Affected homologous chromosome pairing and phosphorylation of testis specific histone, H2AX, in male meiosis under FKBP6 deficiency

A gene for FK506 binding protein 6 (Fkbp6) expresses during a specific stage of male and female meiosis. Disruption of the gene influences male reproduction, i.e. arrests spermatogenesis, but not female reproduction. Using the mouse model (targeted disruption), the role of the gene in homologous chromosome pairing has been demonstrated in a previous study. For further understanding the function of Fkbp6 in chromosome synapsis, we evaluated chromosome pairings during male meiosis in the as/as rat, a spontaneous null mutation, and compared them with those of the mouse model. Electron microscopy of the pachytene nuclei unveiled several types of abnormal chromosome pairing in the rat model, as shown in the mouse previously. The frequencies of aberrant pairings in the knockout mice and mutant rats were 42 of 67 nuclei (62.7%) and 20 out of 74 nuclei (27.0%), respectively. In order to clarify the mechanism of male specific infertility in Fkbp6 deficiency, the localization of gammaH2AX, a marker protein of XY chromosome inactivation during male meiosis, was examined. Immunostaining of gammaH2AX unveiled normal localization of the molecule to XY chromosomes (XY body) in both models, showing the independency of FKBP6 in sex chromosome inactivation. Besides the XY body, focal localization of gammaH2AX was observed in accordance with the unsynapsed chromosomes in both types of null animal. These results indicate the fundamental role of Fkbp6 in homologous chromosome synapsis during male meiosis. In conclusion, male specific infertility under Fkbp6 deficiency remains unsolved.     

Retrospective diagnosis of feline GM2 gangliosidosis variant 0 (Sandhoff-like disease) in Japan: possible spread of the mutant allele in the Japanese domestic cat population

GM2 gangliosidosis variant 0 (human Sandhoff disease) is a lysosomal storage disease caused by simultaneous deficiencies of acid beta-hexosaminidase (Hex) A and Hex B due to an abnormality of beta-subunit, a common component in these enzyme molecules, which is coded by the HEXB gene. In the present study, a retrospective diagnosis was performed in 2 previous suspected cases of feline Sandhoff-like disease using a DNA test to detect the causative mutation identified previously in 4 cats in 2 other families of Japanese domestic cats. Enzymic analysis was also performed using stored leukocytes and plasma collected from the subject families in order to investigate the usefulness of enzymic diagnosis and genotyping of carriers. The DNA test suggested that the 2 cases were homozygous recessive for the mutation. Consequently, 6 cats homozygous for the same mutation have been found in 4 separate locations of Japan, suggesting that this mutant allele may be spread widely in the Japanese domestic cat populations. In enzymic analysis, Hex A and Hex B activities in leukocytes and plasma measured using 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide as a substrate were negligible in affected cats, compared with those in normal and carrier cats. However, there was a wide overlap in enzyme activity between normal and carrier cats. Therefore, it was concluded that enzymic analysis is useful for diagnosis of affected cats, but is not acceptable for genotyping of carriers.     

Molecular typing of sand fly species (Diptera, Psychodidae, Phlebotominae) from areas endemic for Leishmaniasis in Ecuador by PCR-RFLP of 18S ribosomal RNA gene

Surveillance of the distribution of sand fly species is important for prediction of the risk and expansion of Leishmania infection in endemic and surrounding areas. In the present study, a simple and reliable method of typing New World Lutzomyia species circulating in endemic areas in Ecuador was established by using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) technique. PCR-RFLP of 18S ribosomal RNA (rRNA) genes with the restriction enzyme AfaI and subsequently HinfI successfully identified seven sand fly species in nine endemic areas in Ecuador. Although intraspecific genetic-diversity affecting the RFLP-patterns was detected in a species, the patterns were species specific. The method promises to be a powerful tool for the classification of New World Lutzomyia species.     

Improvement of DNA extraction method for dried blood spots and comparison of four PCR methods for detection of Babesia gibsoni (Asian genotype) infection in canine blood samples

To eradicate canine babesiosis in epidemic areas, mass-screening of the infection situation of Babesia gibsoni including occult infection is necessary. The development of cost-effective method for storage and transport of blood samples is required. A highly efficient DNA extraction procedure from dried blood spots (DBS) onto Whatman 3MM filter paper was developed for the diagnosis of B. gibsoni infection in dog by PCR. In 3 extraction methods, Chelex-based method in combination with saponin washing and phenol-chloroform-isoamyl alcohol extraction (Saponin-PCI method) provided the best results. Sensitivity of the 4 previously described PCR methods for detection of B. gibsoni infection was also compared using serially diluted blood samples of B. gibsoni-infected dogs. The PCR method using Gib599F/Gib1270R primer pair provided the best performance. To evaluate the stability of DNA in DBS, DBS of B. gibsoni-infected dogs stored at room temperature for 2 months. The stability was superior to whole blood samples stored at -20 degrees C for 2 months. This highly efficient DNA extraction method on DBS using Whatman 3MM filter paper has potential to be cost-effective and high performance tool for storage, and molecular diagnosis of clinical blood sample from dog. This procedure in combination with the PCR method using Gib599F/Gib1270R primer pair may greatly assist in diagnosis of B. gibsoni infection in dog populations that are geographically distant.     

