Protein Ser/Thr Phosphatases

Continuous exchange of sequence information between dispersed Tc1 transposons in the Caenorhabditis elegans genome

Continuous exchange of sequence information between dispersed Tc1 transposons in the Caenorhabditis elegans genome. show that upon exposure to a brief 2C heat increase, spermatocytes exhibit up to a 25-fold increase in double strand DNA breaks (DSBs) throughout meiotic prophase I and a concurrent reduction in fertility. We demonstrate that these heat-induced DSBs in spermatocytes are independent of the endonuclease SPO-11. Further, we find that the production of these heat-induced DSBs in spermatocytes correlate with heat-induced mobilization of Tc1/transposable elements, K02288 which are known to cause DSBs and alter genome integrity[6, 7]. Moreover, we define the specific sequences and regions of the male genome that preferentially experience these heat-induced Tc1 insertions. In contrast, oocytes do not exhibit changes in DSB formation or Tc1 transposon mobility upon heat increases. Taken together, our data suggests spermatocytes are less tolerant of higher temperatures due an inability to effectively repress the movement of specific mobile DNA elements that cause excessive DNA damage and genome alterations, which can impair fertility. spermatocytes and oocytes, Kurhanewicz transposon mobilization, which compromise genome integrity and male fertility. Results and Discussion Elevated temperatures produce DNA damage in spermatocytes during meiotic prophase I Several studies indicate that spermatocytes in meiotic prophase I, as well as post-meiotic sperm, are particularly susceptible to thermal stress[1, 8, 9]. Further, the dysregulation of DNA damage and repair pathways during meiosis have been implicated as potential mechanisms linking heat-stress and DNA damage in spermatocytes[10]. To investigate this link between heat-stress and DNA damage in spermatocytes we exploited the model system which allows for isolation, visualization, and comparison of both developing oocytes and spermatocytes. To assess DNA damage during oogenesis and spermatogenesis, immunofluorescence for the recombinase RAD-51[11] was K02288 used in the germlines of wild-type adult males (exclusively undergoing spermatogenesis) and adult hermaphrodites (exclusively undergoing oogenesis). Immediately following a 2-hour heat-shock of whole animals at 34C (but not 32C nor extended exposure to 28C), we found that throughout meiotic prophase I, spermatocytes exhibit elevated RAD-51 foci (Figures 1, S1, and S2) that largely disappeared by 3-hours post-heat-shock (Physique S3). In contrast, heat-shocked oocytes did not display any increase in RAD-51 foci (Physique 1). In heat-shocked spermatocytes, RAD-51 foci increased nearly 3-fold in early pachytene nuclei (17 8.7 foci/nucleus (n=119) with heat-shock vs. 6.2 3.6 foci/nucleus (n=150) without heat-shock; p 0.0001; Physique 1) and Mouse monoclonal to FOXA2 late pachytene nuclei exhibited an approximately 25-fold increase in RAD-51 foci (4117 foci/nucleus (n=110) vs. 1.62.7 foci/nucleus without heat-shock (n=146); p 0.0001; Physique 1). In contrast to meiotic prophase I, heat-induced DNA damage was not observed in pre-meiotic S-phase nor in mitotically dividing spermatocytes (Physique S1A). This restriction of heat-induced DNA damage to meiotic prophase I suggests that feature(s) unique to spermatocytes in prophase I permit the formation of heat-induced DNA damage. Further, during the early L4 larval stage of hermaphrodites when their germline is usually exclusively undergoing spermatogenesis, a 34C heat shock also generates DNA damage (Physique S1B). These results in the spermatocytes of the early L4 hermaphrodite indicates the heat-induced DNA damage is not sex-specific but spermatocyte specific. Notably, heat-induced DNA damage in spermatocytes only occurs when the heat threshold of 34C is usually reached. At 36C and 38C, heat-induced DNA damage is usually no longer limited to prophase I and we observe RAD-51 foci in pre-meiotic S-phase region of the germline as well as the mitotically-dividing spermatocytes (Physique S2A). In addition, adult hermaphrodite germlines exposed to 36C and 38C also exhibit DNA damage continuously from the pre-meiotic region through meiotic prophase I (Physique S2B). Thus, once the threshold heat has been exceeded, it is possible that additional mechanisms begin to contribute to the production of heat-induced DNA damage outside of meiotic prophase I. Open in a separate window Physique 1. Acute exposure to heat stress produces DNA damage in male germlines and impairs male fertility. See also Figure S1, S2, and S3. (A) Representative immunofluorescence images of recombinase RAD-51 (green) from early and late pachytene regions of wild-type adult male germlines (spermatogenesis) and adult hermaphrodite germlines (oogenesis) with and without heat-shock (32C or 34C for 2 hours). Numbers on panels K02288 report average number of RAD-51 foci per nucleus for each group. Scale bar represents 5 m. (B) Quantification of RAD-51 foci per nucleus in early and late pachytene for wild-type spermatocytes and oocytes. Violin plots show frequency distribution of the data: center dashed line (median), bottom and top dotted lines (first and third quartiles respectively). Statistical significance between groups was decided using the Kruskal-Wallis non-parametric test, with Dunns test to account for multiple comparisons. Number of nuclei scored from male germlines: early pachytene no heat-shock, n=150; 32C, n=74; 34C, n=119; late pachytene no heat-shock, n=146; 32C, n=49; 34C, n=110. Hermaphrodite germlines: early pachytene no heat-shock, n=20; 32C, n=11; 34C, n=63, late pachytene no heat-shock, n=21; 32C, n=11; 34C, n=45. (C-F.) Male fertility.