During these exposure times, the oocytes and embryos were transferred into the 12-mm column of vitrification solution (oocytes into EAFS 10/10, two-cell into EFS40, four- to eight-cell into EFS30, and morulae into EFS40) in each straw and the end of the straw opposite the polyvinyl alcohol plug was heat sealed. At the conclusion of the defined exposure time, the straw was held for 3 min or more in liquid nitrogen (LN2) vapor at —120 to — 150°C and then plunged into LN2. The measured cooling rate from 20°C to —120°C was 187°C/min. For shipment from Japan, the straws were transferred into a dry cryogenic shipper (Taylor Wharton) that had been precooled with LN2 to — 196°C, and sent to Tennessee by express mail. Upon receipt, the shipping container was checked to make certain it still contained LN2.
For experiments at Tennessee, straws containing the oocytes/embryos were transferred rapidly from LN2 to a water bath at 23°C after a 10-sec hold in room temperature air, and after thawing, the column of cells mixed with the column of 0.5 M sucrose/PB1 in the straw. Some 10 min later, the oocytes were transferred at 25°C to PB1 lacking sucrose, and then to previously prepared droplets of M16 medium in which they were incubated for some 2 h at 37°C under 5% CO2. Several lines of evidence [1, p. 48] support the view that the vitrified-thawed-M16-incubated oocytes and embryos are normal with respect to plasma membrane integrity and osmotic response. canadian healthcare mall
Many of the methods were described in detail in; consequently, here we give details only for those aspects that differed. The procedures for obtaining and manipulating the mouse oocytes and embryos were carried out under the University of Tennessee Institutional Animal Care and Use Committee protocol 911-0607, approved 28 June 2007, and the Animal Ethics Committee of the College of Agriculture, Kochi University.
Collection of Oocytes and Embryos
Mouse oocytes at the MII stage and embryos at the one-cell, two-cell, fourcell, eight-cell, and early morula stages were used in the study. Mature female mice of the ICR strain were induced to superovulate with intraperitoneal injections of 5 IU of equine chorionic gonadotropin (Sigma) and 5 IU of human chorionic gonadotropin (hCG; Sigma) given 48 h later. For the collection of embryos, females were mated with mature males of the same strain. For the collection of oocytes, matured oocytes surrounded by cumulus cells were collected from the ampullar portion of the oviducts at 13 h after hCG injection and were freed from cumulus cells by suspending them in modified phosphate-buffered saline (PB1) containing 0.5 mg/ml hyaluronidase followed by washing with fresh PB1 medium. For the collection of one-cell, two-cell, four-cell, and eight-cell embryos, oviducts of mated animals were flushed with PB1 medium at 25, 44, 55, and 68 h, respectively, after the injection of hCG. For the collection of early morulae, the uteri of mated animals were flushed with PB1 medium 78 h after the injection of hCG. The oocytes and embryos were washed and pooled in fresh PB1 medium in a culture dish under paraffin oil to await each suite of experiments. read only
The formation of ice crystals within cells is almost always lethal. The classical way to avoid it is to cool cells slowly enough so that they lose nearly all their freezable water osmotically before they have cooled to the temperature at which ice nucleation of the remaining supercooled intracellular water can occur. Thus, whether intracellular ice forms or not depends on two factors. One is the rate at which the cells are cooled, for it determines how much supercooled water remains in the cells as a function of temperature. The second is the nucleation temperature of that supercooled water. We have reported that the nucleation temperature of mature mouse oocytes (metaphase II [MII] stage) in 1 M ethylene glycol (EG) is —35°C. The question we are addressing in this report is whether that temperature is the same or different in preimplantation embryos of various developmental stages. this