Abstract
Current approaches for DNA extraction and fragmentation from mammalian spermatozoa provide several challenges for the investigation of the oxidative stress burden carried in the genome of male gametes. Indeed, the potential introduction of oxidative DNA damage induced by reactive oxygen species, reducing agents (dithiothreitol or beta-mercaptoethanol), and DNA shearing techniques used in the preparation of samples for chromatin immunoprecipitation and next-generation sequencing serve to cofound the reliability and accuracy of the results obtained. Here we report optimised methodology that minimises, or completely eliminates, exposure to DNA damaging compounds during extraction and fragmentation procedures. Specifically, we show that Micrococcal nuclease (MNase) digestion prior to cellular lysis generates a greater DNA yield with minimal collateral oxidation while randomly fragmenting the entire paternal genome. This modified methodology represents a significant improvement over traditional fragmentation achieved via sonication in the preparation of genomic DNA from human spermatozoa for downstream applications, such as next-generation sequencing. We also present a redesigned bioinformatic pipeline framework adjusted to correctly analyse this form of data and detect statistically relevant targets of oxidation.
Highlights
High levels of oxidative damage in the DNA of mature humans spermatozoa is a biological feature associated with reduced fertility
Without exposure to DTT and Triton X-100 to permeabilise and relax the chromatin structure before exposure to Micrococcal nuclease (MNase) (Fig 1.12 and 1.15), DNA fragmentation was impaired by restricting digestion to specific chromatin locations leading to the generation of a large number of DNA fragments approximately 50 kb in size, which correspond to the size of toroids; a few DNA fragments below 100bp in size and no fragments in our desired range of 200 to 2,000
We showed that the combination of MNase digestion to generate DNA fragments and removal of oxidising and reducing compounds from cell lysis and DNA extraction processes improved significantly the experimental yield while inflicting the least amount of oxidative damage to DNA
Summary
High levels of oxidative damage in the DNA of mature humans spermatozoa is a biological feature associated with reduced fertility. The oocyte expresses OGG-1 at relatively low levels which limit its capacity to effect the removal of oxidative damage and DNA repair [9] This situation leads to the attendant risk of increasing the mutational load carried by the offspring. A small number of studies have utilised modified chromatin immunoprecipitation techniques (ChIP) [25,26,27] in conjunction with high-throughput sequencing [28, 29] in an attempt to identify genomic sites vulnerable to oxidative attack and subsequently correlate this information with chromatin structure and the arrangement of DNA packaging inside the nucleus of mature spermatozoa [30,31,32,33,34]. The development and implementation of these modified protocols and bioinformatic framework will provide the basis to conduct future analyses into the genetic consequences of oxidative stress in sperm cells of humans and other species
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
