Vitrification of bovine embryos in a mixture of ethylene glycol and dimethyl sulfoxide

The influence of equilibration time before vitrification on the viability of vitrified morula- to blastocyst-stage bovine embryos and in vivo viability of vitrified embryos following transfer to recipients were investigated. In experiment 1, the embryos were exposed to an equilibration solution (50% VSED) containing 12.5% v/v ethylene glycol and 12.5% v/v dimethyl sulfoxide in modified Dulbecco's phosphate buffered saline with 4 mg/ml BSA (m-PBS) for 1, 2 and 5 minutes at room temperature (22 to 24 degrees C). The embryos were then placed in 15mul vitrification solution (VSED) consisting of 25% v/v ethylene glycol and 25% v/v dimethyl sulfoxide in m-PBS and were loaded into 0.25 ml plastic straws at room temperature. After 30 seconds, the straws were placed in liquid nitrogen (LN(2)) vapor for 2 minutes, plunged and stored in LN(2). To thaw, the straws were warmed in water at 20 degrees C for 15 seconds and the contents of the straws were expelled into a plastic dish. The embryos were diluted in 0.5 M sucrose + m-PBS for 5 minutes and were cultured in TCM-199 supplemented with bovine oviductal epithelial tissue. Viability of the embryos was assessed by the forming or reforming of the blastocoele after 24 hours of culture. High in vitro survival rates (73 approximately 90%) of vitrified embryos were obtained after 1 and 2 minute equilibrations, but was reduced (P<0.05) after 5 minute equilibration. In Experiment 2, morula- to blastocyst-stage embryos were vitrified after 1 minute equilibration in 50% VSED and 30 seconds of exposure to VSED. The vitrified-warmed embryos were transferred to recipient heifers at 7 days after estrus (1 embryo per recipient). Five (38%) of 13 (40%) of 10 recipients that had received blastocysts were diagnosed as pregnant using ultrasonography 60 days following transfer.     

W-heterochromatin of chicken; its unusual DNA components,late replication,and chromatin structure

About 65% of DNA in the chicken W chromosome has been shown to consist ofXhoI andEcoRI family repetitive sequences. These sequences showed remarkable delay in the electrophoretic mobility at low temperature on a polyacrylamide gel. Three dimensional structures of the 0.7-kbXhoI and the 1.2-kbEcoRI family repeating units were estimated to be irregular solenoids using a computer program based on wedge angles of all the 16 dinucleotide steps. Fluorescencein situ hybridization demonstrated that these two family sequences were localized in a major heterochromatic body in an interphase nucleus. Incorporation of bromodeoxyuridine into the W chromosome in the synchronous culture of MSB-1 cells occurred about 1 h later than the peak of S phase. The chromatin structure formed alongXhoI andEcoRI family sequences was suggested to be different from the total chromatin or chromatin containing the β-actin gene sequence in that the linker DNA lengths of the former were significantly longer. Fractionation of theHaeIII-digested MSB-1 nuclei yielded a chromatin fraction in whichXhoI family sequences were partially enriched. Several DNA-binding proteins showing higher affinity for theXhoI family sequence were present in this fraction.     

Correction to: Comparison of the usability of an automatic sleep staging program via portable 1-channel electroencephalograph and manual sleep staging with traditional polysomnography

Sleep and Biological Rhythms -     

Some Electrical Properties of a Nuclear Membrane Examined with a Microelectrode

Electrical potential and resistance were measured with microelectrodes in in situ and isolated nuclei of gland cells of Drosophila flavorepleta. The nucleus-cytoplasm boundary was found to be rather impermeable to ion diffusion. It presents a resistance of the order of 1 Ω cm² and sustains a "resting" potential, the nucleoplasm being about 15 mv negative with respect to the cytoplasm. Both the resistance and potential appear to be associated with the nuclear membrane: the potential declines to zero and the resistance to a fraction of its original value, when the membrane is perforated experimentally.     

Endotoxin Induces Severe Inflammatory Reactions with Necrosis at Sites Primed with Delayed-Type Hypersensitivity Reactions in Guinea Pigs

Guinea pigs immunized with Freund's complete adjuvant received challenge injection of the purified protein derivative of Mycobacterium tuberculosis in the flanks and the corneas to prepare delayed-type hypersensitivity (DTH) reactions. The animals were injected subcutaneously with lipopolysaccharide (LPS) or a synthetic lipid A (LA-15-PP). At the skin site primed with DTH reaction, increased swelling and hemorrhagic reaction followed by a definite necrotic reaction occurred. Severe corneal reactions were also observed in the animals. These findings indicate that bacterial endotoxin modulates DTH reactions and induces severe inflammatory reactions.