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

Abstract

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.

Cryopreservation of Spermatozoa from Freeze-Tolerant and -Intolerant Anurans

“…However, recovery of viable spermatozoa in frozen/thawed samples from these species (48 -59%) was lower than that (81%) for R. sylvatica. A similar result was obtained when the viability of spermatozoa frozen to relatively high (ecologically relevant) temperatures was compared between R. sylvatica and R. pipiens (7). This pattern may reflect innate cellular adaptations to tissue freezing in R. sylvatica, which apparently is a normal occurrence during overwintering.…”

“…Glycogenolysis in the liver, which is triggered when freezing begins, provides an ample supply of glucose, the cryoprotectant of its cells and tissues. We recently found that spermatozoa of R. sylvatica survive in vitro freezing at ecologically-relevant temperatures (i.e., ՆϪ4°C), even in the absence of cryoprotectant (7). This capacity is adaptive because R. sylvatica breeds in late winter, when cryoprotectant production capacity is low and freezing may occur (5).…”

Cryopreservation of sperm ofXenopus laevis andXenopus tropicalis

“…To permit cryopreservation under a slow-cooling regime, the freezing point of water is lowered either by solutes that penetrate cells readily (glycerol, glycols, methanol, or dimethyl-sulphoxide) or highly hydrated, nonionic, nonpenetrating solutes such as di-or tri-saccharides (Gao, 2000). We have been unsuccessful (data not shown, also reported by Buchholz, 2004) in cryopreserving sperm of Xenopus species using protocols that involve penetrating cryoprotectants developed for sperm of several amphibian species (Beesley et al, 1998;Browne et al, 1998Browne et al, , 2002aCostanzo et al, 1998). The theoretically optimal concentration of penetrating solute, used successfully in standard protocols with most nonsperm cell types, is very high; for unknown reasons, these conditions usually damage sperm.…”

Amphibian sperm DNA fragmentation

“…Sperm cryopreservation protocols that involve penetrating cryoprotectants developed for other amphibian species (Beesley et al 1998;Browne et al 1998;Costanzo et al 1998) samples showed a significant increase in SDF after 60 minutes of incubation, the proportion of sperm with SDF was still low, being less than 10%. Similarly, spermatozoa of numerous mammals including dog (Prinosilova et al 2012) and stallion ) retain a normal chromatin structure immediately after thawing (Gosálvez et al 2011b).…”

“…This increase in DNA damage could be related to the ageing of spermatozoa stored in the ducts and the presence of immature, apoptotic or necrotic sperm cells (Pérez-Cerezales et al 2010). Costanzo et al (1998) examined freeze tolerance in Rana spp. and reported an increased susceptibility to cryoinjury in the freeze-tolerant wood frog (Rana sylvatica) when exposed to sporadic, but naturally occurring episodes of subzero temperatures shortly prior to the spawning period.…”