Sperm Chromatin Fragmentation Occurs at Specific Chromatin Sites.

We previously demonstrated that when treated with divalent cations mouse epididymal spermatozoa fragment their DNA by inducing Topoisomerase 2B (TOP2B) to create double strand breaks at 25 kb to 50 kb intervals that can be reversed with EDTA. We term this sperm chromatin fragmentation (SCF). Our evidence supports a model that these DNA breaks occur at the sites of attachment of DNA loop domains to the sperm nuclear matrix. This model implies that the break points caused by TOP2B occur at specific sites in the chromatin. We tested this model using genome-wide DNA sequencing of the break points then verifying the specificity of individual break points using PCR. Epididymal spermatozoa were isolated and embedded in low-melting agarose plugs in a buffer containing 0.25% Triton X-100. The plugs were then treated with 10 mM MnCl2 and 10 mM CaCl2 for 1 hr at 37°C, then either placed directly in a digestion buffer containing 0.5% SDS, or incubated further with 50 mM EDTA for 30 min and then placed in a digestion buffer. The sperm DNA was then separated on agarose gels by field inversion gel electrophoresis. Spermatozoa treated with divalent cations fragmented their DNA to 25 kb to 50 kb sizes (SCF-DNA), but those that were subsequently treated with EDTA repaired the double strand breaks and their DNA remained near the top of the gel (SCFrev-DNA) similarly to control DNA from untreated spermatozoa. Sperm DNA was extracted from gel slices from control, divalent cation treated, and cation then EDTA treated samples. SCF-DNA was isolated and end labeled with biotin. The DNA was then fragmented further to much smaller sizes and biotin labeled fragments were isolated with antibodies to biotin using a method called "dDIP" for DNA damage immunoprecipitation. These fragments were sequenced using high throughput analysis, and mapped in the mouse genome. PCR primers were then developed that spanned the break points of the most frequently identified sequences. Control primers were designed to amplify similar sized fragments adjacent to the break points that would not be expected to be cleaved by SCF. These primers were used to test for the presence of the fragments in control DNA, SCF-DNA and SCFrev-DNA. The genome wide sequencing of SCF-DNA identified 3,004 possible break points. Of these, 43.1% (1,296) were identified more than 10 times, and 21.9% (657) were identified more than 100 times. We then focused on two sites that were identified more than 10,000 times, one on chromosome 9 and the other on chromosome 15. We amplified genomic DNA fragments of similar sizes that both crossed the putative break points, and were adjacent to them. In control DNA, both fragments were present. In SCF-DNA, only the regions adjacent to the break point could be amplified. In SCFrev-DNA, both fragments could be amplified, indicating that the break point was repaired correctly. SCF is a controlled breakage of the entire sperm chromatin to small fragments, but the cleavage sites are at specific points. These data support our model that SCF is the result of TOB2B induced double strand DNA breaks at specific sequences, probably at the sites of DNA attachment to the sperm nuclear matrix. They also indicate that the very condensed sperm chromatin retains enough enzymatic machinery to respond to its environment, which may also contribute to normal embryogenesis. This research was supported in part by NIH grants HD060722 (WSW) and HD36512 (SAK). (platform)

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