N-acetylglucosaminyltransferase I activity of bovine oviduct epithelial cells: stimulation by luteinizing hormone, vascular endothelial growth factor and tumor necrosis factor alpha

N-acetylglucosaminyltransferase I (GnT I; EC 2.4.1.101), which catalyzes the first step in the conversion of oligomannose to complex or hybrid N-glycans of glycoproteins, was found in media cultured with bovine oviduct epithelial cells (BOEC) obtained from non-pregnant cows during the follicular phase. Combined treatment with specific hormones increased GnT I release from BOEC. Luteinizing hormone (LH; 10 ng/ml) alone slightly, but together with 17beta-estradiol (E2; 1 ng/ml), synergistically increased GnT I activity. Vascular endothelial growth factor (VEGF) and tumor necrosis factor (TNF) alpha, which have been shown to have their highest activities in the bovine oviduct during the periovulatory period, also increased in GnT I activity. This study provides the first evidence of an increase of GnT I release from BOEC in vitro, and shows that endocrine as well as local factors such as LH, VEGF and TNFalpha increase this activity. The results suggest that GnT I activity in the bovine oviduct may contribute to the induction of glycosylation and thereby contributing to the provision of the optimal microenvironment for fertilization and early development of the embryos.     

Antigenic analysis of avian Chlamydia psittaci using monoclonal antibodies to the major outer membrane protein.

Monoclonal antibodies to the major outer membrane protein (MOMP) of Chlamydia psittaci derived from a parrot were established for antigenic analysis of avian C. psittaci. With 17 monoclonal antibodies to MOMP, 17 reactivity patterns were identified on 112 strains of C. psittaci, C. pneumoniae and C. trachomatis, which were isolated from birds, mammals and humans in Japan, U.S.A., Canada and Taiwan, from 1938 to 1987. Immunological reactivity of budgerigar-derived strains to the monoclonal antibodies was different from that of pigeon-derived strains. Imported bird-derived strains were distinguishable from domestic bird-derived strains by the reactivity to the monoclonal antibodies. A close relationship between the subtypes and geographic origins was indicated on budgerigar-derived strains. On the contrary, various reactivity patterns were shown in pigeon-derived strains isolated in a narrow area. The monoclonal antibodies established in the present work may be useful probes for ecological study of avian C. psittaci.     

Incidence of silent ovulation in dairy cows during post partum period

The present study was undertaken to show the incidence of silent ovulation in high producing dairy cows, by monitoring ovarian cyclicity based on practical milk progesterone assay which was established in this study. The direct milk progesterone enzyme linked immunosorbent assay (ELISA) was developed using anti-progesterone-3(E)-carboxymethyloxime-BSA antibody for the antibody and horse radish peroxidase labeled progesterone for tracer. High sensitivity (26 pg/mL, 0.65 pg/well), high recovery rates (83-97%), high reproducibility (CV of intra-assay 4.8-11.5%; CV of inter-assay 14.3-19.1%) and high correlation between milk progesterone concentrations measured by the direct ELISA and the values obtained by the ELISA after extraction proved the reliability of the assay. In second experiment the incidence of silent ovulation was investigated based on the milk progesterone concentrations in 32 dairy cows within 70 days post partum. The incidences of silent ovulation at the first, second, third and fourth ovulation post partum were 83, 46, 13 and 0%, respectively. Most commonly observed patterns of sequential occurrence of silent ovulation in cows ovulating 2, 3 or 4 times within 70 days post partum were silent-estrus (50%), silent-silent-estrus (60%) and silent-silent-estrus-estrus (67%), respectively. These results suggest that the present ELISA is a reliable and practically applicable method for determination of progesterone in milk and that high producing dairy cows show a high incidence of silent ovulation at the first post partum ovulation as well as the second ovulation, which then decreased with the increased frequency of ovulation.     

A novel repeated sequence located on the bovine Y chromosome: its application to rapid and precise embryo sexing by PCR

A novel repeated sequence specific to male cattle was identified and named S4. S4 is a highly repetitive sequence and is a 1.5 kb repeating unit that contains various internal repeated sequences. FISH analysis showed that S4 is localized on the whole long arm and the proximal region of the short arm of the Y chromosome. We found that a PCR primer set for S4 amplified a male-specific 178 bp product in addition to a 145 bp product common to both male and female cells. Although the origin of the 145 bp product is unknown, it acts as a positive internal control in practical embryo sexing. Due to the high copy number of S4, PCR required only 0.5 pg purified DNA for accurate amplification. This made it possible to reduce the amount of biopsy sample required for embryo sexing and thus result in less damage to embryos manipulated. These studies indicate that embryo sexing based on the S4 sequence is accurate and sensitive.     

