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Title: Mechanisms of reactive oxygen species generation by mammalian spermatozoa
Author: Fisher, Helen M.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1995
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The generation of reactive oxygen species (ROS) is an activity normally associated with phagocytic leucocytes, which employ an enzymatic complex, the NADPH oxidase, to catalyze the univalent reduction of molecular oxygen to superoxide. However, it is now apparent that many other cell types, including mammalian spermatozoa, generate ROS. ROS have been implicated in peroxidative damage associated male sub-fertility, and also more recently, in the regulation of normal sperm function. With the obvious biological importance of ROS generation by spermatozoa in mind, the aims of this PhD have been to identify and characterize the mechanisms, and cellular components involved in the generation of ROS by these cells. Experiments investigating ROS generation by human spermatozoa have shown that the mechanisms involved are functionally similar to those pertaining to the NADPH oxidase of phagocytic leucocytes. ROS generation by human spermatozoa is enzymatic in nature and utilizes NADPH as the electron donor, reducing molecular oxygen to superoxide, which subsequently dismutates to hydrogen peroxide. Further similarities with the NADPH oxidase include the probable involvement of a flavoprotein and a role for protein phosphorylation, as regulated by PKC and protein phosphatases. Biochemical and molecular analyses of human spermatozoa have shown that the cellular components that form the NADPH oxidase of phagocytic leucocytes are not present in the spermatozoon. Spectral analyses of human sperm membranes failed to reveal the presence of the characteristic low potential cytochrome b558, and similarly, western blot analyses of human spermatozoa failed to detect the cytochrome b558 or the presence of the two NADPH oxidase cytosolic components, p47phox and p67phox. ROS generating activity was extracted from human spermatozoa, using the non-ionic detergent n-octyl-β-D-thioglucoside. The solubilized protein extract was resolved by non-denaturing PAGE and shown to contain 6 bands with ROS generating activity. ROS generating activity was then isolated in an active form via 2'-5'ADP affinity chromatography. The individual components of this activity were subsequently separated via SDS-PAGE, which revealed a heterogeneous protein population. One of these proteins was purified, and a polyclonal antibody raised against it.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available