Reproduction, Fertility and Development Reproduction, Fertility and Development Society
Vertebrate reproductive science and technology
RESEARCH ARTICLE (Open Access)

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

S. Morrow A , J. Gosálvez B , C. López-Fernández B , F. Arroyo B , W. V. Holt C and M. J. Guille A D

A School of Biological Sciences and European Xenopus Resource Centre, The University of Portsmouth, Portsmouth, PO1 2DT, UK.

B Department of Biology, Genetics Unit, The Autonomous University of Madrid, 20849 Madrid, Spain.

C Academic Department of Reproductive and Developmental Medicine, The University of Sheffield, Sheffield, S10 2SF, UK.

D Corresponding author. Email: matthew.guille@port.ac.uk

Reproduction, Fertility and Development - https://doi.org/10.1071/RD16190
Submitted: 10 April 2015  Accepted: 4 July 2016   Published online: 3 October 2016

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.

Additional keywords: cryopreservation, DNA fragmentation, sperm chromatin dispersion.


References

Beesley, S. G., Costanzo, J. P., and Lee, R. E. (1998). Cryopreservation of spermatozoa from freeze-tolerant and -intolerant anurans. Cryobiology 37, 155–162.
Cryopreservation of spermatozoa from freeze-tolerant and -intolerant anurans.CrossRef | 1:STN:280:DyaK1cvktlWqsA%3D%3D&md5=7081c871c4709c21509fc2b9d5d2a99bCAS | 9769166PubMed | open url image1

Bennetts, L. E., and Aitken, R. J. (2005). A comparative study of oxidative DNA damage in mammalian spermatozoa. Mol. Reprod. Dev. 71, 77–87.
A comparative study of oxidative DNA damage in mammalian spermatozoa.CrossRef | 1:CAS:528:DC%2BD2MXjtVOqsrk%3D&md5=5decf0ae7e95b9956b055714cc703deaCAS | 15736137PubMed | open url image1

Browne, R. K., Clulow, J., Mahony, M., and Clark, A. (1998). Successful recovery of motility and fertility of cryopreserved cane toad (Bufo marinus) sperm. Cryobiology 37, 339–345.
Successful recovery of motility and fertility of cryopreserved cane toad (Bufo marinus) sperm.CrossRef | 9917350PubMed | open url image1

Clulow, J., Trudeau, V. L., and Kouba, A. J. (2014). Amphibian declines in the twenty-first century: why we need assisted reproductive technologies. Adv. Exp. Med. Biol. 753, 275–316.
Amphibian declines in the twenty-first century: why we need assisted reproductive technologies.CrossRef | 25091914PubMed | open url image1

Cortés-Gutiérrez, E. I., Dávila-Rodríguez, M. I., López-Fernández, C., Fernández, J. L., and Gosálvez, J. (2008). Alkali-labile sites in sperm cells from Sus and Ovis species. Int. J. Androl. 31, 354–363.
Alkali-labile sites in sperm cells from Sus and Ovis species.CrossRef | 17651406PubMed | open url image1

Fernández-Díez, C., González-Rojo, S., Montfort, J., Le Cam, A., Bobe, J., Robles, V., Pérez-Cerezales, S., and Herráez, M. P. (2015). Inhibition of zygotic DNA repair: transcriptome analysis of the offspring in trout (Oncorhynchus mykiss). Reproduction 149, 101–111.
Inhibition of zygotic DNA repair: transcriptome analysis of the offspring in trout (Oncorhynchus mykiss).CrossRef | 25433028PubMed | open url image1

Fish, M. B., Nakayama, T., Fisher, M., Hirsch, N., Cox, A., Reeder, R., Carruthers, S., Hall, A., Stemple, D. L., and Grainger, R. M. (2014). Xenopus mutant reveals necessity of rax for specifying the eye field which otherwise forms tissue with telencephalic and diencephalic character. Dev. Biol. 395, 317–330.
Xenopus mutant reveals necessity of rax for specifying the eye field which otherwise forms tissue with telencephalic and diencephalic character.CrossRef | 1:CAS:528:DC%2BC2cXhsFynsrnE&md5=a468d7d09dca06d7b9b9222456129bd8CAS | 25224223PubMed | open url image1

