Microinsemination with first-wave round spermatids from immature male mice

In several mammalian species, including mice, round spermatids have been used to produce normal offspring by means of microinsemination techniques. In this study, we examined whether mouse round spermatids retrieved from immature testes undergoing the first wave of spermatogenesis had acquired fertilizing ability comparable to cells from mature adults. Microinsemination with round spermatids was performed by direct injection into preactivated oocytes, as previously reported. About 60-85% of the successfully injected oocytes developed to the morula/blastocyst stage after 72 h in culture, irrespective of the age of the males (17-25 days old). After embryo transfer, normal pups were obtained from all age groups, including the day-17 group, the stage at which the first round spermatids appeared. A high correlation (r=0.90) was found between the birth rate and male age (P<0.01, Spearman rank correlation), indicating that the efficiency of producing offspring was dependent on the age of the donor males. Imprinted genes (H19, Igf2, Meg3, and Igf2r) were expressed from the correct parental alleles (maternal, paternal, maternal, and maternal, respectively) in all (n=12) day-9.5 fetuses derived from day-20 spermatids. These results clearly indicate that at least some first-wave spermatogenic cells have a normal haploid genome with the correct paternal imprint and are capable of supporting full-term embryo development, as do mature spermatozoa from adults. The use of male germ cells from immature animals may save time in the production of inbred/congenic strains and rescue male-factor infertility of early onset.     

Oxidative metabolism of tetrachlorocyclohexenes,pentachlorocyclohexenes, and hexachlorocyclohexenes with microsomes from rat liver and house fly abdomen

Microsomal mixed-function oxidase systems from rat liver and house fly abdomen effectively metabolized isomers of 3,4,5,6-tetrachlorocyclohexene, 1,3,4,5,6-pentachlorocyclohexene, and 1,2,3,4,5,6-hexachlorocyclohexene to tetrachlorocyclohexenol isomers, 2,4,5,-trichlorophenol, and 2,3,4,6-tetrachlorophenol, respectively. The $\text{(}\text{346}\text{5}\text{)-isomer}$ of pentachlorocyclohexene gave also an abundant amount of pentachlorocyclohexenol isomers. As the metabolites of ($\text{36}\text{45}$)-, ($\text{35}\text{46}$)-, and $\text{(}\text{34}\text{56}\text{)-hexachlorocyclohexene}$, some compounds such as 1,2,4-trichlorobenzene, 1,2,3,4-tetrachlorobenzene, and pentachlorobenzene were more abundantly formed, respectively, than 2,3,4,6-tetrachlorophenol. These oxidative metabolic reactions were shown to mainly proceed via “ene-like” hydroxylation accompanied by double bond migration. Inhibition by CO, piperonyl butoxide, and SKF 525-A suggested that the “ene-like” hydroxylating enzyme was cytochrome P-450 dependent. The formation of an isomer of pentachlorocyclohexenol from $\text{(}\text{36}\text{45}\text{)-hexachlorocyclohexene}$ was also observed, and this reaction was activated by SKF 525-A.     

Results of metabolic profile test in Japanese black cattle with growth retardation

To evaluate the nutriture of Japanese black cattle with growth retardation, a metabolic profile test was carried out in 8 cattle with growth retardation and in 10 cattle with normal growth. During our observation for 1 month before blood sampling, the cattle with growth retardation ingested their forage completely. They showed lower low-density lipoprotein and albumin concentrations, and higher urea nitrogen, actoacetic acid and beta-hydroxybutyric acid concentrations than the control. There were no significant differences in glucose, free fatty acid, total cholesterol, ammonia, inorganic phosphorus, and calcium concentrations between the cattle with growth retardation and the control. These data suggested that the cattle with growth retardation subjected to energy-negative condition in spite of their good forage intake.     

Ultrastructural study of uterine natural killer cells found in pregnant, interleukin-2 receptor beta-chain overexpressed transgenic mice

We previously reported that all fetuses died or were resorbed on day 12 of pregnancy (Day 1= the day of plug) in interleukin-2 (IL-2) receptor beta-chain overexpressed transgenic (Tg2Rbeta) mice. In this study, to clarify the role of uterine natural killer (uNK) cells in pregnancy, the ultrastructure of Tg2Rbeta mouse uNK cells was analyzed using a transmission electron microscope. uNK cells and their granules on day 10 of pregnancy were larger in Tg2Rbeta mice than control mice, indicating that differentiation of uNK cells in Tg2Rbeta mice progressed rapidly. Additionally, the granules of uNK cells in Tg2Rbeta mice on day 10 of pregnancy had an irregular morphology. The multivesicular regions were present in the cap structure of these granules, suggesting that the uNK cells of the Tg2Rbeta mice had cytotoxic activity.     

