Expression of inhibins, activins, insulin-like growth factor-I and steroidogenic enzymes in the equine placenta

In this study, the expression patterns of inhibins, activins, insulin-like growth factor-I (IGF-I) and steroidogenic enzymes in equine placentae recovered during the latter two-thirds of gestation were examined. Concentrations of inhibin A and inhibin pro-alphaC in endometrial and fetal placental tissue homogenates were very low during the period examined, whereas these tissues contained high concentrations of activin A. In both maternal endometrial and fetal placental tissues, activin A levels decreased as pregnancy progressed. Expression of inhibin alpha-subunit was not observed in the placenta using either immunohistochemistry or in situ hybridization. Inhibin/activin betaA-subunit and its mRNA were confined to maternal endometrial glands, whereas immunopositive betaB-subunit was not detected in either endometrial glands or microcotyledons. Cytochrome P450 side chain cleavage enzyme was detected by immunohistochemistry in both endometrial glands and microcotyledons, whereas cytochrome P450 17alpha-hydroxylase/lyase was absent in these tissues. Immunopositive signals for 3beta-hydroxysteroid dehydrogenase and cytochrome P450 aromatase were localized in microcotyledons but not in endometrial glands. Immunohistochemistry revealed that IGF-I was highly expressed in microcotyledons around Day 130, and decreased as pregnancy progressed. Changes in the expression of IGF-I were correlated with the number of PCNA positive cells in the placenta. The present study demonstrated the presence and localized the site of expression of activin, IGF-I and steroidogenic enzymes in equine placental tissues during the latter two-thirds of gestation; the results suggest that activin and IGF-I may be involved in the regulation of placental development.     

In vitro and in vivo developmental ability of oocytes derived from porcine primordial follicles xenografted into nude mice

We evaluated the developmental ability of oocytes in porcine primordial follicles xenografted into nude mice. Ovarian tissues from 20-day-old piglets, in which most of the follicles were primordial, were transplanted under the kidney capsules of ovariectomized nude mice. Forty-nine to 89 days after grafting (mean +/- SEM, 66.9 +/- 1.9 days; n = 64), the host mice showed the presence of cornified epithelial cells in their vaginal smears for the first time. The mice were then treated with 4 IU of equine chorionic gonadotropin (eCG) 60 days after first detection of vaginal cornification. Oocytes were collected from the host mice 48 h after treatment with eCG, and then matured. The maturation rates, based on the incidence of first polar body, ranged from 25.1% to 42.5%. They were then fertilized in vitro and cultured in vitro for 6 days, or transferred into estrous-synchronized recipients and recovered after 6 days. On Day 6 of culture, 15.4% of the matured oocytes had cleaved to the 2- to 8-cell stage. However, neither the embryos cultured in vitro nor those transferred and recovered developed to advanced embryonic stages, such as morulae or blastocysts. This result suggests that the developmental ability of xenografted oocytes is insufficient, even after in vitro maturation. Further strategies, such as improvement of hormonal treatment for host mice, are required to enable oocytes in xenografted ovarian tissues to acquire the cytoplasmic maturation necessary for embryonic development.     

Footpad reaction induced by Neospora caninum tachyzoite extract in infected BALB/c mice

Little information is available regarding a delayed type hypersensitivity (DTH) reaction in neosporosis. In this study, we examined the elicitation of a DTH reaction in mice infected with Neospora caninum by inoculation of the footpad with tachyzoite antigens. The footpads of BALB/c mice infected with N. caninum and those of non-infected were injected with either the tachyzoite extract, or paraformaldehyde-fixed tachyzoites. In mice inoculated with N. caninum antigens on day 7 p.i. swelling peaked at 6h after injection of the tachyzoite extract. In mice inoculated on days 14, 28 and 56, swelling was observed between 6 and 72 h afterwards. Mice immunized with the tachyzoite extract plus adjuvant showed peak footpad swelling at 6h post injection, and the swelling had decreased at 24h or later. In contrast, mice injected before infection showed no specific swelling. In sections of footpads injected with the tachyzoite extract, exudate had accumulated at 6h post injection and clusters of infiltrated lymphocytes were observed at 48 h post injection. In mice administered anti-CD4+ cell monoclonal antibodies swelling had decreased at 24h post injection of the extract. These results indicate that mice infected with N. caninum produce a DTH reaction, which is a good indicator of the development of type 1 immune responses.     

