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Title: The role of the oocyte activation factor PLCζ in human male factor infertility
Author: Kashir, Junaid
ISNI:       0000 0004 2724 5665
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2011
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General consensus agrees that the oscillations in intracellular calcium (Ca2+) leading to oocyte activation are induced by a testis-specific phospholipase C, PLCzeta (PLCζ). Strong evidence now indicates that certain types of human infertility are caused by failure of the sperm to activate the oocyte in an appropriate manner. Such sperm exhibit absent/reduced levels of PLCζ, while molecular analysis of the PLCζ gene in an infertile patient identified a substitution of a conserved histidine residue at position 398 of the PLCζ protein sequence for a proline residue (H398P), leading to severely reduced PLCζ function and oocyte activation efficiency. Collectively, these studies indicate the therapeutic and diagnostic potential use of PLCζ for assisted reproductive technology (ART). In the present study, a novel mutation was identified in the patient exhibiting the H398P mutation, which caused histidine to be substituted for leucine at position 233 (H233L) of the PLCζ protein sequence. The H233L mutation severely reduced PLCζ functional ability, and investigation of the inheritance patterns of H233L and H398P revealed that the patient inherited H233L from his mother and H398P from his father. Additionally, data suggested that quantitative immunofluorescence of PLCζ in human sperm may not be an informative indicator of oocyte activation capability, while proportional analysis of sperm exhibiting PLCζ immunoreactivity may reflect fertility. Density gradient washing (DGW) increased the proportion of human sperm exhibiting PLCζ immunofluorescence, while cryopreservation reduced total PLCζ immunofluorescence within fertile human sperm. While recombinant human PLCζ protein was successfully purified from a prokaryotic system, this proved to be inactive following mouse oocyte microinjection. Most importantly, the current study describes for the first time, the successful production of active recombinant human PLCζ protein within lysates from transfected transformed human embryonic kidney (HEK293T) cells, which induced characteristic patterns of Ca2+ release when injected into mouse oocytes. The present data indicate that wild type PLCζ was transcribed in an active state within a eukaryotic system, while loss-of-activity PLCζ isoforms may contribute to overall protein instability, possibly resulting in increased rates of protein degradation. It is demonstrated that PLCζ isoforms were localised to the endoplasmic reticulum (ER), with the EF-hand domain possibly determining the localisation of the enzyme. Collectively, these results not only assist with the therapeutic application of PLCζ, but also illustrate the complex nature of the mechanisms underlying its functional activity.
Supervisor: Coward, Kevin ; Parrington, John Sponsor: Nuffield Department of Obstetrics and Gynaecology
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biology ; Pharmacology ; Biochemistry ; Biology (medical sciences) ; Medical Sciences ; Genetics (life sciences) ; Enzymes ; Microscopy ; Obstetrics ; Development (zoology)