Doctor of Philosophy (PhD) / The field of epigenetics emerged primarily from studies in Drosophila, and is now being studied intensively by mammalian biologists. In order to increase our knowledge of epigenetic gene control in the mouse, I have studied modifiers of epigenetic gene silencing. My main method of investigation involved the characterisation of mutants from a sensitised ENU mutagenesis screen performed previously in our laboratory. The screen was carried out in an FVB/NJ strain carrying a variegating GFP transgene expressed in erythrocytes. To date we have recovered 12 dominant (D) and seven recessive (R) mutant mouse lines from this screen that display altered transgene expression. We have named these Mommes (Modifiers of murine metastable epialleles). I investigated the phenotype and attempted to identify the underlying causative mutation of two of these Momme mutants. MommeD6 is a semi-dominant, homozygous lethal mutation that acts as a suppressor of variegation with respect to the GFP transgene. This mutation has a large effect on the level of expression of the transgene in expressing cells, but little effect on the percentage of cells expressing the transgene. MommeD6 is linked to a 2.5 Mbp interval on chromosome 14. MommeD9 is a semi-dominant, homozygous lethal mutation that acts as an enhancer of variegation with respect to the GFP transgene. Mutants have a tendency to become obese as they age, show abnormal haematology profiles, and females develop infertility. MommeD9 is linked to a 17.4 Mbp region on chromosome 7. I produced and studied a strain carrying the same GFP transgene but in a new strain background, C57BL/6J. This strain provided an opportunity to look for strain-specific modifiers of expression of the GFP transgene. Several regions were mapped to chromosomal locations. Further work will be needed to identify the genes involved. This mouse will be useful in future mutagenesis screens of this type.

Research Doctorate - Doctor of Philosophy / On leaving the testis, spermatozoa can neither swim nor fertilize the oocyte. These functional properties are acquired as spermatozoa engage in a process of post-testicular maturation in the epididymis. The studies described in this thesis were designed to elucidate some of the fundamental mechanisms associated with the regulation of epididymal maturation in mouse spermatozoa. The initial studies described in this thesis investigated the expression of a cAMP/PKAdependent, tyrosine phosphorylation signaling pathway that becomes activated during epididymal sperm maturation. It was demonstrated that the entry of spermatozoa into the epididymis was accompanied by the sudden stimulation of this pathway, initially in the principal piece of the cell and subsequently in the midpiece. The competence of these cells to phosphorylate the entire sperm tail, particularly the mitochondria, was accompanied by a capacity to exhibit hyperactivated motility on stimulation with cAMP. A distinctly different pattern of tyrosine phosphorylation involving the acrosomal domain of the sperm head was provoked as spermatozoa entered the caput epididymis and then remained high until these cells entered the distal corpus and cauda. However, tyrosine dephosphorylation of the sperm acrosomal domain during epididymal transit did not appear to be functionally involved in controlling the acrosome reaction. Research into the biochemical basis of sperm epididymal maturation revealed that this process was associated with the activation of sperm mitochondria, leading to the creation of a mitochondrial membrane potential (MMP) and activation of mitochondrial free radical generation. Immature caput spermatozoa displayed a low MMP whereas mature caudal spermatozoa actively maintained a high MMP. Moreover mitochondrial generation of reactive oxygen species (ROS) could be triggered by antimycin A in mature caudal spermatozoa but not in immature caput spermatozoa, suggesting a lack of electron flux in the latter. The molecular mechanisms responsible for regulating mitochondrial function were also found to be reversible, as washing the cells free of epididymal fluid allowed caput spermatozoa to acquire a high MMP and generate ROS while incubating caudal spermatozoa in caput epididymal fluid, suppressed MMP and their ability to generate ROS. Pharmacological suppression of mitochondrial activity was subsequently found to be associated with the inhibition of hyperactivated motility. These results strongly suggested that fluid from the caput epididymis contained a mitochondrial inhibitor and that activation of mitochondrial activity was due to the removal or inactivation of this inhibitor during epididymal transit. This causative factor was not species specific. Incubation of ejaculated human spermatozoa in murine epididymal fluid systematically suppressed their MMP. The characterization of caput epididymal fluid suggested that the putative mitochondrial inhibitor is a heat-resistant protein with a molecular weight larger than 30 kDa. The final results presented in this thesis demonstrate that a full-length Riken protein is a potential candidate for the putative mitochondrial inhibitor that switches off mitochondrial function in caput spermatozoa. Indeed, these results represent the first report suggesting that the epididymal maturation is associated with activation of sperm mitochondria and the first study of a testis specific protein that could be a regulator of mitochondrial function in the male germ line. Further characterization of the mechanisms by which epididymal spermatozoa control mitochondrial function may hold the key to our understanding of sperm maturation. It may also lead us to a clear exposition of the molecular basis of human male infertility, potentially serve as a target for infertility treatment and possibly contribute to the development of novel contraceptive agents.

Thesis (M. Sc.)--University of Canterbury, 2008. / Typescript (photocopy). "May 2008." Includes bibliographical references (p. 55-60). Also available via the World Wide Web.

Thesis (M. Sc.)--University of Canterbury, 2008. / Typescript (photocopy). Includes bibliographical references (leaves 109-118). Also available via the World Wide Web.

Thesis (Ph.D.)--Cleveland State University, 2008. / Abstract. Title from PDF t.p. (viewed on Oct. 8, 2008). Includes bibliographical references. Available online via the OhioLINK ETD Center. Also available in print.

Thesis (Ph. D.)--University of Texas at Austin, 2003. / Supervisor: Jaquelin P. Dudley. Vita. Includes bibliographical references. Also available from UMI.

Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references. Available also in a digital version from UMI/Dissertation Abstracts International.

Thesis (Ph.D.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 182-211) Also available in print.

Thesis (Ph. D.)--University of Texas at Austin, 2005. / Supervisor: Jaquelin P. Dudley. Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2005. / Supervisor: Martin Poenie. Vita. Includes bibliographical references.