A novel genetic marker to differentiate feline herpesvirus type 1 field isolates

Five recent field isolates of feline herpesvirus type 1 (FHV-1) were compared by digestion with a restriction endonuclease, SalI or MluI. The SalI digestion showed a potentially useful difference in one isolate 00-035 that had an approximately 3.0 kbp fragment instead of a 2.6 kbp fragment in the other strains. After cloning the 3.0 and 2.6 kbp fragments, the nucleotide sequences were analyzed. The result showed that the 3.0 kbp fragment of 00-035 included a complete open reading frame of the herpes simplex virus 1 (HSV-1) homologue of the UL17 gene and a 5'-part of UL16 gene and that only one nucleotide substitution was found in the 5'-region of UL17 gene where the SalI site of the 2.6 kbp fragment locates. Based on these nucleotide sequences, two PCR primers were designed to amplify the region around the SalI site in the UL17 gene and the PCR was carried out using 78 field isolates from various parts of Japan. The SalI digestion of the PCR products revealed an interesting profile in that the genotype without the SalI site in UL17 gene was dominant in Tottori and Yamagata prefectures (69% and 75%, respectively) but minor in the other regions of Japan (0-10%). These results suggest that the SalI digestion method described in the present study can be used as a genetic marker to differentiate some FHV-1 field isolates and this is the first report that showed different distributions of FHV-1 genotypes using the novel genetic marker.     

Analysis of porcine cytomegalovirus DNA polymerase by consensus primer PCR

We used a consensus primer PCR method to amplify a region of herpesviral DNA-directed DNA polymerase gene using degenerate primers for initial characterization of the porcine cytomegalovirus (PCMV) genome. The sequence of the PCR product from PCMV DNA template and its alignment with other herpesvirus DNA polymerase counterparts showed that both conserved amino acid residues and conservative amino acid substitutions are in parallel. Phylogenetic analysis revealed that PCMV should be included in the clade comprising human herpesvirus 6 and 7, rather than human and mouse cytomegaloviruses, in Betaherpesvirus subfamily.     

β 1-4 mannobiose enhances Salmonella-killing activity and activates innate immune responses in chicken macrophages

Salmonella spp. is one of the major causes of food-borne illness in humans, and Salmonella enteritidis (SE) infection in commercial poultry is a world-wide problem. Here we have investigated the in vitro immune-modulating effects of β 1-4 mannobiose (MNB), which was previously found to prevent SE infection in vivo in chickens, using chicken macrophage (MQ-MCSU) cells. Treatment of MQ-NCSU cells with MNB dose-dependently increased both phagocytic activity and Salmonella-killing activity of macrophages, with the highest reduction in SE viability observed at a concentration of 40 μg/ml at 48 h post-infection. Likewise, both hydrogen peroxide (H(2)O(2)) and nitric oxide (NO) production were increased in a dose-dependent manner by MNB. Gene expression analysis of MNB-treated macrophages revealed significant increases in the expression of iNOS, NOX-1, IFN-γ, NRAMP1, and LITAF, genes critical for host defense and antimicrobial activity, when compared to untreated cells. This data confirms that MNB possesses potent innate immune-modulating activities and can up-regulate antibacterial defenses in chicken macrophages.     

Effect of the strain combination of the donor and recipient on the production efficiency of W‐bearing sperm in mixed‐sex germline chimeric chickens

Effect of the strain combination of the donor and recipient on production efficiency of W‐bearing sperm in mixed‐sex chimeric testes was analyzed. The combinations of the donors and recipients were White Leghorn (WL) and Rhode Island Red (RIR), and vice versa. Generated mixed‐sex chimeras that had the male phenotype at sexual maturity were classified into four groups: (1) a female WL donor and a male RIR recipient; (2) a male WL donor and a female RIR recipient; (3) a female RIR donor and a male WL recipient; (4) a male RIR donor and a female WL recipient. The mean number of W‐bearing sperm detected by in situ hybridization among 10 000 sperm observed were 147, 165, 30 and 45 in groups 1, 2, 3 and 4, respectively. The numbers in groups 1 and 2 were both significantly higher than those of groups 3 and 4 (P < 0.05). The combination of a WL donor and a RIR recipient produced W‐bearing sperm more efficiently than the reverse combination.     

