, all the AQP3-expressing oocytes became opaque, indicating that intracellular ice formed. Therefore, the dehydration and permeation by propylene glycol were still insufficient. Further studies are necessary to realize the cryopreservation of fish oocytes.

Message to researchers: the characteristic absence of a posterior communicating artery is easily lost in the gerbil.

The Mongolian gerbil has historically been useful for brain ischemia experiments, owing to the gerbil's uniquely underdeveloped circle of Willis (CoW). This led to a gerbil model of cochlear ischemia being generated in our unit. However, we have found that the usual severe hearing loss seen in this model was not being induced consistently in recent experiments using the MON/Jms/GbsSlc gerbil (the sole commercially available gerbil in Japan). We set out to evaluate the posterior communicating artery (PcomA) in MON/Jms/GbsSlc, to re-establish whether this strain is appropriate for ischemia models. Having found that this unique feature is often lost, we then attempted to breed for the characteristic absent PcomA. India-ink perfusion revealed that the percentage of intact bilateral PcomA ("communicating type") in the MON/Jms/GbsSlc gerbil was 57%; unilateral only ("unilateral communicating type") was 39%; and completely absent PcomA ("non-communicating type") was 4%. We were able to obtain few examples of the indigenous old aged Japanese UNG/Mz gerbil strain (at University of Miyazaki). Unfortunately, the pure UNG/Mz female was too elderly for mating. Therefore, selective breeding crosses between MON/Jms/GbsSlc and male UNG/Mz were carried out. After five generations of selective breeding, the percentage of non-communicating type gerbils was significantly higher in the newly generated strain, MON/Jms/SlcMz (F6 generation; 63%) than in the MON/Jms/GbsSlc gerbil. Bilateral common carotid artery occlusion surgery demonstrated that the cerebral blood flow was significantly reduced in MON/Jms/SlcMz compared with MON/Jms/GbsSlc (p < 0.0001) and induced more hippocampal injuries in MON/Jms/SlcMz than in MON/Jms/GbsSlc (p < 0.01). In conclusion, the commercially available MON/Jms/GbsSlc gerbil can easily regain PcomA, and we established a new gerbil strain (MON/Jms/SlcMz) displaying non-PcomA.

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.

Equilibrium vitrification of mouse embryos at various developmental stages.

Previously, we developed a new method by which 2-cell mouse embryos can be vitrified in liquid nitrogen in a near-equilibrium state, and then kept at -80°C for several days. In the present study, we examined whether or not the method was effective for mouse embryos at other developmental stages. Eight-cell embryos, morulae, and expanded blastocysts of ICR mice were vitrified with ethylene glycol-based solutions, named EFSc because of their composition of ethylene glycol (30-40%, v/v) and FSc solution. The FSc solution was PB1 medium containing 30% (w/v) Ficoll PM-70 plus 1.5 M sucrose. The extent of equilibrium was assessed by examining how well vitrified embryos survived after being kept at -80°C. When 8-cell embryos and morulae were vitrified with EFS35c or EFS40c and then kept at -80°C, the survival rate was high even after 4 days in storage and remained high after re-cooling in liquid nitrogen. On the other hand, the survival of vitrified-expanded blastocysts kept at -80°C was low. Therefore, 8-cell embryos and morulae can be vitrified in a near-equilibrium state using the same method as for 2-cell embryos. A high proportion of C57BL/6J embryos at the 2-cell, 8-cell, and morula stages vitrified with EFS35c developed to term after transportation on dry ice, re-cooling in liquid nitrogen, and transfer to recipients. In conclusion, the near-equilibrium vitrification method, which is effective for 2-cell mouse embryos, is also effective for embryos at the 8-cell and morula stages. The method would enable handy transportation of vitrified embryos using dry ice.

Pivotal role of High-Mobility Group Box 2 in ovarian folliculogenesis and fertility.

BACKGROUND: High-Mobility Group Box 1 (HMGB1) and HMGB2 are chromatin-associated proteins that belong to the HMG protein family, and are involved in the regulation of DNA transcription during cell differentiation, proliferation and regeneration in various tissues. However, the role of HMGB2 in ovarian folliculogenesis is largely unknown. METHODS: We investigated the functional role of HMGB1 and HMGB2 in ovarian folliculogenesis and fertilization using C57BL/6 wild type (WT) and HMGB2-knockout (KO) mice. Ovarian tissues were obtained from WT and HMGB2-KO mice at postnatal days 0, 3, 7, and 2, 6 months of age, then performed immunohistochemistry, qPCR and Western blotting analyses. Oocyte fertilization capability was examined by natural breeding and in vitro fertilization experiments. RESULTS: In HMGB2-KO mice, ovary weight was decreased due to reduced numbers of oocytes and follicles. Natural breeding and in vitro fertilization results indicated that HMGB2-KO mice are subfertile, but not sterile. Immunohistochemistry showed that oocytes expressed HMGB2, but not HMGB1, in neonatal and adult WT ovaries. Interestingly, in HMGB2-KO ovaries, a compensatory increase in HMGB1 was found in oocyte nuclei of neonatal and 2-month-old mice; however, this was lost at 6 months of age. CONCLUSIONS: The depletion of HMGB2 led to alterations in ovarian morphology and function, suggesting that HMGB2 plays an essential role in ovarian development, folliculogenesis and fertilization.

