Protamine composition of koala and wombat spermatozoa provides new insights into DNA stability following cryopreservation
S. D. Johnston
A D , C. López-Fernández B , F. Arroyo B , R. Roy B , W. V. Holt C and J. Gosálvez B
+ Author Affiliations
- Author Affiliations
A School of Agriculture and Food Sciences, The University of Queensland, Gatton, Qld 4343, Australia.
B Department of Biology, Autonomous University of Madrid, Cantoblanco, Madrid, 28049, Spain.
C Academic unit of Reproductive and Developmental Medicine, University of Sheffield, Sheffield, S10 2TN, UK.
D Corresponding author. Email: s.johnston1@uq.edu.au
Reproduction, Fertility and Development 31(10) 1558-1566 https://doi.org/10.1071/RD18512
Submitted: 20 December 2018 Accepted: 2 May 2019 Published: 6 June 2019
Abstract
To investigate differences in the post-thaw DNA stability of koala and wombat spermatozoa, protamine amino acid sequences were compared and it was found that there were three more arginine residues for the wombat. Koala and wombat spermatozoa, cryopreserved using identical protocols, were examined for changes in sperm DNA fragmentation (SDF) dynamics over 24 h of post-thaw incubation. Following validation of a wombat sperm chromatin dispersion test, wombat DNA showed a rate of SDF that was 6-fold higher than for koala spermatozoa (P = 0.038). Finally, we examined whether expected differences in chromatin compactness, associated with protamine sequence, had an effect on restriction site accessibility of sperm DNA. Thawed spermatozoa were exposed to Alu I and EcoR1 endonuclease restriction enzymes and the SDF dynamics were observed. Koala spermatozoa exposed to Alu I showed a greater rate of SDF (P = 0.01), whereas wombat spermatozoa exposed to EcoR1 showed a greater rate of SDF (P = 0.032). We conclude that restriction sites in these species are differentially present or exposed and potentially account for differences in SDF dynamics. Although differences in the arginine composition of protamine may explain relative differences in SDF following cryopreservation, they do not support the hypothesis that increased arginine composition increases DNA stability; rather, increased arginine composition in the wombat may reduce post-thaw chromatin swelling.
Additional keywords: arginine, chromatin swelling, marsupials, sperm chromatin dispersion test, sperm DNA fragmentation.
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