Addressing sperm DNA integrity and fertilization; establishment of a PCR based method for detection of DNA damage (the MDDA assay)

• Main Author: Ryan, Jon Håvard
• Format: Dissertation
• Language: English

Humans in industrialized societies are continuously exposed to a plethora of environmental chemicals, of which the long-term consequences are largely unknown. Reduced fertility could be one such undesired consequence, and indeed reduced sperm quality is increasingly reported from many developed countries. Many environmental chemicals induce DNA damage, and sperm DNA damage is associated with reduced sperm quality, disturbed embryo development and early abortions. The present work was aimed at establishing a method to detect sperm DNA damage and to elucidate its impact on fertilization and early embryo development. Specifically we chose to study the widely distributed genotoxic benzo(a)pyrene (B(a)P) and glycidamide (GA; a metabolite of acrylamide), that most of us are exposed to on a daily basis. Determination of sperm DNA damage is a major challenge and at present there are no established protocols available. A less explored strategy is to assess DNA damage by the polymerase chain reaction (PCR). The easily accessible mitochondrial genome is more susceptible to DNA damage than the nuclear genome and thus represents a more sensitive target for the identification of sperm DNA damage. The PCR strategy involves a long amplicon (10 kb) for the determination of DNA damage relative to template number determined by a short fragment PCR assay (117 bp), the mitochondrial DNA damage assay (MDDA). The underlying concept is that DNA lesions will inhibit the DNA polymerase which is utilized in the long PCR assay to detect DNA damage: less PCR product equates to more DNA damage. The short PCR also reveals changes in mitochondrial genome numbers. The optimization of the method included determination of suitable DNA isolation and quantification procedures, along with appropriate PCR conditions, subsequently tested in cells exposed in vitro and frozen mouse tissue samples. Sperm and liver were harvested from mice exposed to B(a)P or GA a few days prior to sacrifice. The level of DNA damage in sperm and in liver was assessed by the MDDA. In parallel the sperm was used for in vitro fertilization experiments to determine fertilization rate and early embryo development. The MDDA proved successful in liver samples although more extensive optimization is required in sperm than was permitted within the scope of this MSc-thesis. Exposure to both B(a)P and GA gave rise to reduced fertilization rates with no indications of disturbance of early embryonal development at the doses used