Temperature increases cause transposon-associated DNA damage specifically in spermatocytes Nicole A. Kurhanewicz, Devin Dinwiddie, Zachary D. Bush, and Diana E. Libuda University of Oregon, Institute of Molecular Biology, Department of Biology Sexually-reproducing organisms use meiosis to generate haploid gametes, such as sperm and eggs, to transmit their genome to the next generation. All tissues are susceptible to dramatic increases in temperature; however, developing sperm in the testes are unusually sensitive to small temperature fluctuations. Failure to thermoregulate spermatogenetic tissue and prolonged exposure to elevated temperatures are linked to male infertility. Further, temperature increases are known to cause DNA damage in spermatocytes, but the molecular mechanisms underlying this damage are unclear. Here we show that upon a brief heat-shock, the spermatocytes (but not oocytes) of Caenorhabditis elegans exhibit an increase in double strand DNA breaks (DSBs), that these temperature-induced DSBs occur via a SPO-11 independent pathway, and occur concurrent with impaired male fertility and temperature-induced transposon activity within the genome. Introduction Double strand DNA break (DSB) formation in Meiosis Figure 2. SPO-11 is a conserved endonuclease that induces double strand DNA breaks (DSBs) during meiosis. Soon after a DSB is formed, the recombinase RAD-51 binds to the 3’ DNA overhangs surrounding the DSB site. RAD-51 is required for DSB repair during meiosis and serves as a marker for meiotic DSB formation. A subset of DSBs are repaired as crossover recombination events, which are required to form the chiasma between homologous chromosomes necessary for proper chromosome segregation during the meiosis I division. Acute exposure to elevated temperature induces DSBs specifically in C. elegans spermatocytes Figure 3. (A) Immunofluorescence for the recombinase RAD-51 (green) and DNA/DAPI (blue) reveals that a two-hour 34°C heat shock causes a dramatic increase in RAD-51 foci (green) per nucleus (DNA in blue) specifically in wild type spermatocytes throughout the meiotic region of the male germ line. (B) Wild type oocytes do not exhibit any temperature-induced RAD-51 foci in any part of the meiotic prophase I. Temperature-induced DSBs progress with repair less than 3 hours after formation in spermatocytes Figure 7. The appearance and disappearance of a RAD-51 focus indicate the initiation and subsequent progression of a DSB through a repair pathway. Measurement of RAD-51 foci counts before heat shock and at 1-hour time points following heat shock reveal that most, if not all, of the temperature-induced DSBs have unloaded RAD-51 and progressed down a repair pathway by 3 hours following heat shock. Funding Sources Increase in Tc1 transposon expression and insertion sites correspond with temperature-induced DSBs Figure 9. Quantitative PCR for Tc1a and Tc3 transposase expression reveals that Tc1a transposase expression and not Tc3 transposase is increased significantly following heat shock. Expression levels were all normalized to act-1 expression levels. 1. Increased temperatures cause RAD-51 marked DNA damage specific to spermatocytes during meiotic prophase I. 2. Temperature-induced DNA damage forms SPO-11 independent double strand DNA breaks that have the capacity to repaired as crossover recombination events. 3. Temperature-induced DNA damage during meiotic prophase I correlates with reduced male fertility 4. Tc1 transposons excise and insert into the genome upon temperature increases in wild-type spermatocytes. Summary Tc1 transposon positions in the spermatocyte genome become altered following temperature increases Figure 10. Tc1 mobility was assessed globally and across individual chromosomes. (A) Inverse PCR paired with amplicon sequencing was used to identify Tc1 locations genome-wide. Representative plots for Chromosome II from adult males and hermaphrodite populations are shown. (B) To evaluate transposo