Reproduction Abstracts

Introduction: Sperm DNA is prone to suffer damage that could have a negative impact on the progeny development. Moreover genes packaged in different chromatin blocks or located in different nuclear areas, could have differential sensitivity to genotoxic agents. Our group has demonstrated that in rainbow trout, with the nucleus homogeneously compacted with protamines – lacking histones –, the distribution of damage is dependent on the genotoxic agent. The present work is aimed to analyze the potential differential susceptibility to DNA cryodamage.

Material and methods: Sperm was cryopreserved following two different protocols: using LDL or egg yolk in the extender and loading in straws or cryovials respectively. The number of lesion per 10 kb in nine different genes, with different relevance for developmental decisions, was analyzed with a qPCR approach. The presence of 8-hydroxyguanosine, DNA strand breaks and their co-localization with nuclear matrix proteins was analyzed by immunofluorescence, TUNEL, and confocal microscopy.

Results and discussion: The two freezing protocols promoted similar level of DNA lesions but significant differences were observed between genes. The immunodetection of specific lesions demonstrated a heterogeneous distribution of 8-hydroxyguanosine into the nucleus, located both in the peripheral and the inner areas of the nucleus. Also some lesions better co-localize with nuclear matrix proteins. The differential susceptibility and location of lesions did not match with the damage promoted by oxidative stress with H₂O₂, suggesting that other factors, apart from the generation of ROS, could render specific nuclear territories more sensitive to cryopreservation practices.

Acknowledgements: Supported by the Spanish Ministry of Economy and Competitiveness (AGL2011-27787).