Water permeability, Lp, of the mouse sperm plasma membrane and its activation energy are strongly dependent on interaction of the plasma membrane with the sperm cytoskeleton.
Two parameters fundamental to cell cryobiology are the water permeability (hydraulic conductivity), Lp, and its activation energy, EA. The Lp can be calculated from two experimental determinations: the critical osmolality, Osmcrit, at which 50% of the cells lyse, and the time, tcrit, to 50% lysis in a highly hyposmotic medium, based on the assumption that the cells swell to lysis with minimal resistance to swelling. We have reported [Cryobiology 32, 220-238 (1995)] that mouse sperm in hyposmotic medium show minimal swelling and so fail to meet this assumption. The concept that resistance to swelling was due to anchoring of the plasma membrane through cytoskeletal interaction was examined by treating mouse sperm with 5 microM cytochalasin D to depolymerize the cytoskeletal filamentous actin (f-actin), whose presence was established by staining with fluorescently labeled phalloidin. Diminution of fluorescence due to loss of f-actin induced by cytochalasin D was shown by flow cytometry. Mouse sperm treated with cytochalasin D showed tail curling in hyposmotic medium, similar to that observed with bovine and human sperm, indicating that the standard swelling model was applicable to these cells. Two sets of Lp values were calculated from tcrit: one using individual means of Osmcrit and one using the mean of means of Osmcrit between 37 and 4 degrees C, as these individual means were not significantly different. Values (micron.min-1.atm-1), respectively, were 9.95, 7.15 (37 degrees C); 1.51, 0.91 (22 degrees C); 0.54, 0.78 (12 degrees C); 0.47, 0.50 (4 degrees C); 0.33 (0 degree C); and 0.36 (-3 degrees C). Arrhenius plots gave EA = 13.7 and 11.7 kcal/mol, respectively. Values of t1/2 were calculated from the first-order rate constants characterizing the kinetics of cell lysis at the higher four temperatures; Lp values calculated from these, and the two sets of Osmcrit values described were 5.70, 4.09 (37 degrees C); 1.18, 0.71 (22 degrees C); 0.62, 0.90 (12 degrees C); and 0.34, 0.37 (4 degrees C). Arrhenius plots gave EA = 14.2 and 11.0 kcal/mol, respectively. We propose that these EA values are characteristic of the plasma membrane relatively unperturbed by cytoskeletal interactions. In untreated sperm, decrease of Lp with decreasing temperature and presence of cryoprotectant and the cytoskeletal interactions all act to hamper the sperm cells' ability to respond to osmotic stress encountered during freezing and thawing, such that these cells are especially sensitive to cryodamage.
Noiles
E E
EE
Center for Research on Reproduction and Women's Health, University of Pennsylvania Medical Center, Philadelphia 19104, USA.
Thompson
K A
KA
Storey
B T
BT
D000199:Actins / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability; D015925:Cryopreservation; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D051379:Mice; D009997:Osmotic Pressure; D012662:Semen Preservation; D013094:Spermatozoa / Q000187:drug effects / Q000378:metabolism*; D013816:Thermodynamics; D014867:Water / Q000378:metabolism
D000199:Actins; D014867:Water; D015638:Cytochalasin D