Regional variations in the distribution of small intestinal intraepithelial lymphocytes in three inbred strains of mice

The regional variation in the intraepithelial lymphocytes (IELs) in the small intestine was examined in BALB/c male and female mice and C3H/He and C57BL/6 male mice. The small intestines were taken from 11 to 12-week-old mice and divided equally into 3 parts (the proximal, middle and distal parts). IELs were isolated from each part of the intestine and analyzed with flow cytometer. The number of IELs was highest in the proximal part and lowest in the distal part. The distribution of IEL subsets was markedly different between the proximal and the distal parts, and that in the middle part showed the intermediate pattern. The percentage of alphabeta T cells were higher in the distal part. In alphabeta T cell subset, the percentage of CD8alphaalpha T cells was higher in the proximal part, whereas those of CD4 and CD4CD8alphaalpha double positive T cells were higher in the distal part. In gammadelta T cell subset, no regional variations were found. The regional variations in the number and subsets of IELs showed almost the same patterns between male and female BALB/c mice and similar patterns among three strains of mice. This strongly suggests that the regional variations in the small intestinal IELs are common to mouse species.     

Utilization of digital differential display to identify differentially expressed genes related to rumen development

This study aimed to identify the genes associated with the development of the rumen epithelium by screening for candidate genes by digital differential display (DDD) in silico. Using DDD in NCBI's UniGene database, expressed sequence tag (EST)‐based gene expression profiles were analyzed in rumen, reticulum, omasum, abomasum and other tissues in cattle. One hundred and ten candidate genes with high expression in the rumen were derived from a library of all tissues. The expression levels of 11 genes in all candidate genes were analyzed in the rumen, reticulum, omasum and abomasum of nine Japanese Black male calves (5‐week‐old pre‐weaning: n = 3; 15‐week‐old weaned calves: n = 6). Among the 11 genes, only 3‐hydroxy‐3‐methylglutaryl‐CoA synthase 2 (HMGCS2), aldo‐keto reductase family 1, member C1‐like (AKR1C1), and fatty acid binding protein 3 (FABP3) showed significant changes in the levels of gene expression in the rumen between the pre‐ and post‐weaning of calves. These results indicate that DDD analysis in silico can be useful for screening candidate genes related to rumen development, and that the changes in expression levels of three genes in the rumen may have been caused by weaning, aging or both. © 2015 Japanese Society of Animal Science     

Ligand-binding characteristics of feline insulin-binding immunoglobulin G

Polyclonal immunoglobulin (Ig) G autoantibodies against insulin have been identified in sera of healthy cats. We purified and fractionated insulin-binding IgGs from cat sera by affinity chromatography and analyzed affinity of insulin-binding IgGs for insulin and their epitopes. Following the passing of fraction A, which did not bind to insulin, insulin-binding IgGs were eluted into two fractions, B and C, by affinity chromatography using a column fixed with bovine insulin. Dissociation constant (KD) values between insulin-binding IgGs and insulin, determined by surface plasmon resonance analysis (Biacore™system), were 1.64e⁻⁴ M for fraction B (low affinity IgGs) and 2e⁻⁵ M for fraction C (high affinity IgGs). Epitope analysis was conducted using 16 peptide fragments synthesized in concord with the amino acid sequence of feline insulin by an enzyme-linked immunosorbent assay. Fractions B and C showed higher absorbance (affinity) of the peptide fragment of 10 amino acid residues at the carboxyl-terminal of the B chain (peptide No. 19), followed by peptide fragments of 6 to 15 amino acid residues of the B chain (peptide No. 8). Fraction C showed a higher absorbance to 7 to 16 amino acid residues of the B chain (peptide No. 5) compared with the absorbance of fraction B. Polyclonal insulin-binding IgGs may form a macromolecule complex with insulin through the multiple affinity sites of IgG molecules. Feline insulin-binding IgGs are multifocal and may be composed of multiple IgG components and insulin.     

