Instability of Trinucleotidic Repeats During Chromatin Remodeling in Spermatids

Abstract

Transient DNA breaks and evidence of DNA damage response have recently been reported during the chromatin remodeling process in haploid spermatids, creating a potential window of enhanced genetic instability. We used flow cytometry to achieve separation of differentiating spermatids into four highly purified populations using transgenic mice harboring 160 CAG repeats within exon 1 of the human Huntington disease gene (HTT). Trinucleotic repeat expansion was found to occur immediately following the chromatin remodeling steps, confirming the genetic instability of the process and pointing to the origin of paternal anticipation observed in some trinucleotidic repeats diseases.

Progressive Polyglutamine Repeat Expansion in Peripheral Blood Cells and Sperm of Transgenic Huntington’s Disease Monkeys

“…the increase of CAG repeat length in lymphocytes over time similar to that of sperm, which has not been reported in prior reports [29]. Additionally, the discrepancy between studies in rodent models and human HD patients, especially in sperm, could be due to the fundamental differences in spermatogenesis that impact CAG repeat instability in HD [16,19,[30][31][32]. Rodent goes through many developmental stages (19 steps) with multiple replication steps resulting in 10 times more sperm production compared to humans, while human only undergoes 6 developmental stages [30].…”

Step-specific Sorting of Mouse Spermatids by Flow Cytometry

“…Flow sorted spermatids obtained as described here can be used for a variety of experiments. DNA from these cells can easily be extracted using commercial genomic DNA purification kits and demonstrated to be of proper quality for PCR analyses 9 and next generation sequencing (data not shown). Sorted spermatids can also be used for protein analyses 9 where we performed several immunoblots against stage-specific markers to confirm the high purity of the sorted spermatids.…”

“…DNA from these cells can easily be extracted using commercial genomic DNA purification kits and demonstrated to be of proper quality for PCR analyses 9 and next generation sequencing (data not shown). Sorted spermatids can also be used for protein analyses 9 where we performed several immunoblots against stage-specific markers to confirm the high purity of the sorted spermatids. We verified the cellular integrity of the sorted spermatids and found that the nucleus and cytoplasm of all populations remain intact.…”

Repeat instability during DNA repair: Insights from model systems

“…For HD, which has a paternal bias for inheritance of expanded alleles, an illuminating study of repeat size in human male spermatocytes concluded that the majority of expansions occur as a pre-meiotic event, although some additional expansions were observed to occur post-meiotically (Yoon et al ., 2003). Yet in a mouse model of HD, it was concluded that most expansions arise after meiosis in the haploid gamete (Kovtun & McMurray, 2001), and recent results indicate that these post-meiotic expansions arise during remodeling of spermatid chromatin (Simard et al ., 2014). A factor that could explain the difference between the mouse and human data is that the human spermatogonial cells studied were estimated to have undergone many more cell divisions (~600–800 over 40 years) compared with those in the mice (~35 over <3 months).…”