González-Marín, C., Gosálvez, J., and Roy, R. (2012). Types, causes, detection and repair of DNA fragmentation in animal and human sperm cells. Int. J. Mol. Sci. 13, 14 026–14 052.
Types, causes, detection and repair of DNA fragmentation in animal and human sperm cells.CrossRef | open url image1

González-Rojo, S., Fernández-Díez, C., Guerra, S. M., Robles, V., and Herraez, M. P. (2014). Differential gene susceptibility to sperm DNA damage: analysis of developmental key genes in trout. PLoS One 9, e114161.
Differential gene susceptibility to sperm DNA damage: analysis of developmental key genes in trout.CrossRef | 25479606PubMed | open url image1

Gosálvez, J., Cortés-Gutiérrez, E. I., Nuñez, R., Fernández, J. L., Caballero, P., López-Fernández, C., and Holt, W. V. (2009). A dynamic assessment of sperm DNA fragmentation versus sperm viability in proven fertile human donors. Fertil. Steril. 92, 1915–1919.
A dynamic assessment of sperm DNA fragmentation versus sperm viability in proven fertile human donors.CrossRef | 18980765PubMed | open url image1

Gosálvez, J., López-Fernández, C., Fernández, J. L., Gouraud, A., and Holt, W. V. (2011a). Relationships between the dynamics of iatrogenic DNA damage and genomic design in mammalian spermatozoa from eleven species. Mol. Reprod. Dev. 78, 951–961.
Relationships between the dynamics of iatrogenic DNA damage and genomic design in mammalian spermatozoa from eleven species.CrossRef | 21919111PubMed | open url image1

Gosálvez, J., Ramirez, M. A., López-Fernández, C., Crespo, F., Evans, K. M., Kjelland, M. E., and Moreno, J. F. (2011b). Sex-sorted bovine spermatozoa and DNA damage: I. Static features. Theriogenology 75, 197–205.
Sex-sorted bovine spermatozoa and DNA damage: I. Static features.CrossRef | 20932559PubMed | open url image1

Gosálvez, J., Holt, W. V., and Johnston, S. D. (2014). Sperm DNA fragmentation and its role in wildlife conservation. Adv. Exp. Med. Biol. 753, 357–384.
Sperm DNA fragmentation and its role in wildlife conservation.CrossRef | 25091917PubMed | open url image1

Gurdon, J. B. (1977). Methods for nuclear transplantation in amphibia. Methods Cell Biol. 16, 125–139.
Methods for nuclear transplantation in amphibia.CrossRef | 1:CAS:528:DyaE2sXlsVyjt7c%3D&md5=edff58b791337b1abc374a700498fc3eCAS | 329056PubMed | open url image1

Harland, R. M., and Grainger, R. M. (2011). Xenopus research: metamorphosed by genetics and genomics. Trends Genet. 27, 507–515.
Xenopus research: metamorphosed by genetics and genomics.CrossRef | 1:CAS:528:DC%2BC3MXhsVKhur7K&md5=123f4c51f39fbd57ea90ddff43182070CAS | 21963197PubMed | open url image1

Hellsten, U., Harland, R. M., Gilchrist, M. J., Hendrix, D., Jurka, J., Kapitonov, V., Ovcharenko, I., Putnam, N. H., Shu, S., Taher, L., et al. (2010). The genome of the Western clawed frog Xenopus tropicalis. Science 328, 633–636.
The genome of the Western clawed frog Xenopus tropicalis.CrossRef | 1:CAS:528:DC%2BC3cXlt1Crt70%3D&md5=59f6918eaa0ed6c85218501c9848e1b1CAS | 20431018PubMed | open url image1

Holt, W. V. (2000). Fundamental aspects of sperm cryobiology: the importance of species and individual differences. Theriogenology 53, 47–58.
Fundamental aspects of sperm cryobiology: the importance of species and individual differences.CrossRef | 1:STN:280:DC%2BD3c7pvFagtA%3D%3D&md5=026e3d4311245704c504bc37328f8d05CAS | 10735061PubMed | open url image1