Reduced mitotic activity and increased apoptosis of fetal sertoli cells in rat hypogonadic (hgn/hgn) testes

Sterility in male hypogonadic (hgn/hgn) rats results from congenital testicular dysplasia caused by a single recessive gene hgn on rat chromosome 10. We recently identified an insertion mutation in the Spag5/astrin gene of hgn/hgn rats that may cause defective proliferation of immature Sertoli cells in the postnatal hgn/hgn testis. Since the pathological alterations were present in the testes at birth, we examined the involvement of defective mitosis and apoptotic cell death in embryonic development of hgn/hgn testes. Testicular hypoplasia was apparent at embryonic day (ED) 18.5. Immunostaining of hgn/hgn testes at ED 21.5 with antibody to GATA-4, which is specific for fetal Sertoli cells in the seminiferous cords, showed that the significant decrease in the number of fetal Sertoli cells was accompanied by a two fold increase in their mitotic index and abnormal mitosis and apoptosis. Prior to this, we observed a decrease in the number of BrdU-labeled cells, an increase in the number of TUNEL-positive apoptotic cells, and presence of MIS-positive apoptotic cells in hgn/hgn testes on ED 17.5 and 18.5. These results suggest that the Spag5 mutation may cause a reduction in mitotic activity and an increase in apoptosis of fetal Sertoli cells in hgn/hgn testes.     

Intermediate Filament Keratin Dynamics During Oocyte Maturation Requires Maturation/M‐Phase Promoting Factor and Mitogen‐Activated Protein Kinase Kinase Activities in the Hamster

Research related to intermediate filaments in mammalian oocytes remains poorly advanced. We investigated keratin reorganization in oocytes during meiotic maturation using immunofluorescence, and examined effects of inhibitors for cdc2 and mitogen‐activated protein kinase kinase (MAPKK) on keratin assembly. In germinal vesicle (GV) oocytes (n = 26), large and oval‐shaped aggregates of non‐fibrillar keratin were found in the cortical ooplasm (designated as a ‘cortical’ pattern). The delicate network of keratin filaments was concentrated in the GV periphery. The large keratin aggregates began to divide into small fragments at the pro‐MI/MI stage (n = 22, designated as a ‘fragmented’ pattern). Some keratin fragments were occasionally broken down into several granules at the peripheral region. In the MII oocytes (n = 24), the filament network was extended over the ooplasm and numerous keratin granules were scattered across the oocyte (designated as a ‘granular’ pattern). After 12 h of incubation with roscovitine, 66.7% of the oocytes (20/30) were at the GV stage and showed a cortical pattern of keratin. After incubation with U0126, most oocytes (83.9%, 26/31) were at the MII stage; most of them (76.9%, 20/26) showed a fragmented pattern of keratin. The increasing complexity of keratin filament network from the GV to MII stages suggests a possible role in maintaining cell integrity under physical stress after ovulation. In fact, maturation/M‐phase promoting factor is necessary for such keratin reorganization, as is meiotic nuclear progression. In addition, MAPKK is involved in keratin reorganization from a fragmented pattern to a granular pattern.     

Secretion of inhibin in female Japanese quails (Coturnix japonica) from hatch to sexual maturity

To clarify the cellular source and secretory pattern of inhibin in the Japanese quail during follicular development, the plasma concentrations of immunoreactive (ir) inhibin were measured from 1 to 7 weeks after hatching. Localization of the inhibin/activin alpha, beta A and beta B subunits was investigated by immunohistochemistry. To monitor development of the pituitary and ovarian functions, the plasma luteinizing hormone (LH) and progesterone concentrations were also measured. Ovarian weight increased gradually until 6 weeks of age and then abruptly increased at 7 weeks of age just at the onset of egg production. Plasma concentrations of LH increased significantly at 6 weeks of age. The plasma concentrations of ir-inhibin and progesterone and the pituitary contents of LH also increased significantly at 7 weeks of age. Immunohistochemically, the inhibin/activin alpha, beta A and beta B subunits were localized in the granulosa cells of all follicles during different stages of development from 1 to 7 weeks after hatching. The inhibin alpha, beta A and beta B subunits were also found in the interstitial cells but not theca cells of all follicles. These results demonstrated that the plasma concentrations of ir-inhibin of the female Japanese quails rose with ovarian development. The immunohistochemical results suggested that granulosa and interstitial cells are the major source of ovarian inhibins in female Japanese quails.     