Chicken peripheral blood CD3+CD4-CD8- cells are regulated by endocrine and nerve systems

The existence of CD3(+)CD4(-)CD8(-) T cells in thymus and spleen has already been known. However, because of the presence of large amounts of thrombocytes in peripheral blood (PB), the proportion of CD3(+)CD4(-)CD8(-) T cells in PB has yet to be investigated. Therefore, the proportion of peripheral T cell-subsets was investigated in 6-week-old chickens. The percentage of CD3(+) cells, CD4(+) cells, CD8 alpha(+) cells, CD8 beta(+), and CD3(+)CD4(-)CD8(-) cells was 76%, 41%, 14%, 5%, and 15%, respectively. The proportion of CD3(+)CD4(-)CD8(-) cells in PB increased during egg-laying periods and in chickens treated with an analog of estrogen, while it decreased with age and in response to restraint stress. All of the CD3(+)CD4(-)CD8(-) cells expressed TCR1, and did not have NK activity. CD3(+)CD4(-)CD8(-) cells represent about 60% of peripheral TCR1(+) cells. These findings indicate that the proportion of CD3(+)CD4(-)CD8(-) cells is regulated by the endocrine and nerve systems.     

Maternal exposure to low doses of bisphenol a has no effects on development of female reproductive tract and uterine carcinogenesis in Donryu rats

Effects of maternal exposure to low doses of bisphenol A (BPA), including those comparable with human exposure levels, on growth and development of the female reproductive system and uterine carcinogenesis in Donryu rats were investigated. Dams were administered BPA (0, 0.006 and 6 mg/kg/day) daily by gavage from gestation day 2 up to the day before weaning (postnatal day 21 at offspring). The serum levels of BPA were significantly elevated in the dams receiving 6 mg/kg/day, however, BPA levels in the milk of dams, and those in the serum and liver of offspring were similar between control and treated groups. The treatment did not exert any influences on uterine development including weight, gland genesis and estrogen receptor alpha expression, vaginal opening and gonadotropin secretion in the female offspring up to puberty. After maturation, no effects were evident with regard to estrous cyclicity in female offspring treated with BPA. In addition, the treatment had no effects on age-related morphological changes of the reproductive and endocrine organs and uterine carcinogenesis until 15 months of age. The results demonstrate that maternal exposure to BPA at levels comparable to human exposure did not have any effects on the female reproductive system of offspring in rats. In addition, BPA was also found in the serum, milk and liver of control dams and pups, and low levels of BPA were detected in drinking water and pellet diet. The present study showed that the experimental animals were also exposed to environmental BPA in the animal room.     

Design of hammerhead ribozymes that cleave murine Sry mRNA in vitro and in vivo

As the first step in investigating the possiblity of applying ribozyme technology to artificial control of the sex ratios at birth in farm animals, where the demand for females exceeds that for males, we designed a hammerhead ribozyme (HHRz) and 2 tRNA(val)-hammerhead ribozyme complexes (tRNARz3 and tRNARz4), and examined their effects upon murine Sry mRNA in vitro and in cells. We demonstrated that HHRz and tRNARz3 could effectively cleave the target Sry mRNA in vitro. For the purpose of experiments in vivo, HHRz was cloned into the highly efficient pUC-CAGGS mammalian expression vector (pCAG/HHRz), and the tRNA ribozyme complexes were cloned into the pol III promoter-driven pPUR-KE vector (pPUR/tRNARz3 and pPUR/tRNARz4); the ribozyme vectors were co-transfected with the target vector (pCAG/Sry). A suppressive action (up to approx. 60%) was confirmed for pCAG/HHRz and pPUR/tRNARz3 upon the transiently expressed exogenously introduced Sry in M15 cultured cells.     

Characterization of a new Maillard type reaction product generated by heating 1-deoxymaltulosyl-glycine in the presence of cysteine

The reaction between Amadori compounds and cysteine was investigated. When 1-deoxymaltulosyl-glycine (glycyl-fructosyl-glucose) was heated at 100 degrees C with cysteine in a neutral aqueous solution, a novel intermediate composed of 1-deoxyosone and cysteine was detected. NMR and mass spectrometry studies revealed the structure of the isolated intermediate to be 7,8a-dihydroxy-4a-methyl-8-(alpha-d-glucopyranosyloxy)hexahydro-5-oxa-4-thia-1-azanaphthalene-2-carboxylic acid. This intermediate easily generated isomaltol and acetylfuran as volatile compounds in 1 mol/L HCl at 100 degrees C.