Date of Award
Doctor of Philosophy
Biochemistry and Cellular and Molecular Biology
Mary Ann Handel, Jeff Becker, Cynthia Peterson, Bem Culiat, Sundar Venkatachalam
Maintenance of genomic stability is of crucial importance for all organisms. Cells are continually exposed to exogenous and endogenous agents that can damage DNA. Of special interest is the manner in which male germ cells respond to and repair induced DNA double strand breaks (DSBs), as the propagation of cells carrying damaged DNA can lead to the production of heritable mutations, genetic diseases, and sterility. In addition, treatment of male germ cells with DNA damaging agents, such as many chemotherapeutic drugs, can ultimately result in infertility. This research focused on investigating the mechanisms by which male germ cells, specifically those in late prophase of meiosis I, from two species respond to exogenously induced DNA damage. The laboratory mouse and Drosophila melanogaster were chosen for our study primarily due to the differential requirements for repair proteins during normal spermatogenesis between the two species.
Two DNA-damaging agents were employed for in-vitro treatment of mouse pachytene spermatocytes: γ-irradiation and etoposide, a topoisomerase II inhibitor that results in persistent un-ligated DSBs. Drosophila late prophase spermatocytes were treated in-vivo with γ-irradiation and methyl methanesulfonate (MMS). Chromatin modifications associated with DSBs were monitored after exposure by examining immunolocalization of RAD51 (homologous recombination repair protein) and the phosphorylated variant of histone H2AX, γH2AX (mouse), or H2Av, γH2Av (Drosophila) (which modifies chromatin associated with DSBs).
An extensive comparative analysis of results from these two model systems showed that spermatocytes from the laboratory mouse and Drosophila melanogaster respond to the induction of DSBs by phosphorylating histone H2A variants, H2AX and H2Av, respectively. These results suggest that this is an evolutionarily conserved response in spermatocytes. In contrast, RAD51 was recruited to damaged chromatin only in mouse spermatocytes, indicating that spermatocytes from these two species repair DNA DSBs by different pathways.
Matulis, Shannon Marie, "Cross-Species Comparison of Spermatocyte Responses to Induced DNA Damage: the Laboratory Mouse and Drosophila melanogaster. " PhD diss., University of Tennessee, 2006.