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Title: The oocyte activation factor, phospholipase C zeta (PLCζ) : potential mechanisms of action and scope for human infertility treatment
Author: Ramadan, Walaa M.
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2013
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It is widely believed that the sperm-specific protein phospholipase C zeta (PLCζ) is released into the oocyte upon fusion and initiates calcium (Ca2+) oscillations which regulate oocyte activation. Reduced amounts, abnormal localisation patterns, and aberrant forms of PLCζ underlie certain types of male-factor infertility, prompting significant interest in the use of this fundamental protein as a clinical therapeutic agent or diagnostic marker. This thesis systematically addressed a range of outstanding questions relating to the expression, localisation, and therapeutic/diagnostic application of PLCζ. Using immunohistochemical and quantitative immunofluorescence techniques, the current study investigated the effect of paternal age and epididymal maturation upon PLCζ expression and localisation in mouse sperm. Neither age nor epididymal maturity exhibited significant effect upon the total level of PLCζ in mouse sperm, although there was a significant trend towards post-acrosomal localisation as sperm matured during transit in the epididymis. Immunohistochemical analysis of mouse testis showed that the expression of PLCζ first appeared at the roundspermatid stage of spermatogenesis. Paternal age did not influence total levels of PLCζ expression or localisation pattern in human sperm samples (n=29), with PLCζ being predominantly localised to the post-acrosomal region. A significant reduction (34%) was detected in the proportion of sperm exhibiting PLCζ from an oocyte-activation deficient patient compared with fertile controls. Novel immunohistochemical analyses suggested, for the first time, that PLCζ is first expressed from the round spermatid stage in humans, and that the analysis of PLCζ expression in human testicular biopsies (n=29) may provide credible indication of the oocyte activation ability of surgically-retrieved sperm samples from infertile patients. Initial data also suggest that flow cytometry may serve as a useful prognostic/diagnostic tool for the quantitative analyses of PLCζ expression in human sperm samples. Mammalian and bacterial expression systems were utilised to generate recombinant human PLCζ protein as a therapeutic agent and for the generation of a novel monoclonal anti-PLCζ antibody respectively. Expression studies using HEK293T cells demonstrated that human PLCζ appeared to localise to the endoplasmic reticulum and that the EF hand domain appeared to be involved in regulating this pattern of localisation. Collectively, these studies extend our fundamental knowledge of how PLCζ is expressed in developing sperm and assist in the translation of PLCζ as a clinical therapeutic and diagnostic biomarker for oocyte activation ability.
Supervisor: Coward, Kevin Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available