Journal Article

The Activation of DNA Damage Detection and Repair Responses in Cleavage-Stage Rat Embryos by a Damaged Paternal Genome

Lisanne Grenier, Bernard Robaire and Barbara F. Hales

in Toxicological Sciences

Volume 127, issue 2, pages 555-566
Published in print June 2012 | ISSN: 1096-6080
Published online March 2012 | e-ISSN: 1096-0929 | DOI: http://dx.doi.org/10.1093/toxsci/kfs120

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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.

Keywords: cyclophosphamide; γH2AX; 53BP1; poly(ADP-ribose) polymers; spermatozoa; embryo; epigenetic marks

Journal Article.  8213 words.  Illustrated.

Subjects: Medical Toxicology ; Toxicology (Non-medical)

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