Na?ve-like conversion enhances the difference in innate in vitro differentiation capacity between rabbit ES cells and iPS cells

Quality evaluation of pluripotent stem cells using appropriate animal models needs to be improved for human regenerative medicine. Previously, we demonstrated that although the in vitro neural differentiating capacity of rabbit induced pluripotent stem cells (iPSCs) can be mitigated by improving their baseline level of pluripotency, i.e., by converting them into the so-called “naïve-like” state, the effect after such conversion of rabbit embryonic stem cells (ESCs) remains to be elucidated. Here we found that naïve-like conversion enhanced the differences in innate in vitro differentiation capacity between ESCs and iPSCs. Naïve-like rabbit ESCs exhibited several features indicating pluripotency, including the capacity for teratoma formation. They differentiated into mature oligodendrocytes much more effectively (3.3–7.2 times) than naïve-like iPSCs. This suggests an inherent variation in differentiation potential in vitro among PSC lines. When naïve-like ESCs were injected into preimplantation rabbit embryos, although they contributed efficiently to forming the inner cell mass of blastocysts, no chimeric pups were obtained. Thus, in vitro neural differentiation following naïve-like conversion is a promising option for determining the quality of PSCs without the need to demonstrate chimeric contribution. These results provide an opportunity to evaluate which pluripotent stem cells or treatments are best suited for therapeutic use.     

Aureusidin synthase: a polyphenol oxidase homolog responsible for flower coloration

Aurones are plant flavonoids that provide yellow color to the flowers of some popular ornamental plants, such as snapdragon and cosmos. In this study, we have identified an enzyme responsible for the synthesis of aurone from chalcones in the yellow snapdragon flower. The enzyme (aureusidin synthase) is a 39-kilodalton, copper-containing glycoprotein catalyzing the hydroxylation and/or oxidative cyclization of the precursor chalcones, 2',4',6',4-tetrahydroxychalcone and 2',4',6',3,4-pentahydroxychalcone. The complementary DNA encoding aureusidin synthase is expressed in the petals of aurone-containing varieties. DNA sequence analysis revealed that aureusidin synthase belongs to the plant polyphenol oxidase family, providing an unequivocal example of the function of the polyphenol oxidase homolog in plants, i.e., flower coloration.     

Assignment of the CD Cotton Effect to the Chiral Center in Pseurotins,and the Stereochemical Revision of Pseurotin A2

Pseurotins A₁ (1) and A₂ (2) were isolated from a culture broth of the fungal strain Aspergillus fumigatus WFZ-25 as stereoisomers of pseurotin A (3) in 2011. We also isolated 1 and 2 together with 3 from A. fumigatus OUPS-T106B-5 separated from the marine fish Mugil cephalus. In this study, we re-examined the stereochemistry of 1 and 2 using chemical transformation and the CD spectra, and found the relationship between the CD Cotton effect and the absolute configurations of 1 and 2, which led us to revise the stereostructure of pseurotin A₂.     

Guided bone regeneration with beta-tricalcium phosphate and poly L-lactide-co-glycolide-co-epsilon-caprolactone membrane in partial defects of canine humerus

This study was performed to evaluate the effect of beta-tricalcium phosphate and poly L-lactide-co-glycolide-co-epsilon-caprolactone (TCP/PLGC) membrane in the repair of partial bone defects in canine proximal humerus. Three adult mixed-breed dogs were used during the experimental period. The length of the defect was quarter of the full length of humerus, and width of the defect was quarter of middle diameter of the lateral aspect of humerus. The humeri of each dog were divided into treatment (TCP/PLGC) and control groups. The defect was covered with TCP/PLGC membrane in treatment group. To evaluate regeneration of the bone, computerized tomography (CT) and histopathologic examination were performed. The radiopaque lines were appeared at the original defect sites in TCP/PLGC group but below the original site in control at 4th week. Radiopacity and thickness of the defect sites, and radiopaque lines were more increased at 8th week than those of 4th week. Histopathologic findings revealed fibrous connective tissue migration into the defect and the migration inhibited the structure of new cortex to be placed in the original level in control whereas new cortex growth was found in the level of original line in TCP/PLGC group. However, the new cortical bone in the TCP/PLGC group was thinner and less organized than the adjacent intact cortex, and the amount of new cancellous bones were also scanty. The result suggested that TCP/PLGC membrane is a good guided bone regeneration material to restore the original morphology of humerus in partial defect.     

