DNA Double Strand Break Response and Limited Repair Capacity in Mouse Elongated Spermatids

Abstract

Spermatids are extremely sensitive to genotoxic exposures since during spermiogenesis only error-prone non homologous end joining (NHEJ) repair pathways are available. Hence, genomic damage may accumulate in sperm and be transmitted to the zygote. Indirect, delayed DNA fragmentation and lesions associated with apoptotic-like processes have been observed during spermatid elongation, 27 days after irradiation. The proliferating spermatogonia and early meiotic prophase cells have been suggested to retain a memory of a radiation insult leading later to this delayed fragmentation. Here, we used meiotic spread preparations to localize phosphorylate histone H2 variant (γ-H2AX) foci marking DNA double strand breaks (DSBs) in elongated spermatids. This technique enabled us to determine the background level of DSB foci in elongated spermatids of RAD54/RAD54B double knockout (dko) mice, severe combined immunodeficiency SCID mice, and poly adenosine diphosphate (ADP)-ribose polymerase 1 (PARP1) inhibitor (DPQ)-treated mice to compare them with the appropriate wild type controls. The repair kinetics data and the protein expression patterns observed indicate that the conventional NHEJ repair pathway is not available for elongated spermatids to repair the programmed and the IR-induced DSBs, reflecting the limited repair capacity of these cells. However, although elongated spermatids express the proteins of the alternative NHEJ, PARP1-inhibition had no effect on the repair kinetics after IR, suggesting that DNA damage may be passed onto sperm. Finally, our genetic mutant analysis suggests that an incomplete or defective meiotic recombinational repair of Spo11-induced DSBs may lead to a carry-over of the DSB damage or induce a delayed nuclear fragmentation during the sensitive programmed chromatin remodeling occurring in elongated spermatids.

Keywords: DNA repair; NHEJ; PARP1-inhibited mice; Rad54/Rad54B deficient mice; SCID mice; elongated spermatids.

Figure 1

Localization of the phosphorylated-H2 variant (γ-H2AX, green) (A), 53 binding protein1 (53BP1, red, B,C and green, D), Ku70 (red, E,F), X-ray repair cross-complementing protein 1 (PARP1) (G) and X-ray repair cross-complementing protein1 (XRCC1) (H) in non-irradiated testis sections; (I) Meiotic spreads of elongated spermatids from RAD54/RAD54B deficient mice stained for γ-H2AX and counterstained with DAPI (blue). Elongated spermatids (Esp), Leptotene (Lep), Pachytene (Pachy), Sertoli (Ser). Scale bars: 10 µm.

Figure 2

Representative images from non-irradiated meiotic spreads of pachytene spermatocytes and elongated spermatids stained for γ-H2AX (red) from wild type (A,B); RAD54/RAD54B double knockout mice (C,D); Severe Combined Immunodeficiency (SCID) mice (E,F) and PARP1 inhibitor (DPQ)-treated mice (G,H). In pachytene, spermatocytes of Rad54/Rad54B dko mice more large (L) foci can be seen (arrows, C), more foci in elongated spermatids are also shown in addition to the staining background (D). Scale bars: 10 µm.

Figure 3

Representative images from irradiated meiotic spreads of pachytene spermatocytes and elongated spermatids stained for γ-H2AX, at 1 h and 8 h after ionized radiation (IR) in wild type (A,B); RAD54/RAD54B double knockout mice (C,D); SCID mice (E,F) and PARP1 inhibitor (DPQ)-treated mice (G,H). Scale bars: 10 µm.

Figure 4

The kinetics of γ-H2AX foci formation and removal after IR in elongated spermatids. Number of γ-H2AX foci per cell in RAD54/RAD54B double knockout mice (A), SCID mice (B) and PARP1 inhibitor (DPQ)-treated mice (C) relative to the wild type mice. Between 40 and 50 cells were counted per mouse, data are presented as mean ± SEM, n = 3. p < 0.01, ^a compared to 8 h irradiated B6.129, ^b compared to the elongated spermatids in all non-irradiated mice. Percentages of repaired breaks after 8 h post- IR in wild type and other mice (D).

Figure 4

The kinetics of γ-H2AX foci formation and removal after IR in elongated spermatids. Number of γ-H2AX foci per cell in RAD54/RAD54B double knockout mice (A), SCID mice (B) and PARP1 inhibitor (DPQ)-treated mice (C) relative to the wild type mice. Between 40 and 50 cells were counted per mouse, data are presented as mean ± SEM, n = 3. p < 0.01, ^a compared to 8 h irradiated B6.129, ^b compared to the elongated spermatids in all non-irradiated mice. Percentages of repaired breaks after 8 h post- IR in wild type and other mice (D).