General consensus agrees that the oscillations in intracellular calcium (Ca2+) leading to oocyte activation are induced by a testis-specific phospholipase C, PLCzeta (PLCS). 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 PLCS, while molecular analysis of the PLCS gene in an infertile patient identified a substitution of a conserved histidine residue at position 398 of the PLCS protein sequence for a proline residue (H398P), leading to severely reduced PLCS function and oocyte activation efficiency. Collectively, these studies indicate the therapeutic and diagnostic potential use of PLCs 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 PLCS protein sequence. The H233L mutation severely reduced PLCS 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 PLCS in human sperm may not be an informative indicator of oocyte activation capability, while proportional analysis of sperm exhibiting PLCS immunoreactivity may reflect fertility. Density gradient washing (DGW) increased the proportion of human sperm exhibiting PLCS immunofluorescence, while cryopreservation reduced total PLCS immunofluorescence within fertile human sperm. While recombinant human PLCS protein was successfully purified from a prokaryotic system, this proved to be inactive following mouse oocyte micro injection. Most importantly, the current study describes for the first time, the successful production of active recombinant human PLCS 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 PLCS was transcribed in an active state within a eukaryotic system, while loss-of-activity PLCS isoforms may contribute to overall protein instability, possibly resulting in increased rates of protein degradation. It is demonstrated that PLCS 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 PLCs, but also illustrate the complex nature of the mechanisms underlying its functional activity.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:558444 |
| Date | January 2011 |
| Creators | Kashir, Junaid |
| Contributors | Coward, Kevin ; Parrington, John |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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