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

Junaid Kashir, Celine Jones, Hoi Chang Lee, Katja Rietdorf, Dimitra Nikiforaki UGent, Claire Durrans, Margarida Ruas, Sze Tian Tee, Björn Heindryckx UGent and Antony Galione, et al. (2011) HUMAN REPRODUCTION. 26(12). p.3372-3387
abstract
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 zeta). 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 zeta gene of a male patient with oocyte activation deficiency has previously identified a point mutation causing a histidine to proline substitution at PLC zeta residue 398 (PLC zeta(H398P)), leading to abnormal Ca(2+) 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 zeta (PLC zeta(WT)) protein which, upon microinjection into mouse oocytes, induced Ca(2+) oscillations characteristic of oocyte activation. Injection of recombinant PLC zeta H398P was unable to elicit Ca(2+) oscillations in mouse oocytes. Loss of activity mutations, such as PLC zeta(H398P) and an artificially induced frameshift mutation (PLC zeta DYC2) did not affect Ca(2+) release when over-expressed in HEK293T cells, whereas PLC zeta(WT) inhibited adenosine triphosphate-activated Ca(2+) release. Confocal imaging of fluorescently tagged PLC zeta isoforms in HEK293T cells suggested a cytoplasmic pattern of localization, while quantitative analysis of fluorescence levels showed that PLC zeta(WT). PLC zeta(H398P). PLC zeta Delta(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 zeta immunofluorescence from the patient exhibiting PLC zeta(H398P) compared with fertile controls. CONCLUSIONS: We demonstrate, for the first time, the production of active recombinant human PLC zeta protein which retained the ability to elicit characteristic Ca(2+) oscillations in mouse oocytes, an ability which was eliminated by an infertility-linked mutation. These findings advance our understanding of PLC zeta, and provide a critical step forward in obtaining purified PLC zeta protein as a potential therapeutic agent for oocyte activation deficiency.
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author
organization
alternative title
Loss of activity mutations in phospholipase C zeta (PLC zeta) abolishes calcium oscillatory ability of human recombinant protein in mouse oocytes
year
type
journalArticle (original)
publication status
published
subject
keyword
MAMMALIAN EGG ACTIVATION, TRIGGERS CA2+ OSCILLATIONS, PHOSPHATIDYLINOSITOL 4, EF-HAND DOMAINS, NUCLEAR TRANSLOCATION, INTRACYTOPLASMIC SPERM INJECTION, male infertility, phophospholipase C zeta (PLCzeta), sperm, assisted oocyte activation, oocyte activation, DYNAMICS, INFERTILITY, FERTILIZATION, EMBRYO DEVELOPMENT, 5-BISPHOSPHATE
journal title
HUMAN REPRODUCTION
Hum. Reprod.
volume
26
issue
12
pages
3372 - 3387
Web of Science type
Article
Web of Science id
000297058000021
JCR category
OBSTETRICS & GYNECOLOGY
JCR impact factor
4.475 (2011)
JCR rank
3/78 (2011)
JCR quartile
1 (2011)
ISSN
0268-1161
DOI
10.1093/humrep/der336
language
English
UGent publication?
yes
classification
A1
copyright statement
I have transferred the copyright for this publication to the publisher
id
1984503
handle
http://hdl.handle.net/1854/LU-1984503
date created
2012-01-12 12:14:32
date last changed
2012-01-18 11:22:42
@article{1984503,
  abstract     = {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 zeta). 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 zeta gene of a male patient with oocyte activation deficiency has previously identified a point mutation causing a histidine to proline substitution at PLC zeta residue 398 (PLC zeta(H398P)), leading to abnormal Ca(2+) 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 zeta (PLC zeta(WT)) protein which, upon microinjection into mouse oocytes, induced Ca(2+) oscillations characteristic of oocyte activation. Injection of recombinant PLC zeta H398P was unable to elicit Ca(2+) oscillations in mouse oocytes. Loss of activity mutations, such as PLC zeta(H398P) and an artificially induced frameshift mutation (PLC zeta DYC2) did not affect Ca(2+) release when over-expressed in HEK293T cells, whereas PLC zeta(WT) inhibited adenosine triphosphate-activated Ca(2+) release. Confocal imaging of fluorescently tagged PLC zeta isoforms in HEK293T cells suggested a cytoplasmic pattern of localization, while quantitative analysis of fluorescence levels showed that PLC zeta(WT). PLC zeta(H398P). PLC zeta Delta(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 zeta immunofluorescence from the patient exhibiting PLC zeta(H398P) compared with fertile controls. 