Effects of polyethylene glycol and a synthetic ice blocker during vitrification of immature porcine oocytes on survival and subsequent embryo development

We evaluated the effects of polyethylene glycol (PEG) and Supercool X‐1000 (SC) as supplements during the vitrification of immature cumulus‐enclosed porcine oocytes in a solution based on 17.5% ethylene glycol + 17.5% propylene glycol. After warming, the oocytes were subjected to in vitro maturation, fertilization and embryo culture. In Experiment 1, equilibration and vitrification solutions were supplemented with or without 2% (w/v) PEG (PEG+ and PEG‐, respectively). The survival rate, cleavage and blastocyst development were similar between PEG+ and PEG‐ groups; however, all values were lower than those in the non‐vitrified control. In Experiment 2, vitrification solution was supplemented with or without 1% (v/v) SC (SC+ and SC‐, respectively). The percentages of survival and blastocyst development were similar between SC+ and SC‐ groups but lower than those in the non‐vitrified control. The percentage of cleavage in the SC‐ group was significantly lower than the control and the SC+ groups, which were in turn similar to one another. In both experiments, the cell numbers in blastocysts were not significantly different among the non‐vitrified and vitrified groups. In conclusion, PEG did not improve oocyte survival and embryo development, whereas SC improved the ability of surviving oocytes to cleave but not to develop into blastocysts.     

Gene response in rice plants treated with continuous fog influenced by pH,was similar to that treated with biotic stress

Background

Throughout Asia, including Japan, rice plants are cultivated in a wide range of areas from lowlands to highlands and are frequently exposed to fog, including acid fog. Some physiological studies have shown that acid fog can be a stress factor for plants. We analyzed the gene expression profiles of rice plants treated with artificially prepared simulated acid fog (SiAF) or simulated neutral fog (SiNF) for 1 or 7 days.

Results

Microarray analysis results suggested that both the SiAF and the SiNF treatments induced the expression of genes involved in the defense and stress responses in rice plants. Induction of such genes was detected in plants treated with SiAF for 1 day, and the number of induced genes increased in plants treated with SiAF for 7 days. The genes for defense and stress responses were also induced by SiNF for 7 days, although they were not induced by SiNF for 1 day. The gene expression profiles of the SiAF-treated and the SiNF-treated plants were compared to those of plants treated with other stress factors. The comparison revealed that both SiAF and SiNF treatments have similar effects to biotic stresses and ozone stress. The genes encoding NADPH oxidase and germin, which function in apoplasts, were also induced by SiAF, SiNF and biotic stresses.

Conclusions

These findings suggest that both the SiAF and the SiNF treatments may result in oxidative stress through the apoplastic production of reactive oxygen species.     

Sucrose assists selection of high‐quality oocytes in pigs

We investigated whether high‐quality in vitro matured (IVM) oocytes can be distinguished from poor ones based on the morphological changes after treatment with hyperosmotic medium containing 0.2 mol/L sucrose in pigs. We hypothesize that IVM oocytes maintaining round shape have higher quality than mis‐shapened oocytes following dehydration. Oocyte quality was verified by determining embryonic developmental competence using in vitro fertilization, nuclear transfer and parthenogenetic activation. In all cases, the round oocytes had greater (p < .05) developmental competence than that of mis‐shapened oocytes in terms of blastocyst rate and total cell number in blastocysts obtained after 6 days of in vitro culture. We also confirm that round aged oocytes are higher in quality than mis‐shapened aged oocytes. In an attempt to find out why high‐quality oocytes maintain a round shape whereas poorer oocytes become mis‐shapened following sucrose treatment, we examined the arrangement of actin microfilaments and microtubules. Abnormal organization of these cytoskeletal components was higher (p < .05) in mis‐shapened oocytes compared to round oocytes after 52 hr of IVM. In conclusion, sucrose treatment helps selection of high‐quality oocytes, including aged oocytes, in pigs. Abnormal cytoskeleton arrangements partly explain for low developmental competence of mis‐shapened oocytes.     

