Osmotic and freezing tolerance in spermatozoa of freeze-tolerant and -intolerant frogs.

The wood frog ( Rana sylvatica) is a freeze-tolerant species that encounters subzero temperatures during its winter breeding season, whereas the leopard frog ( R. pipiens) is freeze intolerant and breeds in spring. Osmotic and freezing tolerances of spermatozoa from these species were inferred from spermolysis rate, integrity of the plasma membrane as judged using vital dye assay, and motility rate. Sperm of R. sylvatica became motile in hypotonic media (≤220 mosmol/kg) and tolerated in vitro exposure to osmotic concentrations spanning nearly three orders of magnitude. Relative to sperm from R. sylvatica, which were unaffected by freezing at temperatures of -4°C or greater, R. pipiens sperm were more susceptible to osmotic damage and cryoinjury. These differences likely reflect cellular adaptations to somatic freezing in R. sylvatica. Unprotected sperm from both species were extensively damaged by freezing at -8°C, but the presence of glucose, the cryoprotectant used by R. sylvatica, or the permeant glycerol markedly diminished cryoinjury. These data suggest the feasibility of developing gamete cryopreservation protocols to aid efforts in conserving amphibian populations.