Effects of freezing and activation on membrane quality and DNA damage in Xenopus tropicalis and Xenopus laevis spermatozoa

Abstract

Abstract. There is growing concern over the effect of sperm cryopreservation on DNA integrity and the subsequent development of offspring generated from this cryopreserved material. In the present study, membrane integrity and DNA stability of Xenopus laevis and Xenopus tropicalis spermatozoa were evaluated in response to cryopreservation with or without activation, a process that happens upon exposure to water to spermatozoa of some aquatic species. A dye exclusion assay revealed that sperm plasma membrane integrity in both species decreased after freezing, more so for X. laevis than X. tropicalis spermatozoa. The sperm chromatin dispersion (SCD) test showed that for both X. tropicalis and X. laevis, activated frozen spermatozoa produced the highest levels of DNA fragmentation compared with all fresh samples and frozen non-activated samples (P , 0.05). Understanding the nature of DNA and membrane damage that occurs in cryopreserved spermatozoa from Xenopus species represents the first step in exploiting these powerful model organisms to understand the developmental consequences of fertilising with cryopreservation-damaged spermatozoa.

An optimized method for cryogenic storage of Xenopus sperm to maximise the effectiveness of research using genetically altered frogs

“…activated sperm), suggests that X. laevis sperm retain the capability to fertilise under optimal conditions even when they have significant plasma membrane and DNA damage [44]. The lack of an effect of activation on the state of frozen-thawed sperm is in agreement with Morrow et al.…”

Husbandry, General Care, and Transportation of Xenopus laevis and Xenopus tropicalis

“…Sperm can be used immediately or stored for extended period of time either at −80 °C or in liquid nitrogen. In general, fertilization from cryopreserved sperm is efficient and animals generated develop normally, however, it has also been demonstrated that cryopreserving sperm does cause some plasma membrane damage and DNA fragmentation in the spermatozoa [7, 8]. …”

Sperm collection and storage for the sustainable management of amphibian biodiversity

“…The sperm of Cryptobranchus proved amenable to cryopreservation with the slow lowering of straws into a LN2 vapour (Dale McGinnity personal communication), but using a similar freezing method with Andrias only recovered <10% motility [47]. In fish optimal cooling rates are membrane lipid dependent [114,115] and this is expected to be the case with amphibian sperm.…”

“…Many morphological deformations can be found in post-thaw amphibian spermatozoa, such as swelling or rupture of the plasma membrane, loss of the nuclear envelope, fracture of the perforatorium and axoneme, degeneration of the undulating membrane and disappearance of the mitochondrial ridge [48,115]. Morphological damage may be associated with impacts on post-thaw activation mechanisms where fish [116,117] or Anuran [53,112] spermatozoa are intact but unable to activate.…”