Johnston, S. D., Zee, Y. P., López-Fernández, C., and Gosálvez, J. (2012). The effect of chilled storage and cryopreservation on the sperm DNA fragmentation dynamics of a captive population of koalas. J. Androl. 33, 1007–1015.
The effect of chilled storage and cryopreservation on the sperm DNA fragmentation dynamics of a captive population of koalas.CrossRef | 1:CAS:528:DC%2BC38XhsFSqt7%2FM&md5=091b8fb40f25fd87c4c9bc67b6256717CAS | 22282436PubMed | open url image1

Karpinka, J. B., Fortriede, J. D., Burns, K. A., James-Zorn, C., Ponferrada, V. G., Lee, J., Karimi, K., Zorn, A. M., and Vize, P. D. (2015). Xenbase, the Xenopus model organism database; new virtualized system, data types and genomes. Nucleic Acids Res. 43, D756–D763.
Xenbase, the Xenopus model organism database; new virtualized system, data types and genomes.CrossRef | 25313157PubMed | open url image1

Khokha, M. K. (2012). Xenopus white papers and resources: folding functional genomics and genetics into the frog. Genesis (New York, N.Y.: 2000) 50, 133–142.
Xenopus white papers and resources: folding functional genomics and genetics into the frog.CrossRef | 1:CAS:528:DC%2BC38XjtFygtLc%3D&md5=1babcc827f856a8dcde2678873ea4b43CAS | open url image1

Kopeika, J., Kopeika, E., Zhang, T., Rawson, D. M., and Holt, W. V. (2004). Effect of DNA repair inhibitor (3-aminobenzamide) on genetic stability of loach (Misgurnus fossilis) embryos derived from cryopreserved sperm. Theriogenology 61, 1661–1673.
Effect of DNA repair inhibitor (3-aminobenzamide) on genetic stability of loach (Misgurnus fossilis) embryos derived from cryopreserved sperm.CrossRef | 1:CAS:528:DC%2BD2cXhslWnu7w%3D&md5=4f2ed5906848b14a6098901db676523bCAS | 15019462PubMed | open url image1

Kopeika, J., Thornhill, A., and Khalaf, Y. (2015). The effect of cryopreservation on the genome of gametes and embryos: principles of cryobiology and critical appraisal of the evidence. Hum. Reprod. Update 21, 209–227.
The effect of cryopreservation on the genome of gametes and embryos: principles of cryobiology and critical appraisal of the evidence.CrossRef | 25519143PubMed | open url image1

Li, P., Li, Z.-H., Dzyuba, B., Hulak, M., Rodina, M., and Linhart, O. (2010). Evaluating the impacts of osmotic and oxidative stress on common carp (Cyprinus carpio, L.) sperm caused by cryopreservation techniques. Biol. Reprod. 83, 852–858.
Evaluating the impacts of osmotic and oxidative stress on common carp (Cyprinus carpio, L.) sperm caused by cryopreservation techniques.CrossRef | 1:CAS:528:DC%2BC3cXhtlGls7%2FK&md5=ab2767c5792174d7eca873992d03e4a5CAS | 20668258PubMed | open url image1

Lopes, S., Jurisicova, A., Sun, J. G., and Casper, R. F. (1998). Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa. Hum. Reprod. 13, 896–900.
Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa.CrossRef | 1:CAS:528:DyaK1cXjs12gtbo%3D&md5=411c4bbfcf36849dba5590b22056a0e1CAS | 9619544PubMed | open url image1

Mansour, N., Lahnsteiner, F., and Patzner, R. A. (2009). Optimization of the cryopreservation of African clawed frog (Xenopus laevis) sperm. Theriogenology 72, 1221–1228.
Optimization of the cryopreservation of African clawed frog (Xenopus laevis) sperm.CrossRef | 1:CAS:528:DC%2BD1MXhtlCgtr3J&md5=4225a6d70e1101216b43eba047adc4e8CAS | 19766299PubMed | open url image1

