Prolonged exposure to hyaluronidase decreases the fertilization and development rates of fresh and cryopreserved mouse oocytes

Hyaluronidase is generally used to remove cumulus cells from mouse oocytes before oocyte cryopreservation, intracytoplasmic sperm injection or DNA injection. In general, use of cumulus-free mouse oocytes decreases in vitro fertilizing ability compared with cumulus-surrounded oocytes. The effect of hyaluronidase exposure on the quality of mouse oocytes is not fully understood. Here, we investigated the effect of hyaluronidase exposure time on the fertilization rate of fresh and vitrified mouse oocytes and their subsequent developmental ability in vitro. We found that the fertilization rate decreased with hyaluronidase treatments. This reduction in the fertilization rate following treatment with hyaluronidase was fully reversed by removal of the zona pellucida. In addition, oocytes treated with hyaluronidase for 5 min or longer had a reduced capacity to develop to the morula and blastocyst stage. The survival, fertilization, and developmental rates of vitrified-warmed oocytes were also reduced by longer exposure to hyaluronidase. In conclusion, these results suggest that prolonged exposure to hyaluronidase decreases the quality of mouse oocytes and shorter hyaluronidase treatment times may help achieve a stable and high fertilization rate in fresh and cryopreserved oocytes.     

Expression profiles of perforin,granzyme B and granulysin genes during the estrous cycle and gestation in the bovine endometrium

The conceptus is susceptible to destruction by maternal cytotoxic lymphocytes, which have cytotoxic potential. Therefore, it is expected that mechanisms for regulating cytotoxic lymphocytes exist, but little is known about the expression of cytotoxic genes in the endometrium. In the present study, we examined the spatial and temporal expression patterns of the cytotoxic genes perforin, granzyme B, and granulysin during the estrous cycle and gestation in the bovine endometrium. Endometrial tissues were collected from cows during the estrous cycle and gestation. The gene expression patterns of the three cytotoxic genes were examined using quantitative polymerase chain reaction and in situ hybridization, and cytotoxic lymphocyte subsets were characterized using immunohistochemistry. During mid‐ to late gestation in the intercaruncular (ICAR), granulysin expression was significantly increased, and a large number of granulysin‐expressing cells were localized in the luminal epithelium. Perforin and granzyme B displayed similar expression profiles and were highly expressed in the peri‐implantation endometrium, but few cells expressing these genes were found in the endometrial stroma. In conclusion, these findings suggest that in the ICAR epithelium granulysin may play important roles in the establishment and maintenance of gestation during normal pregnancy.     

Comparative study of morphological changes in hardwoods treated with the ionic liquid, 1-ethyl-3-methylimidazolium chloride

Three hardwoods of varying vessel arrangement were treated with the ionic liquid, 1-ethyl-3-methylimidazolium chloride ([C2mim][Cl]), which can dissolve cellulose, to investigate its influence on wood tissue morphology. Characterization was carried out by light and scanning electron microscopy. The wood fibers of all species swelled significantly during [C2mim][Cl] treatment. The swelling behavior varied according to wood species and differed from other cell types such as ray parenchyma cells. Morphological changes of the pits also varied between wood species. Treatment with [C2mim][Cl] affects wood tissues differently depending on wood species and cell type. These differences are not due to the vessel arrangement and its presence, but possibly from the chemical component and the microfibril angle of various wood tissues.     

Occurrence of Spontaneous Tumors in the Central Nervous System (CNS) of F344 and SD Rats

In order to accurately assess the carcinogenicity of chemicals with regard to rare tumors such as rat CNS tumors, sufficient information about spontaneous tumors are very important. This paper presents the data on the type, incidence and detected age of CNS tumors in F344/DuCrlCrlj (a total of 1363 males and 1363 females) and Crl:CD(SD) rats (a total of 1650 males and 1705 females) collected from in-house background data-collection studies and control groups of carcinogenicity studies at our laboratory, together with those previously reported in F344 and SD rats. The present data on F344/DuCrlCrlj rats (F344 rats) and Crl:CD(SD) rats (SD rats) clarified the following. (1) The incidences of all CNS tumors observed in F344 rats were less than 1%. (2) The incidences of malignant astrocytoma and granular cell tumor were higher in male SD rats than in female SD rats. (3) The incidences of astrocytoma and granular cell tumor were higher in SD rats than in F344 rats. (4) Among astrocytoma, oligodendroglioma and granular cell tumor, oligodendroglioma was detected at the youngest age, followed by astrocytoma, and ultimately, granular cell tumor developed in both strains. The incidences observed in our study were almost consistent with those previously reported in F344 and SD rats.     

