The aim of this study was to compare the effects of spermatozoa separation techniques on sperm quality and in-vitro fertilization (IVF) results for cryopreserved bovine semen. Sephadex, glass wool and Percoll gradient separation techniques were used for sperm separation and sperm motility, morphology and membrane integrity were evaluated before and after separation. Also, cleavage and blastocyst developmental rate were investigated after IVF with sperm recovered by each separation technique. The motility of samples obtained by the three separation techniques were greater compared to the control samples (p < 0.05). The percentage of spermatozoa with intact plasma-membrane integrity, identified by 6-carboxyfluoresceindiacetate/propidium iodide fluorescent staining and the hypo-osmotic swelling test, was highest in the glass wool filtration samples (p < 0.05). The cleavage and blastocyst rate of total oocytes produced from glass wool filtration samples were also higher than the control and Sephadex filtration samples (p < 0.05), but were not significantly different from Percoll separation samples. However, a significantly greater number of cleaved embryos produced by glass wool filtration developed to blastocyst stage than those produced by Percoll separation (p < 0.05). These results indicate that spermatozoa with good quality can be achieved by these three separation techniques and can be used for bovine IVF. In particular, it suggests that glass wool filtration would be the most effective method of the three for improving sperm quality and embryo production for cryopreserved bovine spermatozoa.
Animals ; Cattle ; Cryopreservation/methods/*veterinary ; Cytological Techniques/methods/*veterinary ; *DEAE-Dextran ; Female ; Fertilization in Vitro/methods/*veterinary ; *Glass ; Male ; Semen Preservation/methods/*veterinary ; Spermatozoa/*physiology ; Zygote/cytology

BACKGROUND: Chlamydia pneumoniae, a new species of the obligate intracellular Chlamydia, has been recognized as a significant pathogen that causes infection of the human respiratory tract and has recently been associated with coronary atherosclerosis. Diagnosis of infections with C. pneumoniae is problematic, because the syndrome usually presents few distinguishing features and culture of the organism is far more difficult than other Chlamydia species. To further improve the cell culture isolation and passage of C. pneumoniae organisms. we have studied several chemical and physical factors that might affect their viability and growth. METHODS: C. pneumoniae strain (TW-183) was obtained from the Centers for Disease Control, Atlanta. Ga. First we compared McCoy HeLa-229, and HEp-2 cells in the search for a more efficient and practical cell culture system. The growth rate of C. pneumoniae was assessed by the effects of diethylaminoethyl-dextrin, by the adequate centrifugation force and time, by the growth promoting effect of cycloheximide, and by the optimal incubation time. All of the results were evaluated by the indirect immunofluorescent stain using the genus-specific monoclonal antibody(HYMo 1-1) to Chlamydia. RESULTS: The HEp-2 cell was the most efficient for culturing C. pneumoniae and the inclusion bodies in monolayer were increased with DEAE-dextran pretreatment at 30microgram/ml. Also application of a centrifugal force of 1.500 xg for at least 15 minute during inoculation enhanced the growth of C. pneumoniae. The best concentration of cycloheximide in the culture medium for host cell cytostasis was 1microgram/ml. The yields of organisms were greater when the cultures were harvested at 48 hours. CONCLUSIONS: We suggest that this system may make it more practical for laboratories to culture for C. pneumoniae.
Cell Culture Techniques ; Centers for Disease Control and Prevention (U.S.) ; Centrifugation ; Chlamydia* ; Chlamydophila pneumoniae* ; Coronary Artery Disease ; Cycloheximide ; DEAE-Dextran ; Diagnosis ; Humans ; Inclusion Bodies ; Pneumonia ; Respiratory System

To investigate the interaction of stimulatory GTP binding protein (G(s)) pathways with others, we overexpressed wild type alpha subunit of G(s) (G(s) alpha), constitutively activated R201E G(s) alpha, and dominant negative G226A G(s) alpha in COS-1 cells by transfection with DEAE-dextran, respectively, The expression of various G proteins in the transfected cells was analyzed after 72 h by quantitative Western blots, and cAMP production by stimulation with isoproterenol and forskolin was quantitated using cAMP binding proteins, The expression of Gs alpha increased about 5-fold in the transfected cells, with concomitant increase in the small forms. However, there was no significant alteration the in the level of the alpha subunit of inhibitory G protein (G(i)) and G(q), and the beta subunits of G proteins. The cAMP level without stimulation increased in the cells transfected with G(s) alpha regardless to the type of mutation, Treatment with either isoproterenol or forskolin resulted in comparable increase of the cAMP level in all the transfected cells, though the ratio to its respective basal level was smaller in the G(s) alpha-transfected cells, From this experiment, we found that the expression of the other G proteins and the signaling pathway producing cAMP did not change significantly by transiently expressing wild type, constitutively activated type, and dominant negative type of G(s) alpha. Analysis of the effects of long-term expression of Gs alpha would contribute to better understanding on how the G(s) alpha signaling system interacts with other signaling pathways and how it adapts to the changed environments.
Animals ; Blotting, Western ; Carrier Proteins ; Colforsin ; COS Cells* ; Cyclic AMP ; DEAE-Dextran ; GTP-Binding Proteins* ; Isoproterenol ; Protein Engineering ; Recombinant Proteins ; Transfection