Daily incremental lines in sika deer (Cervus nippon) dentine

This work was designed to observe the dentine incremental lines of the sika deer (Cervus nippon) fawns and to investigate their periodicity using the chronological labeling method with fluorochromes. The incremental lines were observed in decalcified specimens stained by Bodian's silver technique, and the fluorescence-labeled lines were observed in undecalcified and ground specimens. In the silver stained specimens, there were two types of lines, deeply stained thick lines and faintly stained minute regular incremental lines. The intervals and staining intensities of the deeply stained thick lines were very similar to those of the fluorescence-labeled lines in the ground specimens obtained from the same tooth, and hence, it appeared that the both lines were identical. The number of minute incremental lines between the deeply stained thick lines was the same as that of days between the time when each fluorescent labeling injection was made. Therefore, it seemed that each minute incremental line was formed each day. The possibility of age estimation in days using diurnal dentine increments was discussed.     

Mechanical transmission of porcine reproductive and respiratory syndrome virus throughout a coordinated sequence of events during cold weather

Using a field-based model, mechanical transmission of porcine reproductive and respiratory syndrome virus (PRRSV) was assessed throughout a coordinated sequence of events that replicated common farm worker behavior during cold weather (< 0°C). The model involved fomites (boots and containers), vehicle sanitation, transport, and the movement of personnel. A field strain of PRRSV was inoculated into carriers consisting of snow and water, and carriers were adhered to the undercarriage of a vehicle. The vehicle was driven approximately 50 km to a commercial truck washing facility where the driver's boots contacted the carriers during washing, introducing the virus to the vehicle interior. The vehicle was then driven 50 km to a simulated farm site, and the driver's boots mechanically spread virus into the farm anteroom. Types of containers frequently employed in swine farms (styrofoam semen cooler, metal toolbox, plastic lunch pail, and cardboard animal health product shipping parcel) contacted drippings from footwear on the anteroom floor. The truck wash floor, vehicle cab floor mats, boot soles, anteroom floor, and the ventral surface of containers were sampled to track the virus throughout the model. Ten replicates were conducted, along with sham-inoculated controls. At multiple sampling points PRRSV nucleic acid was detected in 8 of 10 replicates. In each of the 8 PCR-positive replicates, infectious PRRSV was detected on the surfaces of containers by virus isolation or swine bioassay. All sham-inoculated controls were negative. These results indicate that mechanical transmission of PRRSV can occur during coordinated sequence of events in cold weather.     

Nuclear transfer using a bovine mammary epithelial cell line (BMEC)

The developmental potential of nuclei from a bovine mammary epithelial cell line (BMEC) in nuclear transfer was investigated. For nuclear transfer donors, BMEC cells (passage 15) were cultured for 4–5 days after seeding at cell densities of 1.0 × 10⁵ cells/cm² (high‐density group) or 0.8 × 10⁴ cells/cm² (low‐density group). First, the effective electric stimulation for fusion of enucleated oocytes with BMEC cells was examined. Fusion rates reached maximum with two DC pulses of 30 V/150 µm for 20 µs in the high‐density group and with two DC pulses of 25 V/150 µm for 10 µs in the low‐density group. The fusion rate (37.5%) in the high‐density group was significantly (P < 0.005) lower than in the low‐density group (71.4%). Second, the in vitro developmental potential of nuclear transfer embryos derived from BMEC cells was examined. In the high‐density and low‐density groups, 18.8% and 24.1% of fused oocytes, respectively, developed to blastocyst stage. The results of this study indicate that nuclei from BMEC cells support the development of nuclear transfer embryos to the blastocyst stage and that the efficiency of oocyte–cell fusion is affected by the culture conditions of the donor BEMC cells before nuclear transfer.     

A Case of Feline Gastrointestinal Eosinophilic Sclerosing Fibroplasia

Feline gastrointestinal eosinophilic sclerosing fibroplasia was diagnosed in an 8-month-old Scottish fold that had a primary gastrointestinal mass involving the stomach, duodenum and mesenteric lymph nodes. Histopathologically, the most characteristic feature of this mass was granulation tissue with eosinophil infiltration and hyperplasia of sclerosing collagen fiber. Immunohistochemically, large spindle-shaped cells were positive for smooth muscle actin and vimentin. This case emphasizes the importance of feline gastrointestinal eosinophilic sclerosing fibroplasia as a differential diagnosis of gastrointestinal neoplastic lesions such as osteosarcoma and mast cell tumor in cats.