Distribution of lignin and lignin precursors in differentiating xylem of Japanese cypress and poplar

Lignin is an integral component of the cell wall of vascular plants. The mechanism of supply of lignin precursors from the cytosol into the cell wall of differentiating xylem has not yet been elucidated. The present study showed that a certain amount of coniferyl alcohol glucoside (coniferin) occurred in the differentiating xylem of Japanese cypress (Chamaecyparis obtusa), as previously reported in gymnosperms. Coniferin content peaked in the early stages of secondary wall formation and decreased during lignification. In contrast to gymnosperms, coniferin content was limited in the differentiating xylem of poplar (Populus sieboldii × Populus grandidentata). Moreover, coniferyl alcohol was not detected in all specimens. In the differentiating xylem of poplar, a higher amount of sinapyl alcohol occurred than glucoside (syringin). However, the phloem contained syringin and not sinapyl alcohol. The sinapyl alcohol content in the xylem peaked in the cells with ceasing cell wall formation, and decreased gradually towards the boundary of the annual ring, where the lignin content kept increasing. Sinapyl alcohol in the differentiating xylem of poplar may be used for the lignification of the xylem.     

Vaginal rhabdomyosarcoma in a dog

A 10-year-old, female, mongrel showed hemorrhage from vulva. By magnetic resonance image (MRI) and endoscopic examination, a multipapillary mass with a grape-like appearance was found around the urethral opening. Histologically, the mass consisted of variable-sized round-, spindle-to-polygonal-shaped tumor cells including many multinuclear cells. Mitotic figures were also frequently observed. In some areas, that tumor cells were loosely arranged, with intercellular myxoid components. Immunohistochemically, these tumor cells were strongly positive for vimentin and focally positive for desmin but negative for myoglobin. Thus, the case was diagnosed as a relatively poorly differentiated botryoid rhabdomyosarcoma by the macroscopic, histopathologic, and immunohistochemical identification. This is the first report of botryoid rhabdomyosarcoma developing in the vagina of a dog.     

Distinct antigen recognition pattern during zoonotic visceral leishmaniasis in humans and dogs

Leishmania infantum is a causative agent of endemic zoonotic visceral leishmaniasis (VL) in regions of South America and the Mediterranean. Dogs are the major reservoirs for L. infantum in these regions, and control of disease in dogs could have a significant impact on human disease. Although dogs share many symptoms of VL with humans as a result of L. infantum infection, they also show some unique clinical manifestations, which are often a combination of visceral and cutaneous leishmaniasis, suggesting different mechanisms of disease development in dogs and humans. Here, we compare antibody responses of dogs and humans with VL to various defined leishmanial antigens. Parasite lysate and K39, the two most commonly used antigens for serodiagnosis of VL, detected the highest levels of antibodies in both humans and dogs with VL, whereas the recognition patterns of these antigens were distinct between the hosts. Among other defined antigens tested, LmSTI1 and CPB detected higher levels of antibodies in dogs and humans, respectively. These results indicate there is a difference between humans and dogs in antigen recognition patterns during VL. We infer that different strategies may need to be used in development of vaccines and diagnostics for humans and for dogs. In addition, we show a correlation between antibody titers to several antigens and severity of clinical symptoms during canine VL.     

Ultrasonographic imaging of abomasal curd in preruminant calves

The aim of this study was to determine whether ultrasonography could be used to evaluate curd formation in the abomasum of preruminant calves. Holstein-Friesian calves were fed one of three milk replacers: clotting (five calves), non-clotting (four calves) and pH-dependent clotting (clots form at pH 5.5, but not at pH 6.5; six calves). Ultrasonography was performed until 6h after feeding the milk replacers. In calves fed the clotting milk replacer, a large clot of curd was visualised by ultrasonography as a clearly outlined echogenic image and whey as an anechoic image. In calves fed the non-clotting milk replacer, abomasal contents were visualised as a uniform, entirely echogenic image, indicating the absence of curd formation. In calves fed milk replacer with pH-dependent clotting properties, several small curds and whey were visualised by ultrasonography. It was concluded ultrasonography can be used to visualise abomasal curd and to distinguish the presence and absence of curds in the abomasum of calves.     

Immunohistochemical localization of metallothionein in plant tissues

The localization of metallothionein ( MT ) in the seeds and roots of soybean was investigated by immunohistochemistry. The germinating seeds at 2 hr, 1, 2, 3, 4 and 6 d including 1-mo root tips of soybean ( c.v. Toyosuzu ) with and without heavy metals ( Cu 400 μg Lor Zn 3 μg ml^?1) treatment were used to demonstrate the localization of MT by the indirect immunoperoxidase technique using polyclonal rabbit antirat MT conjugated to ascaris as a primary antibody. Metallothionein was localized in the proliferating regions such as the embryo in seeds, and root and shoot apices of both the control and heavy metals-treated plants. The intensity of MT staining in the proliferating regions generally increased as the soybean seeds germinate. Starting at about 1 day after germination, MT was found in the veins and vascular bundles suggesting its translocation to other organs. Similar observation hold true in the case of plants treated with heavy metals. This means that heavy metals treatment had no effect on MT localization. However, the heavy metals-treated plants showed higher concentration of MT over the control with respect to the growth stage of soybean seeds. These indicate that MT found in soybean plays a physiological role in heavy metal transport, detoxification and cell division in a similar manner to mammalian MT.