Species relationships between a wild tetraploid potato species,Solanum acaule bitter, and its related species as revealed by rflps of chloroplast and nuclear DNA

Solanum acaule Bitter is a wild, disomic tetraploid potato species widely distributed in the South America. In this study, species relationships between eight accessions ofS. acaule and 108 accessions of 27 morphologically closely related species were investigated using chloroplast and nuclear DNA RFLPs. Chloroplast DNA types were determined for most of the accessions based on restriction digestion patterns of entire chloroplast DNA. C type chloroplast DNA was common inS. acaule andS. albicans (a hexaploid derivative fromS. acaule), which was also found inS. megistacrolobum, S. sanctae-rosae,S. raphanifolium, and several other Peruvian species in seriesTuberosa. Nuclear DNA was analyzed by Southern hybridization with 31 single-copy RFLP probes. One hundred and thirty-four bands were scored, from which a UPGMA dendrogram was constructed to show overall similarities among accessions.Solanum acaule andS. albicans (andS. demissum) formed a cluster with a high similarity, and then, this group was clustered withS. megistacrolobum andS. sanctae-rosae. The resulting cluster was well separated from other clusters. Thus, we suggest that an initial tetrasomic tetraploid arose from eitherS. megistacrolobum orS. sanctae-rosae. Interestingly, a Mexican hexaploid speciesS. demissum showed the closest similarity toS. acaule andS. albicans, suggesting that they shared a common ancestry.     

Quantitation of tocotrienol and tocopherol in various rice brans

Rice bran is abundant in bioactive compounds including tocotrienol (T3, unsaturated vitamin E). T3 has been reported about its potential functionalities (i.e., antiangiogenic effect), so much attention has been paid on usability of rice bran T3. Hence, we developed a rapid screening method for T3-rich rice bran by one-step equilibrium direct solvent extraction followed by normal phase high-performance liquid chromatography (HPLC). The method gave high-extraction rate of rice bran T3 and tocopherol (above 90%), and the determination of vitamin E by HPLC was completed within 15 min. Using the method, an average of total T3 content in 109 kinds of rice bran samples was 830 mug/g dry wt. Kouchi-Akamai, Joushuu, and Wataribune were found as the T3-rich rice bran varieties (1350-1430 microg T3/g dry wt). According to T3 ratio against total vitamin E (wt %), the average ratio was 61%. Hirayama, Moritawase, and Kaneko were found as the varieties having the highest T3 ratio (80-86%). Since T3 content in Koshihikari rice bran (the leading variety in Japan) was a little above the average, we cross-fertilized Koshihikari with T3-rich varieties and found that T3 content or ratio in F1 was improved compared with Koshihikari. The varieties found rich in T3 could be used for nutraceutical purpose.     

Chloroplast DNA variation in the wild potato species, Solanum acaule and S. albicans

The chloroplast DNA of Solanum acaule (109 accessions) and S. albicans (9 accessions) was investigated by restriction endonuclease analysis. Unexpectedly, all the accessions analyzed had C type chloroplast DNA in common. This suggested that S. acaule originated from a species with C type chloroplast DNA. DraI restriction digestion revealed further differentiation of C type chloroplast DNA into 8 types. The DraI polymorphism indicated the province of Salta in Argentina and the nearby regions to be a center of diversity for S. acaule. Surprisingly, S. albicans as well as S. acaule both ssp. acaule and ssp. punae, from Peru were virtually indistinguishable, although by morphology and/or cytology all three taxa are easily distinguished. This revised version was published online in July 2006 with corrections to the Cover Date.     

Overcoming hybrid lethality in a cross between Japanese pear and apple using gamma irradiation and confirmation of hybrid status using flow cytometry and SSR markers

Although there have been some attempts to obtain intergeneric hybrids between Japanese pear (Pyrus pyrifolia Nakai) and apple (Malus × domestica Borkh.) mature hybrids have not been generated due to hybrid lethality. Shoots from immature hybrid embryos were gamma irradiated and cultured at normal temperature conditions to obtain viable intergeneric plants. Gamma irradiation doses of 50 and 100 Gy cultured at 25 °C killed all shoots but there was one survivor at 150 Gy and 25 °C. Hybrid status of the shoot was confirmed by using morphology, flow cytometry and SSR markers. The intergeneric plant had similar leaf serrations to apple and similar petioles to Japanese pear. Flow cytometric analysis revealed that the intergeneric shoot had a DNA content intermediate between the parents. SSR analysis showed one allele from each parent for all 16 SSR loci investigated. These data strongly suggest that the obtained shoot is a true hybrid between Japanese pear and apple.