Meistrich, M. L., Mohapatra, B., Shirley, C. R., and Zhao, M. (2003). Roles of transition nuclear proteins in spermiogenesis. Chromosoma 111, 483–488.
Roles of transition nuclear proteins in spermiogenesis.CrossRef | 12743712PubMed | open url image1

Men, N. T., Kikuchi, K., Nakai, M., Fukuda, A., Tanihara, F., Noguchi, J., Kaneko, H., Linh, N. V., Nguyen, B. X., Nagai, T., and Tajima, A. (2013). Effect of trehalose on DNA integrity of freeze-dried boar sperm, fertilization, and embryo development after intracytoplasmic sperm injection. Theriogenology 80, 1033–1044.
Effect of trehalose on DNA integrity of freeze-dried boar sperm, fertilization, and embryo development after intracytoplasmic sperm injection.CrossRef | 1:CAS:528:DC%2BC3sXhsVKktLvN&md5=99e3a797d2118a0a302e10d7a5392958CAS | 24041826PubMed | open url image1

Michael, S. F., and Jones, C. (2004). Cryopreservation of spermatozoa of the terrestrial Puerto Rican frog, Eleutherodactylus coqui. Cryobiology 48, 90–94.
Cryopreservation of spermatozoa of the terrestrial Puerto Rican frog, Eleutherodactylus coqui.CrossRef | 1:CAS:528:DC%2BD2cXht1yiurg%3D&md5=b29454023d8a3f39e23e9dc9056c2526CAS | 14969686PubMed | open url image1

Nakayama, T., Fisher, M., Nakajima, K., Odeleye, A. O., Zimmerman, K. B., Fish, M. B., Yaoita, Y., Chojnowski, J. L., Lauderdale, J. D., Netland, P. A., and Grainger, R. M. (2015). Xenopus pax6 mutants affect eye development and other organ systems, and have phenotypic similarities to human aniridia patients. Dev. Biol. 408, 328–344.
Xenopus pax6 mutants affect eye development and other organ systems, and have phenotypic similarities to human aniridia patients.CrossRef | 1:CAS:528:DC%2BC2MXjs1Gqu7g%3D&md5=dba935d03eae58fbb79f4ce817eb84adCAS | 25724657PubMed | open url image1

O’Neill, A. C., and Ricardo, S. D. (2013). Human kidney cell reprogramming: applications for disease modeling and personalized medicine. J. Am. Soc. Nephrol. 24, 1347–1356.
Human kidney cell reprogramming: applications for disease modeling and personalized medicine.CrossRef | 1:CAS:528:DC%2BC3sXhsFCqsr3N&md5=f1e9b94b4448b37455ff38672dad43f3CAS | 23949797PubMed | open url image1

Paasch, U., Sharma, R. K., Gupta, A. K., Grunewald, S., Mascha, E. J., Thomas, A. J., et al. (2004). Cryopreservation and thawing is associated with varying extent of activation of apoptotic machinery in subsets of ejaculated human spermatozoa. Biol. Reprod. 71, 1828–1837.
Cryopreservation and thawing is associated with varying extent of activation of apoptotic machinery in subsets of ejaculated human spermatozoa.CrossRef | 1:CAS:528:DC%2BD2cXhtVWgsr%2FF&md5=e863b2dcca52220c9fa21bec63a68dc2CAS | 15286043PubMed | open url image1

Pérez-Cerezales, S., Gutiérrez-Adán, A., Martínez-Páramo, S., Beirão, J., and Herráez, M. P. (2011). Altered gene transcription and telomere length in trout embryo and larvae obtained with DNA cryodamaged sperm. Theriogenology 76, 1234–1245.
Altered gene transcription and telomere length in trout embryo and larvae obtained with DNA cryodamaged sperm.CrossRef | 21741697PubMed | open url image1

