Spermatogenesis and oogenesis create genetically unique haploid gametes in most sexually reproducing organisms. In comparison to oogenesis, spermatogenesis must occur in a narrow temperature range, typically 2-7°C below body temperature. In mammalian spermatocytes subjected to acute thermal stress, an increase in DNA damage occurs as well as a reduction in fertility. Despite these well documented effects on genome integrity and fertility, the exact mechanisms that cause temperature-induced heat stress in spermatogenesis remain unclear. Recent work in Caenorhabditis elegans revealed a large increase in DNA double-strand breaks (DSBs) marked by the recombinase protein RAD-51 when spermatocytes are exposed to a two-hour heat shock at 34°C. The temperature-induced DSBs are SPO-11 independent, suggesting other mechanisms are responsible for DSB formation. While a small portion of the temperature-induced DSBs were attributed to the transposition of the DNA transposable element Tc1, the origin of the remaining DSBs are unclear. To determine the other mechanisms that generate the temperature-induced DSBs, we will use RAD-51 chromatin immunoprecipitation followed by sequencing (ChIP-seq) on C. elegans males before and after heat-shock. From these experiments, we will define the wild type DSB landscape in C. elegans males. Also, we will determine where temperature-induced DSBs are found across the genome, therefore indicating how these DSBs are formed, including whether other transposable elements are activated upon heat shock. In addition, we will investigate the repair pathways required for resolving temperature-induced DSBs. In hermaphrodites, interhomolog recombination requires the protein RAD-50 to load RAD-51 onto DSB sites. To determine if interhomolog recombination is required to repair temperature-induced DSBs in males, we are utilizing a
rad-50 null mutation. Immunofluorescence of RAD-51 in
rad-50 mutant males before and after heat-shock will determine whether males require RAD-50 dependent RAD-51 loading during meiotic prophase I and if the RAD-50 pathway is required for repair of temperature-induced DNA damage in spermatocytes. Together, this work will establish the DSB landscape in C. elegans males and provide insight into how temperature-induced DSBs are both formed and repaired.