Pathway for the movement of water and cryoprotectants in bovine oocytes and embryos.

The permeability of cells is important for cryopreservation. Previously, we showed in mice that the permeability to water and cryoprotectants of oocytes and embryos at early cleavage stages (early embryos) is low because these molecules move across the plasma membrane predominantly by simple diffusion through the lipid bilayer, whereas permeability of morulae and blastocysts is high because of a water channel, aquaporin 3 (AQP3). In this study, we examined the pathways for the movement of water and cryoprotectants in bovine oocytes/embryos and the role of AQP3 in the movement by determining permeability, first in intact bovine oocytes/embryos, then in bovine morulae with suppressed AQP3 expression, and finally in mouse oocytes expressing bovine AQP3. Results suggest that water moves through bovine oocytes and early embryos slowly by simple diffusion, as is the case in mice, although channel processes are also involved in the movement. On the other hand, water appears to move through morulae and blastocysts predominantly by facilitated diffusion via channels, as in mice. Like water, cryoprotectants appear to move through bovine oocytes/early embryos mostly by simple diffusion, but channel processes could also be involved in the movement of glycerol and ethylene glycol, unlike that in mice. In bovine morulae, although glycerol and ethylene glycol would move predominantly by facilitated diffusion, mostly through AQP3, as in mice, dimethylsulfoxide appears to move predominantly by simple diffusion, unlike in mice. These results indicate that permeability-related properties of bovine oocytes/embryos are similar to those of mouse oocytes/embryos, but species-specific differences do exist.

Cryobiological properties of immature zebrafish oocytes assessed by their ability to be fertilized and develop into hatching embryos.

As a step to develop a cryopreservation method for zebrafish oocytes, we investigated the cryobiological properties of immature oocytes at stage III by examining their ability to mature and to develop into hatching embryos after fertilization. When oocytes were chilled at -5°C for 30min, the maturation rate decreased, but the rates of fertilization and hatching were not significantly different from those of controls. When oocytes were exposed to hypotonic solutions for 60min at 25°C, the rates of maturation, fertilization, and hatching decreased in a solution with 0.16Osm/kg or below. When oocytes were exposed to hypertonic solutions (containing sucrose) at 25°C for 30min, the maturation rate decreased in solution with 0.51Osm/kg, whereas the hatching rate decreased with lower osmolality (0.40Osm/kg). In an experiment on the toxicity of cryoprotectants (∼10%, at 25°C), it was found that glycerol and ethylene glycol were toxic both by the assessment of maturation and hatching. Propylene glycol, DMSO and methanol were less toxic by the assessment of maturation, but were found to be toxic by the assessment of hatching. Methanol was the least toxic, but it was less effective to make a solution vitrify than propylene glycol. Therefore, a portion of methanol was replaced with propylene glycol. The replacement increased the toxicity, but could be effective to reduce chilling injury at -5°C. These results clarified the sensitivity of immature oocytes to various cryobiological properties accurately, which will be useful for realizing cryopreservation of zebrafish oocytes.

Developmental ability of vitrified mouse oocytes expressing water channels.

Previously, we showed that the exogenous expression of aquaporin 3 (AQP3), an aquaglyceroporin, improved the tolerance of mouse oocytes to vitrification with a glycerol-based solution. In the present study, we examined conditions suitable for the expression of AQP3 and the ability of vitrified oocytes to develop in vitro and in vivo after fertilization. After only partial remove of cumulus cells, immature mouse oocytes (germinal vesicle stage) were injected with 5, 10 or 20 pg of AQP3 cRNA and cultured for 12 h for maturation. When matured oocytes were vitrified with a glycerol-based solution, 57-61% survived regardless of the amount of cRNA injected (5-20 pg). By contrast, no oocytes injected with water (control) survived. When the zona pellucida was removed from the vitrified oocytes and the oocytes were then fertilized in vitro and cultured, the proportions that were fertilized and developed into blastocysts were higher when the amount of cRNA injected was 5 pg than 10-20 pg. When 16 blastocysts were transferred to a pseudopregnant mouse, 5 developed to term, demonstrating that oocytes vitrified after injection of AQP3 cRNA retained the ability to develop to term. The water-permeability of cRNA-injected oocytes was higher than that of control oocytes from the maturing stage to the 1-cell zygote stage, whereas glycerol-permeability was higher only at metaphase II. This indicates that AQP3 was expressed for a relatively short period of time. These results suggest that the transient expression of water/cryoprotectant channels is effective for cryopreserving cells that have low membrane-permeability, such as mammalian oocytes.

Translocator protein imaging with 18F-FEDAC-positron emission tomography in rabbit atherosclerosis and its presence in human coronary vulnerable plaques.

