Biol Reprod Keystone Symposia Conference on Frontiers in Reproductive Biology & Regulation of Fertility.
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BOR - Papers in Press, published online ahead of print March 16, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.036939
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Submitted October 5, 2004
Returned for revision November 16, 2004
Accepted March 9, 2005

Gamete Biology


On the Nature and Origin of DNA Strand Breaks in Elongating Spermatids

Rémi-Martin Laberge and Guylain Boissonneault *

* To whom correspondence should be addressed. E-mail: guylain.boissonneault{at}usherbrooke.ca.

Abstract
Transient DNA strand breaks are generated in the whole population of elongating spermatids and are perfectly coincident with histone H4 hyperacetylation at chromatin-remodeling steps. Given the limited DNA repair capacity of elongating spermatids, chromatin remodeling may present a threat to genetic integrity of the male gamete. The nature of the DNA strand breakage, the enzymes involved and the role of H4 hyperacetylation in the process must be determined to further investigate the potential mutagenic consequences of this important transition. We used the metachromatic dye acridine orange in combination with fluorescence-activated cell sorting (FACS) in order to achieve separation of spermatids according to their condensation state. Using single-cell electrophoresis (comet assay), in both alkaline and neutral conditions, we demonstrated that double-stranded breaks account for most of the DNA fragmentation observed in purified elongating spermatids. DNA strand breaks were generated in round spermatids as a result of de novo histone hyperacetylation induced by TSA whereas an increase in endogenous DNA strand breaks was observed in elongating spermatids. Using a short-term culture of testicular cells, we demonstrated that DNA strand breaks in spermatids were abolished upon incubation with two functionally different topoisomerase II inhibitors. Hence, topoisomerase II appears as the unique enzyme responsible for the transient double-stranded breaks in elongating spermatids but depends on histone hyperacetylation for its activity.

Key words: Gamete Biology • Developmental biology • Gametogenesis • Spermatid • Spermatogenesis


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