Vol. 275, Issue 3, R713-R719, September 1998
Osmotic and freezing tolerance in spermatozoa of
freeze-tolerant and -intolerant frogs
Jon P.
Costanzo1,
John A.
Mugnano1,2,
Heidi M.
Wehrheim1, and
Richard E.
Lee Jr. 1
1 Department of Zoology, Miami
University, Oxford, Ohio 45056; and
2 Department of Veterinary
Clinical Sciences, Purdue University, West Lafayette, Indiana 47906
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.
cryoprotection; reproduction; amphibian; anuran
