Loss of activity mutations in phospholipase C zeta (PLC?) abolishes calcium oscillatory ability of human recombinant protein in mouse oocytes  

Oleh:

Kashir, Junaid ; Jones, Celine ; Lee, Hoi Chang ; Rietdorf, Katja

Jenis: Article from Journal - ilmiah internasional
Dalam koleksi: Human Reproduction vol. 26 no. 12 (Dec. 2011), page 3372-3387.
Topik: Oocyte activation; assisted oocyte activation; sperm; phophospholipase C zeta (PLCzeta); male infertility

Ketersediaan

Perpustakaan FK Nomor Panggil: H07.K.2011.02 Non-tandon: 1 (dapat dipinjam: 0) Tandon: tidak ada

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Isi artikel

BACKGROUND Mammalian oocyte activation occurs via a series of intracellular calcium (Ca2+) oscillations thought to be induced by a sperm-specific phospholipase C zeta (PLC?). There is now strong evidence to indicate that certain types of human male infertility are caused by failure of the sperm to activate the oocyte in an appropriate manner. Molecular analysis of the PLC? gene of a male patient with oocyte activation deficiency has previously identified a point mutation causing a histidine to proline substitution at PLC? residue 398 (PLC?H398P), leading to abnormal Ca2+ release profiles and reduced oocyte activation efficiency. METHODS AND RESULTS In the present study, we used HEK293T cells to produce recombinant human wild-type PLC? (PLC?WT) protein which, upon microinjection into mouse oocytes, induced Ca2+ oscillations characteristic of oocyte activation. Injection of recombinant PLC?H398P was unable to elicit Ca2+ oscillations in mouse oocytes. Loss of activity mutations, such as PLC?H398P and an artificially induced frameshift mutation (PLC??YC2) did not affect Ca2+ release when over-expressed in HEK293T cells, whereas PLC?WT inhibited adenosine triphosphate-activated Ca2+ release. Confocal imaging of fluorescently tagged PLC? isoforms in HEK293T cells suggested a cytoplasmic pattern of localization, while quantitative analysis of fluorescence levels showed that PLC?WT > PLC?H398P > PLC??YC2, indicating that loss of activity mutations may lead to protein instability. This was further indicated by the low proportion of sperm and the lower levels of total PLC? immunofluorescence from the patient exhibiting PLC?H398P compared with fertile controls. CONCLUSIONS We demonstrate, for the first time, the production of active recombinant human PLC? protein which retained the ability to elicit characteristic Ca2+ oscillations in mouse oocytes, an ability which was eliminated by an infertility-linked mutation. These findings advance our understanding of PLC?, and provide a critical step forward in obtaining purified PLC? protein as a potential therapeutic agent for oocyte activation deficiency.

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