BACKGROUND AND AIMS: This study aimed to investigate whether N-benzyl-N-methyl-2-[7,8-dihydro-7-(2-[18F]fluoroethyl)-8-oxo-2-phenyl-9H-purin-9-yl]acetamide (18F-FEDAC), a probe for translocator protein (TSPO), can visualize atherosclerotic lesions in rabbits and whether TSPO is localized in human coronary plaques. METHODS: 18F-FEDAC-PET of a rabbit model of atherosclerosis induced by a 0.5% cholesterol diet and balloon injury of the left carotid artery (n = 7) was performed eight weeks after the injury. The autoradiography intensity of 18F-FEDAC in carotid artery tissue sections was measured, and TSPO expression was evaluated immunohistochemically. TSPO expression was examined in human coronary arteries obtained from autopsy cases (n = 16), and in human coronary plaques (n = 12) aspirated from patients with acute myocardial infarction (AMI). RESULTS: 18F-FEDAC-PET visualized the atherosclerotic lesions in rabbits as high-uptake areas, and the standard uptake value was higher in injured arteries (0.574 ± 0.24) than in uninjured arteries (0.277 ± 0.13, p < 0.05) or myocardium (0.189 ± 0.07, p < 0.05). Immunostaining showed more macrophages and more TSPO expression in atherosclerotic lesions than in uninjured arteries. TSPO was localized in macrophages, and arterial autoradiography intensity was positively correlated with macrophage concentration (r = 0.64) and TSPO (r = 0.67). TSPO expression in human coronary arteries was higher in AMI cases than in non-cardiac death, or in the vulnerable plaques than in early or stable lesions, respectively. TSPO was localized in macrophages in all aspirated coronary plaques with thrombi. CONCLUSIONS: 18F-FEDAC-PET can visualize atherosclerotic lesions, and TSPO-expression may be a marker of high-risk coronary plaques.

Maintenance of fertility in cryopreserved Indian gerbil (Tatera indica) spermatozoa.

This study is the first attempt at sperm cryopreservation, as well as a further examination of frozen sperm fertility by the hamster test, applied to the maintenance of an Indian gerbil (Tatera indica) colony, which is a newly developing experimental animal. The osmotic tolerance of the spermatozoa was initially investigated by subjection to hypertonicity, up to 620mOsm/kg, for 5min at room temperature prior to freezing. Although the percentage of total motile sperm was not affected, that of progressive motile spermatozoa began to drop at 400mOsm/kg, and a significant decrease was observed at 620mOsm/kg (p<0.01). According to these results, the osmolality of the solutions for the freezing experiment, in which 6-22% raffinose was present, was fixed at approximately 400mOsm/kg. Sperm, suspended in a plastic straw, were frozen in liquid nitrogen vapor for 5min, followed by immersion in liquid nitrogen. Motile sperm were recovered from all freezing conditions, and high survival was obtained when sperm were frozen in the presence of 14% and 18% raffinose, with a normalized motility higher than 40%. Fertility of cryopreserved Indian gerbil sperm was examined by the zona-free hamster test. Thawed sperm adhered to 88% of the zona-free hamster oocyte surface, and some oocytes were penetrated and exhibited swollen sperm heads or male pronuclei, which we used to define fertilization. Although the fertilization rate of cryopreserved sperm to zona-free hamster eggs was significantly lower than that of fresh sperm (6% vs. 30%, p<0.01), we demonstrated that thawed Indian gerbil spermatozoa have the ability to maintain their fertility.

Comparison of the gut microbiotas of laboratory and wild Asian house shrews (Suncus murinus) based on cloned 16S rRNA sequences.

The Asian house shrew, Suncus murinus, is an insectivore (Eulipotyphla, Mammalia) and an important laboratory animal for life-science studies. The gastrointestinal tract of Suncus is simple: the length of the entire intestine is very short relative to body size, the large intestine is quite short, and there are no fermentative chambers such as the forestomach or cecum. These features imply that Suncus has a different nutritional physiology from those of humans and mice, but little is known about whether Suncus utilizes microbial fermentation in the large (LI) or small (SI) intestine. In addition, domestication may affect the gastrointestinal microbial diversity of Suncus. Therefore, we compared the gastrointestinal microbial diversity of Suncus between laboratory and wild Suncus and between the SI and LI (i.e., four groups: Lab-LI, Lab-SI, Wild-LI, and Wild-SI) using bacterial 16S rRNA gene library sequencing analyses with a sub-cloning method. We obtained 759 cloned sequences (176, 174, 195, and 214 from the Lab-LI, Lab-SI, Wild-LI, and Wild-SI samples, respectively), which revealed that the gastrointestinal microbiota of Suncus is rich in Firmicutes (mostly lactic acid bacteria), with few Bacteroidetes. We observed different bacterial communities according to intestinal region in laboratory Suncus, but not in wild Suncus. Furthermore, the gastrointestinal microbial diversity estimates were lower in laboratory Suncus than in wild Suncus. These results imply that Suncus uses lactic acid fermentation in the gut, and that the domestication process altered the gastrointestinal bacterial diversity.