Using LN2 vapor for cooling and electrical resistors for heating, the Linkam cryostage with its associated control hardware and Pax-it software allows samples to be subjected to sequential ramps in which cooling rate, limiting temperature, holding time, and warming rate can be specified. The ramps used here are shown in Table 2. The procedure was as follows: the oocytes/embryos were cooled rapidly to —5.0°C, and cooled slowly to —8.0°C (ramps 1 and 2).
TABLE 2. Cooling and warming rates programmed into the Linkam cryostage for oocytes and embryos frozen in 1.0 M ethylene glycol/PBS.
External ice formation (EIF) occurred at a mean of —7.2 6 0.06°C. The sample was then warmed (ramp 3) to —3.2°C, which is just at the melting point of the medium. Most, but not all, of the external ice melted. Recooling was then initiated in ramp 4 after a 10-sec hold at the end of ramp 3. The purpose of ramp 3 was to provide time for the external liquid medium, the external ice, and the supercooled water in the cell to come to near equilibrium before recooling began. If ramp 3 was omitted, the observed temperatures of IIF were about 20°C higher. Most cases of IIF occurred during the rapid cool (20°C/min) in ramp 5. IIF was manifested by abrupt blackening of the cell. Above —30°C, cells completely blackened in a fraction of a second. Below that temperature, blackening could take a number of seconds. The nucleation or IIF temperature was taken to be the temperature at which the blackening first became evident. comments
Glycyrrhetinic Acid Experiments
The compound 18p-glycyrrhetinic acid (GA) is a known gap junction blocker. To determine whether it affected sequential IIF in morulae, 30 iM GA (Sigma) was added to the final 1.0 M EG/PBS solution in which the morulae were suspended for 15 min at 25°C prior to freezing in the Linkam cryostage. In some experiments, we used a PBS that lacked the normal Ca++ and Mg++. The absence of Ca++ in particular is supposed to disaggregate the compacted blastomeres, and that action would be expected to disrupt gap junctions.
Plus/minus values in tables and error bars in figures are standard errors (standard deviations of the mean). Two-tailed t-tests and chi-square analysis were used to assess significant differences.