Histopathological characteristics of hepatic lipogranulomas with portosystemic shunt in dogs

In the canine liver with portosystemic shunts (PSS), focal lesions consisting of cells with cytoplasmic brown pigments and lipid vacuoles are often observed in the hepatic parenchyma. Termed lipogranulomas, their histopathological characteristics have been little studied. In the present study, we examined liver biopsy samples from 144 dogs (age: 3 months-16 years; 65 PSS and 79 non-PSS cases), and investigated the histopathological characteristics, incidence, and density of lipogranulomas. Lipogranulomas were detected histopathologically in 55.4% of PSS dogs. The lesions were then grouped into 3 types according to the amount of cytoplasmic lipid vacuoles and brown pigments. The pigments were positive for Berlin blue, PAS, and Sudan black B, but negative with the Hall method. The majority of the cells were immunohistochemically positive for macrophage scavenger receptor, class A (MSR-A), while no cells were positive for hepatocyte-antigen and albumin. The cytoplasmic pigments were recognized as electron-dense microgranular materials by electron microscopy. The incidence of lipogranulomas was significantly higher in the PSS group than non-PSS group when dogs less than 1 year old were excluded. The lipogranuloma density in the liver was significantly higher in the PSS group. It is concluded that lipogranulomas are frequently observed in liver biopsies of canine PSS especially in dogs more than 1 year old. The lesions consisted of Kupffer cells and/or macrophages, and the cytoplasmic brown pigments are ceroid and hemosiderin. The pathogenesis of lipogranuloma in PSS needs to be clarified.     

Parent‐of‐origin effects on carcass traits in Japanese Black cattle

Variances caused by the differential expression of paternally and maternally imprinted genes controlling carcass traits in Japanese Black cattle were estimated in this study. Data on marbling score (BMS), carcass weight, rib thickness, rib‐eye area (REA) and subcutaneous fat thickness (SFT) were collected from a total of 13,115 feedlot steers and heifers in a commercial population. A sire–maternal grandsire model was used to analyse the data, and then, imprinting parameters were derived by replacing the genetic effect of the dam with the effect of the maternal grandsire in the imprinting model to calculate the genetic parameter estimates. The proportions of the total genetic variance attributable to imprinted genes ranged from 8.7% (SFT) to 35.2% (BMS). The remarkably large imprinting variance of BMS was mainly contributed by maternally expressed inheritance because the maternal contribution of the trait was much larger than that of the paternal trait. The parent‐of‐origin effect originating from maternal gene expression was also observed for REA. The results suggested the existence of genomic imprinting effects on the traits of the Japanese Black cattle. Hence, the parent‐of‐origin effect should be considered for the genetic evaluation of Japanese Black cattle in breeding programmes.     

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.     

Production of androgenetic amago salmon<Emphasis Type="Italic"> Oncorhynchus masou ishikawae</Emphasis> with dispermy fertilization

With the aim of improving artificial androgenesis in teleost fishes, we tested two methods for producing androgenetic diploids of amago salmon (Oncorhynchus masou ishikawae), namely, fertilization of gamma-ray irradiated eggs with fused spermatozoa (sperm-fusion method) and the fertilization of irradiated eggs with untreated sperm followed by the blocking of cell division (mitosis-inhibition method). Our results showed that the optimal condition for sperm fusion was to treat the sperm with 50% polyethylene glycol (molecular weight 7500) for 100 s. The efficiency of the two methods of androgenesis was compared in terms of fertilization rate, hatching rate, and larval survival after hatching. The rate of fertilization was lower with the sperm-fusion method than with the mitosis-inhibition method, but the reverse was true for the hatching rate. The survival rate of hatched larvae was the same with the two methods. Androgenesis was confirmed with a recessive albino color marker, and all viable offspring were found to be heterozygous based on analysis of the microsatellite markers. Our results suggest that androgenesis with the sperm-fusion method is a promising approach with potential applications in both aquaculture breeding programs and the preservation of endangered freshwater fishes.     

Nutritional evaluation of rotifers in rearing tanks without water exchange during seed production of amberjack <Emphasis Type="Italic">Seriola dumerili</Emphasis>

We evaluated the nutrient contents of rotifers sampled from larval-rearing tanks (tank rotifers) without water exchange during the seed production of amberjack Seriola dumerili at three facilities (Kamiura, Kagoshima, and Miyazaki) and compared them with the nutrient contents of freshly enriched rotifers. Compared to the enriched rotifers, the lipid contents, especially neutral lipids and proportion of 22:6n-3, tended to decrease in the tank rotifers. These trends were clearer at Miyazaki where the tank rotifers were sampled before daily supplementation of microalgae (Nannochloropsis). Crude protein content of the tank rotifers did not decrease markedly although the proportion of lysine tended to decrease. Vitamin C and E contents of the tank rotifers decreased significantly only at Miyazaki. Calcium content of the tank rotifers increased at Kamiura and Miyazaki, and the increases in iron and manganese contents of the tank rotifers at Miyazaki and zinc content at Kagoshima were pronounced. These results suggest that the nutritional value of rotifers in larval-rearing tanks without water exchange can be maintained by appropriate supplementation of microalgae. The effect of certain minerals that became high in tank rotifers on subsequent larval development requires further investigation.