Parp1-XRCC1 and the repair of DNA double strand breaks in mouse round spermatids.
The repair of DNA double strand breaks (DSBs) in male germ cells is slower and differently regulated compared to that in somatic cells. Round spermatids show DSB repair and are radioresistant to apoptosis induction. Mutation induction studies using ionizing irradiation, indicated a high frequency of chromosome aberrations (CA) in the next generation. Since they are in a G1 comparable stage of the cell cycle, haploid spermatids are expected to repair DSBs by the non-homologous end-joining pathway (NHEJ). However, immunohistochemical evidence indicates that not all components of the classical NHEJ pathway are available since the presence of DNA-PKcs cannot be shown. Here, we demonstrate that round spermatids, as well as most other types of male germ cells express both Parp1 and XRCC1. Therefore, we have determined whether the alternative Parp1/XRCC1 dependent NHEJ pathway is active in these nuclei and also have tested for classical NHEJ activity by a genetic method. To evaluate DSB repair in SCID mice, deficient for DNA-PKcs, and to study the involvement of the Parp1/XRCC1 dependent NHEJ pathway in round spermatids, the loss of gamma-H2AX foci after irradiation has been determined in nucleus spreads of round spermatids of SCID mice and in nucleus spreads and histological sections of Parp1-inhibited mice and their respective controls. Results show that around half of the breaks in randomly selected round spermatids are repaired between 1 and 8h after irradiation. The repair of 16% of the induced DSBs requires DNA-PKcs and 21% Parp1. Foci numbers in the Parp1-inhibited testes tend to be higher in spermatids of all epithelial stages reaching significance in stages I-III which indicates an active Parp1/XRCC1 pathway in round spermatids and a decreased repair capacity in later round spermatid stages. In Parp1-inhibited SCID mice only 14.5% of the breaks were repaired 8h after irradiation indicating additivity of the two NHEJ pathways in round spermatids.
Ahmed
Emad A
EA
Department of Endocrinology and Metabolism, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
de Boer
Peter
P
Philippens
Marielle E P
ME
Kal
Henk B
HB
de Rooij
Dirk G
DG
D000818:Animals; D015153:Blotting, Western; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded / Q000528:radiation effects; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005720:Gamma Rays; D005854:Germ Cells / Q000528:radiation effects; D006657:Histones / Q000378:metabolism; D007124:Immunoenzyme Techniques; D008297:Male; D051379:Mice; D016513:Mice, SCID; D009687:Nuclear Proteins / Q000378:metabolism; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D013087:Spermatids / Q000378:metabolism* / Q000528:radiation effects*; D013091:Spermatogenesis; D000076105:X-ray Repair Cross Complementing Protein 1
D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D006657:Histones; D009687:Nuclear Proteins; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D000076105:X-ray Repair Cross Complementing Protein 1; C000619352:Xrcc1 protein, mouse; C499321:Parp1 protein, mouse; C497264:Prkdc protein, mouse; C475961:gamma-H2AX protein, mouse