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Vertebrate reproductive science and technology
RESEARCH ARTICLE

Directional mapping of DNA nicking in ejaculated and cauda epididymidal spermatozoa of the short-beaked echidna (Tachyglossus aculeatus: Monotremata)

S. D. Johnston A D , C. López-Fernández B , A. Gosálbez B , W. V. Holt C and J. Gosálvez B
+ Author Affiliations
- Author Affiliations

A School of Animal Studies, The University of Queensland, Gatton, Qld 4343, Australia.

B Autonomous University of Madrid, Cantoblanco 20849, Spain.

C Institute of Zoology, Zoological Society of London, London NW1 4RY, UK.

D Corresponding author. Email: stevejohnston@uqconnect.net

Reproduction, Fertility and Development 21(8) 1008-1014 https://doi.org/10.1071/RD09079
Submitted: 30 March 2009  Accepted: 7 June 2009   Published: 30 October 2009

Abstract

Prototherian spermatozoa are unique amongst the Mammalia in terms of their filiform morphology, tandem arrangement of chromosomes and formation of sperm bundles. In the present study, we provide observations of echidna spermatozoa and note that the superstructure of the bundle is engineered around the shape of the individual sperm head and that this in turn may be a consequence of the unusual circumferential and helicoidal condensation of the DNA during spermiogenesis. Frozen–thawed ejaculated echidna spermatozoa were incubated and examined for the presence of non-typical DNA conformation by means of in situ labelling of DNA breaks using Klenow polymerase and via alkaline single-cell comet assays for detection of fragmented DNA. Both techniques successfully revealed the presence of what appeared to be directional DNA nicking, co-localised with the presence of highly sensitive alkali sites along the length of the sperm nucleus. It was not possible to define whether these alternative DNA configurations were associated with a failure of the sperm nucleus to condense appropriately during spermiogenesis or were evidence of DNA fragmentation following post-thaw incubation or a sequential structural chromatin rearrangement necessary for fertilisation.

Additional keywords: comet assay, sperm chromatin dispersion assay.


Acknowledgements

The authors thank Ms Paqui Arroyo for her technical help. This work was supported by the Ministry of Education and Science, Spain – Grants BFU 2007–66340/BFI; CGL2005–02898/BOS.


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