Pollock, K., Gosálvez, J., Arroyo, F., López-Fernández, C., Guille, M., Noble, A., and Johnston, S. D. (2015). Validation of the sperm chromatin dispersion (SCD) test in the Amphibian Xenopus laevis using in situ nick translation and comet assay. Reprod. Fertil. Dev. 27, 1168–1174.
Validation of the sperm chromatin dispersion (SCD) test in the Amphibian Xenopus laevis using in situ nick translation and comet assay.CrossRef | 1:CAS:528:DC%2BC2MXhs1Glu7jI&md5=f841cbf8fbe967b20234e46b6a1a05e3CAS | 25482041PubMed | open url image1

Pratt, K. G., and Khakhalin, A. S. (2013). Modeling human neurodevelopmental disorders in the Xenopus tadpole: from mechanisms to therapeutic targets. Dis. Model. Mech. 6, 1057–1065.
Modeling human neurodevelopmental disorders in the Xenopus tadpole: from mechanisms to therapeutic targets.CrossRef | 23929939PubMed | open url image1

Said, T. M., Gaglani, A., and Agarwal, A. (2010). Implication of apoptosis in sperm cryoinjury. Reprod. Biomed. Online 21, 456–462.
Implication of apoptosis in sperm cryoinjury.CrossRef | 20800544PubMed | open url image1

Sargent, M. G., and Mohun, T. J. (2005). Cryopreservation of sperm of Xenopus laevis and Xenopus tropicalis. Genesis (New York, N.Y.: 2000) 41, 41–46.
Cryopreservation of sperm of Xenopus laevis and Xenopus tropicalis.CrossRef | open url image1

Schmitt, S. M., Gull, M., and Brändli, A. W. (2014). Engineering Xenopus embryos for phenotypic drug discovery screening. Adv. Drug Deliv. Rev. 69–70, 225–246.
Engineering Xenopus embryos for phenotypic drug discovery screening.CrossRef | 24576445PubMed | open url image1

Shi, Z., Wang, F., Cui, Y., Liu, Z., Guo, X., Zhang, Y., Deng, Y., Zhao, H., and Chen, Y. (2015). Heritable CRISPR/Cas9-mediated targeted integration in Xenopus tropicalis. FASEB J. 29, 4914–4923.
Heritable CRISPR/Cas9-mediated targeted integration in Xenopus tropicalis.CrossRef | 1:CAS:528:DC%2BC28Xns1Cis7w%3D&md5=415deabfeda1b6a00296bbeecab2ff51CAS | 26268927PubMed | open url image1

Wright, C., Milne, S., and Leeson, H. (2014). Sperm DNA damage caused by oxidative stress: modifiable clinical, lifestyle and nutritional factors in male infertility. Reprod. Biomed. Online 28, 684–703.
Sperm DNA damage caused by oxidative stress: modifiable clinical, lifestyle and nutritional factors in male infertility.CrossRef | 1:CAS:528:DC%2BC2cXmtlajsLc%3D&md5=dcd63d5c6b64658833124e5d28c2e4d1CAS | 24745838PubMed | open url image1

Yániz, J. L., Palacín, I., Vicente-Fiel, S., Gosalvez, J., López-Fernández, C., and Santolaria, P. (2013). Comparison of membrane-permeant fluorescent probes for sperm viability assessment in the ram. Reproduction in Domestic Animals 48, 598–603.
Comparison of membrane-permeant fluorescent probes for sperm viability assessment in the ram.CrossRef | 23293961PubMed | open url image1

Zini, A., San Gabriel, M., and Baazeem, A. (2009). Antioxidants and sperm DNA damage: a clinical perspective. J. Assist. Reprod. Genet. 26, 427–432.
Antioxidants and sperm DNA damage: a clinical perspective.CrossRef | 19768529PubMed | open url image1

Zribi, N., Feki Chakroun, N., El Euch, H., Gargouri, J., Bahloul, A., and Ammar Keskes, L. (2010). Effects of cryopreservation on human sperm deoxyribonucleic acid integrity. Fertil. Steril. 93, 159–166.
Effects of cryopreservation on human sperm deoxyribonucleic acid integrity.CrossRef | 1:CAS:528:DC%2BC3cXit1Wmt74%3D&md5=136a066bf66ea16ce8a5c537d0980fa7CAS | 19027111PubMed | open url image1


Full Text PDF (1.1 MB) Export Citation Cited By (1)