BACKGROUND: Gamma-interferon (gamma-IFN) is known to suppress thyroperoxidase (TPO), thyroglobulin (Tg), thyrotropin receptor (TSHR) mRNA expression via unclarified mechanism. Thyroid transcription factor-1 (TTF-1) is a nuclear protein involved in the maximal expression and tissue specific expression of thyroid-specific antigens (TPO, Tg, TSHR, NIS) in thyrocytes. Although It's plausible that gamam-IFN induced suppression of thyroid-specific antigen expression may be mediated by decrease of TTF-1 expression, such an effect has not been documented yet. In this study we investigated the effect of gamma-IFN on the expression of TTF-1 in the rat thyroid cell, FRTL-5, and determined whether such an effect is mediated by sclass 2 transactivator (CIITA). METHEODS: The mRNA expression of TTF-1 was quantitated by northern blot analysis after treatment of gamma-IFN, and after expression of CIITA in FRTL-5 cells. Four different promoter constructs were made by cloning into the pRSV-luciferase vector, each contained 5'flanking sequence of different lengths (-5.18 kb, -4.11 kb, -1.94 kb, -1.15 kb) of rat TTF-1 gene. Effects of gamma-IFN and CIITA on promoter activities were analyzed by luciferase assay in FRTL-5 cells into which each promoter construct had been transfected by DEAE-dextran method. RESULTS: Steady state TTF-1 mRNA level at 48 h after gamma-IFN treatment (100 U/mL) was significantly decreased from that of the pre-treatment level (1.65+/-0.16 vs. unit, p<0.05). In all 4 promoter constructs gamma-IFN significantly suppressed promoter activities compared to the vector only transfected cells. CIITA expression in FRTL-5 cells significantly decreased the steady state TTF-1 mRNA level when compared to that in mock-transfected cells (1.69+/-0.31 vs. 1.17+/-0.44 arbitrary unit, p<0.05). CIITA expression in FRTL-5 cells caused suppression of promoter activities in -5.18 kb and -4.11 kb constructs, but had no effects on those activities in -1.94 kb; and -1.15 kb constructs. CONCLUSION: gamma-IFN, directly and indirectly via CIITA expression, suppressed the transcription of TTF-1 gene in the FRTL-5 cells. It may be one of the mechanisms involved in the gamma-IFN-induced suppression of thyroid-specific protein expressions in thyrocytes 1.25+/-0.27 arbitrary
Animals ; Blotting, Northern ; Clone Cells ; Cloning, Organism ; DEAE-Dextran ; Interferon-gamma ; Luciferases ; Nuclear Proteins ; Rats ; Receptors, Thyrotropin ; RNA, Messenger ; Thyroglobulin ; Thyroid Gland* ; Trans-Activators*

OBJECTIVESTo establish and evaluate the anti-human seminal plasma phospholipase A2 (PLA2) monoclonal antibody (McAb).

METHODSAfter having been separated and purified from human seminal plasma by PEG precipitation, Sephacryl S-300 column chromatography, DEAE-Sephadex A-25 column chromatography and HA column chromatography, PLA2 was regarded as an antigen to immune BALB/C mouse to produce anti-human seminal plasma PLA2 McAb. The PLA2 McAb sensitivity and specificity were performed by ELISA technique and Western-blot analysis, respectively.

RESULTSThe molecular weight of PLA2 depurated with 245 fold purification from human seminal plasma was about 34,900, while the sensitivity and typing of its McAb were 1:5(6)-1:5(8) and IgM (kappa) with a satisfied Western-Blot results.

CONCLUSIONSThe PLA2, which had not been reported in international and domestic papers, may be a new type of PLA2. The establish of its McAb will provide significant tools for the research of the relationship between PLA2 in human seminal plasma and male fertility.

Antibodies, Monoclonal ; immunology ; DEAE-Dextran ; analogs & derivatives ; chemistry ; Electrophoresis, Polyacrylamide Gel ; Humans ; Molecular Weight ; Phospholipases A ; immunology ; isolation & purification ; metabolism ; Phospholipases A2 ; Semen ; enzymology