CONCLUSIONS: We demonstrate, for the first time, the production of active recombinant human PLC zeta protein which retained the ability to elicit characteristic Ca(2+) oscillations in mouse oocytes, an ability which was eliminated by an infertility-linked mutation. These findings advance our understanding of PLC zeta, and provide a critical step forward in obtaining purified PLC zeta protein as a potential therapeutic agent for oocyte activation deficiency.},
  author       = {Kashir, Junaid and Jones, Celine and Lee, Hoi Chang and Rietdorf, Katja and Nikiforaki, Dimitra and Durrans, Claire and Ruas, Margarida and Tee, Sze Tian and Heindryckx, Bj{\"o}rn and Galione, Antony and De Sutter, Petra and Fissore, Rafael and Parrington, John and Coward, Kevin},
  issn         = {0268-1161},
  journal      = {HUMAN REPRODUCTION},
  keyword      = {MAMMALIAN EGG ACTIVATION,TRIGGERS CA2+ OSCILLATIONS,PHOSPHATIDYLINOSITOL 4,EF-HAND DOMAINS,NUCLEAR TRANSLOCATION,INTRACYTOPLASMIC SPERM INJECTION,male infertility,phophospholipase C zeta (PLCzeta),sperm,assisted oocyte activation,oocyte activation,DYNAMICS,INFERTILITY,FERTILIZATION,EMBRYO DEVELOPMENT,5-BISPHOSPHATE},
  language     = {eng},
  number       = {12},
  pages        = {3372--3387},
  title        = {Loss of activity mutations in phospholipase C zeta (PLC\ensuremath{\zeta}) abolishes calcium oscillatory ability of human recombinant protein in mouse oocytes},
  url          = {http://dx.doi.org/10.1093/humrep/der336},
  volume       = {26},
  year         = {2011},
}

Chicago
Kashir, Junaid, Celine Jones, Hoi Chang Lee, Katja Rietdorf, Dimitra Nikiforaki, Claire Durrans, Margarida Ruas, et al. 2011. “Loss of Activity Mutations in Phospholipase C Zeta (PLCζ) Abolishes Calcium Oscillatory Ability of Human Recombinant Protein in Mouse Oocytes.” Human Reproduction 26 (12): 3372–3387.
APA
Kashir, Junaid, Jones, C., Lee, H. C., Rietdorf, K., Nikiforaki, D., Durrans, C., Ruas, M., et al. (2011). Loss of activity mutations in phospholipase C zeta (PLCζ) abolishes calcium oscillatory ability of human recombinant protein in mouse oocytes. HUMAN REPRODUCTION, 26(12), 3372–3387.
Vancouver
1.
Kashir J, Jones C, Lee HC, Rietdorf K, Nikiforaki D, Durrans C, et al. Loss of activity mutations in phospholipase C zeta (PLCζ) abolishes calcium oscillatory ability of human recombinant protein in mouse oocytes. HUMAN REPRODUCTION. 2011;26(12):3372–87.
MLA
Kashir, Junaid, Celine Jones, Hoi Chang Lee, et al. “Loss of Activity Mutations in Phospholipase C Zeta (PLCζ) Abolishes Calcium Oscillatory Ability of Human Recombinant Protein in Mouse Oocytes.” HUMAN REPRODUCTION 26.12 (2011): 3372–3387. Print.