Infrared spectroscopic study of alkaline oxygen treatment of lignin with ATR technique in aqueous state 1: method for determining quantitative spectra of oxygen-degraded lignin

For the fundamental study of oxygen delignification of kraft pulp, structural changes of kraft lignin during alkaline oxygen treatment were investigated with the use of infrared measurement with attenuated total reflectance (ATR) technique. In the neutralized reaction mixture of alkaline oxygen-treated kraft lignin, there is a significant amount of NaCl, so that the spectral changes of water due to the coexistence of NaCl was investigated, and how to remove the huge absorption of NaCl solution is discussed. Sodium vanillate–NaCl solutions were employed as model solutions for the reaction mixture. Partial least square (PLS) regression was applied for the prediction of NaCl concentration, and the spectrum of NaCl solution was subtracted from the spectrum of sodium vanillate–NaCl solution as background measurement. This allowed us to obtain the vanillate spectra free from the absorption of NaCl solution. In addition, the mathematical method for reconstructing the spectrum of NaCl solution is discussed. The spectrum of NaCl solution is reconstructed as the linear combination of basic spectra calculated by singular value decomposition (SVD), and it was subtracted from that of the sodium vanillate–NaCl solution. By this procedure, the vanillate spectra were also obtained quantitatively, as has been demonstrated in PLS regression study. It was also confirmed that the quantitative spectra of high molecular weight fraction of alkaline oxygen-treated kraft lignin were obtained by the use of this reconstruction technique.Parts of this report were presented at the 52nd Annual Meeting of the Japan Wood Research Society, Gifu, Japan, April 2002 and the 12th International Symposium on Wood and Pulping Chemistry, Madison, USA, June 2003     

Purification of N-acetyllactosamine-binding activity from the porcine sperm membrane: possible involvement of an ADAM complex in the carbohydrate-binding activity of sperm

Although the importance of carbohydrate recognition by sperm during egg zona pellucida binding has been widely reported, the sperm molecular species that recognize the carbohydrates are poorly characterized. Our previous cytochemical study indicated that two kinds of carbohydrate-binding proteins are expressed on porcine sperm heads-one recognizes N-acetyllactosamine (Galβ1-4GlcNAc-), and the other recognizes the Lewis X structure (Galβ1-4(Fucα1-3)GlcNAc-). For this report, we used proteomic techniques to characterize the sperm proteins that bind N-acetyllactosamine. Porcine sperm plasma membrane was solubilized with a detergent solution and subjected to sequential chromatography with dextran sulfate agarose, affinity, and hydroxyapatite, and the binding activities in the eluates were monitored by a solid-phase binding assay. The tryptic peptides of two proteins most likely associated with the binding activities were subjected to tandem mass spectrometry sequencing. A subsequent database search identified one of the two proteins as predicted disintegrin and metalloprotease domain-containing protein 20-like (XP_003128672). The other protein was identified as disintegrin and metalloprotease domain-containing protein 5 (AB613817) by database searches for homologous amino acid sequences, cDNA cloning, nucleotide sequencing and nucleotide database searches. Furthermore, two-dimensional blue native/SDS-PAGE demonstrated that they formed a variety of non-covalent complexes. Therefore, these ADAM complexes probably are responsible for the N-acetyllactosamine-binding activity. An affinity-purified fraction containing these ADAM complexes showed zona pellucida-binding activity, though the activity was relatively weak, and the presence of another zona pellucida-binding protein that probably works in concert with these ADAM complexes was suggested. Immunofluorescence testing suggested that ADAM20-like was localized on the anterior part of the sperm plasma membrane.