Chicken peripheral blood CD3+CD4-CD8- cells are regulated by endocrine and nerve systems

The existence of CD3(+)CD4(-)CD8(-) T cells in thymus and spleen has already been known. However, because of the presence of large amounts of thrombocytes in peripheral blood (PB), the proportion of CD3(+)CD4(-)CD8(-) T cells in PB has yet to be investigated. Therefore, the proportion of peripheral T cell-subsets was investigated in 6-week-old chickens. The percentage of CD3(+) cells, CD4(+) cells, CD8 alpha(+) cells, CD8 beta(+), and CD3(+)CD4(-)CD8(-) cells was 76%, 41%, 14%, 5%, and 15%, respectively. The proportion of CD3(+)CD4(-)CD8(-) cells in PB increased during egg-laying periods and in chickens treated with an analog of estrogen, while it decreased with age and in response to restraint stress. All of the CD3(+)CD4(-)CD8(-) cells expressed TCR1, and did not have NK activity. CD3(+)CD4(-)CD8(-) cells represent about 60% of peripheral TCR1(+) cells. These findings indicate that the proportion of CD3(+)CD4(-)CD8(-) cells is regulated by the endocrine and nerve systems.     

Essential role of Fkbp6 in male fertility and homologous chromosome pairing in meiosis

Meiosis is a critical stage of gametogenesis in which alignment and synapsis of chromosomal pairs occur, allowing for the recombination of maternal and paternal genomes. Here we show that FK506 binding protein (Fkbp6) localizes to meiotic chromosome cores and regions of homologous chromosome synapsis. Targeted inactivation of Fkbp6 in mice results in aspermic males and the absence of normal pachytene spermatocytes. Moreover, we identified the deletion of Fkbp6 exon 8 as the causative mutation in spontaneously male sterile as/as mutant rats. Loss of Fkbp6 results in abnormal pairing and misalignments between homologous chromosomes, nonhomologous partner switches, and autosynapsis of X chromosome cores in meiotic spermatocytes. Fertility and meiosis are normal in Fkbp6 mutant females. Thus, Fkbp6 is a component of the synaptonemal complex essential for sex-specific fertility and for the fidelity of homologous chromosome pairing in meiosis.     

Endocrine status and development of porcine testicular tissues in host mice

Clarification of the endocrine status of host mice provides us with basic knowledge with which we can manipulate the growth and function of xenografted testicular tissues. We investigated the hormonal profiles of castrated mice grafted with porcine immature testicular tissues from 30 to 210 or more days after grafting (day 0=castration and grafting). The serum follicle stimulating hormone (FSH) concentrations of the host mice declined (P<0.05) from day 60 compared with those of the castrated, ungrafted mice. The serum inhibin and testosterone levels were higher (P<0.05) than those in the castrated, ungrafted mice from days 30 and 90 days, respectively. The inhibin levels further increased (P<0.05) from day 90, during which time the levels were higher (P<0.05) than those in the intact male mice. In the grafts, formation of lumens occurred in the seminiferous cords on day 90 and spermatozoa appeared in the lumens from day 120. However, spermatogenesis in the grafts did not reach the qualitatively normal levels observed in adult boars. The intensity of the immune reaction to inhibin alpha subunits in the Sertoli cells of the grafts decreased with differentiation of the seminiferous tubules. The present findings indicate that a feedback loop was established between the mouse hypothalamo-pituitary axis and the grafted porcine tissues from 60 days post-grafting. The results also indicate that the serum inhibin levels in the host mice remained high even after the appearance of lumens in the seminiferous tubules of the grafted tissues; this is strikingly different to the situation in normal male animals, in which the serum inhibin levels decline at around the time of tubular differentiation. The lack of efferent ducts in the tubules of the grafted tissues probably caused the accumulation of inhibin to be released into the lumens, resulting in high concentrations of circulating inhibin. These high levels of inhibin may directly affect spermatogenic activity and suppress FSH secretion.