title: The activation of DNA damage detection and repair responses in cleavage-stage rat embryos by a damaged paternal genome.
abstract: Male germ cell DNA damage, after exposure to radiation, exogenous chemicals, or chemotherapeutic agents, is a major cause of male infertility. DNA-damaged spermatozoa can fertilize oocytes; this is of concern because there is limited information on the capacity of early embryos to repair a damaged male genome or on the fate of these embryos if repair is inadequate. We hypothesized that the early activation of DNA damage response in the early embryo is a critical determinant of its fate. The objective of this study was to assess the DNA damage response and mitochondrial function as a measure of the energy supply for DNA repair and general health in cleavage-stage embryos sired by males chronically exposed to an anticancer alkylating agent, cyclophosphamide. Male rats were treated with saline or cyclophosphamide (6 mg/kg/day) for 4 weeks and mated to naturally cycling females. Pronuclear two- and eight-cell embryos were collected for immunofluorescence analysis of mitochondrial function and biomarkers of the DNA damage response: γH2AX foci, 53BP1 reactivity, and poly(ADP-ribose) polymer formation. Mitochondrial activities did not differ between embryos sired by control- and cyclophosphamide-exposed males. At the two-cell stage, there was no treatment-related increase in DNA double-strand breaks; by the eight-cell stage, a significant increase was noted, as indicated by increased medium and large γH2AX foci. This was accompanied by a dampened DNA repair response, detected as a decrease in the nuclear intensity of poly(ADP-ribose) polymers. The micronuclei formed in cyclophosphamide-sired embryos contained large γH2AX foci and enhanced poly(ADP-ribose) polymer and 53BP1 reactivity compared with their nuclear counterparts. Thus, paternal cyclophosphamide exposure activated a DNA damage response in cleavage-stage embryos. Furthermore, this damage response may be useful in assessing embryo quality and developmental competence.
authors.affiliation: Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec H3G 1Y6, Canada.
authors.forename: Lisanne
authors.initials: L
authors.lastname: Grenier
authors.forename: Bernard
authors.initials: B
authors.lastname: Robaire
authors.forename: Barbara F
authors.initials: BF
authors.lastname: Hales
mesh_terms: D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000633:toxicity*; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D002970:Cleavage Stage, Ovum / Q000378:metabolism* / Q000473:pathology; D003520:Cyclophosphamide / Q000633:toxicity*; D004249:DNA Damage; D004260:DNA Repair; D004734:Energy Metabolism; D005260:Female; D005306:Fertilization; D006657:Histones / Q000378:metabolism; D008297:Male; D048629:Micronuclei, Chromosome-Defective; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism* / Q000473:pathology; D018812:Paternal Exposure; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D013094:Spermatozoa / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology
chemical_list: D018906:Antineoplastic Agents, Alkylating; D002868:Chromosomal Proteins, Non-Histone; D006657:Histones; D011064:Poly Adenosine Diphosphate Ribose; D003520:Cyclophosphamide