Noiles E E, Bailey J L, Storey B T
Department of Obstetrics and Gynecology, University of Pennsylvania Medical Center, Philadelphia 19104, USA.
Cryobiology. 1995 Jun;32(3):220-38. doi: 10.1006/cryo.1995.1022.
Cryopreservation of sperm from transgenic mice would make possible the economical perpetuation of these genetic models. In order to design a protocol for this process, it was first necessary to determine the hydraulic conductivity, Lp, or water permeability, of the plasma membrane as a function of temperature to calculate its activation energy, Ea. This was done with sperm from the caudae epididymides of CD-1 mice, using the hyposmotic cell lysis method, in which the critical osmolality, Osmcrit, defined as the osmolality at which 50% of the cells lyse, was first determined, and then the time, t, to lysis of 50% of the cells in a medium of Osm < Osmcrit was obtained. Values (mean +/- SEM, n = 10) of Osmcrit at 37, 22, 4, and 0 degrees C were 32.1 +/- 2.7, 33.7 +/- 4.1, 30.8 +/- 3.4, and 101.0 +/- 10.8 mOsm, and of t in 17 mOsm medium were 40.6 +/- 2.4, 33.8 +/- 5.7, 25.0 +/- 4.4, and 4.7 +/- 1.2 s, respectively. Values of Lp were calculated from Osmcrit and t by two different algorithms, one based on the high-amplitude swelling model used with sperm from other mammalian species and the other based on a low-amplitude swelling model. Values of Lp in micron.min-1.atm-1 from the high-amplitude swelling model at 37, 22, 4, and 0 degrees C were 1.41 +/- 0.08, 1.88 +/- 0.35, 2.12 +/- 0.19, and 1.13 +/- 0.1, respectively. From the low-amplitude swelling model, the Lp values were 0.025 +/- 0.001, 0.040 +/- 0.008, 0.082 +/- 0.029, and 1.66 +/- 0.23. Examination of mouse sperm in hyposmotic media by microscopy revealed little swelling of the cells, indicating that the low-amplitude swelling model may be the one more applicable to these cells. The temperature dependence, and hence Ea, of Lp shows a marked discontinuity between 4 and 0 degrees C with values calculated from either model. This suggests a membrane phase transition to a more brittle structure in this temperature range, consistent with the observed marked increase in Osmcrit (P < 0.0003) and decrease in t (P < 0.0001) at 0 degrees C compared to the other temperatures. In the presence of 1 M glycerol, there was no discontinuity between 4 and 0 degrees C in the values of Osmcrit, consistent with the ability of glycerol to fluidize the membrane. Low hydraulic conductivity and low-temperature embrittlement of the plasma membrane are proposed as two factors leading to mouse sperm hypersensitivity to cryodamage.
