Jose Raul Gonzalez-Garcia

Cardiff University, Cardiff, Wales, United Kingdom

Are you Jose Raul Gonzalez-Garcia?

Claim your profile

Publications (4)17.41 Total impact

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Egg activation at fertilization in mammals is initiated by prolonged Ca(2+) oscillations that trigger the completion of meiosis and formation of pronuclei. A late fall in MAPK activity is essential for pronuclear formation, but the precise timing and mechanism of decline are unknown. Here, we have measured the dynamics of MAPK inactivation in fertilizing mouse eggs using novel chemiluminescent MAPK activity reporters. This reveals that the MAPK activity decrease begins during the Ca(2+) oscillations, but MAPK does not completely inactivate until after pronuclear formation. MAPK in eggs consists of Mos, MEK and ERK1/2. Notably, the MAPK activity decline at fertilization is not explained by upstream destruction of Mos, because a decrease in Mos-luciferase signal is not associated with egg activation. Further, Mos over-expression does not affect the timing of MAPK inactivation or pronuclear formation. However, the late decrease in MAPK could be rapidly reversed by the protein phosphatase inhibitor, okadaic acid. These data suggest that the completion of meiosis in mouse zygotes is driven by an increased phosphatase activity and not by a decline in Mos levels, or MEK activity.
    Preview · Article · Apr 2014 · Journal of Cell Science
  • [Show abstract] [Hide abstract]
    ABSTRACT: Phospholipase C-zeta (PLCζ) is a sperm-specific protein believed to cause Ca(2+) oscillations and egg activation during mammalian fertilization. PLCζ is very similar to the somatic PLCδ1 isoform but is far more potent in mobilising Ca(2+) in eggs. To investigate how discrete protein domains contribute to Ca(2+) release, we assessed the function of a series of PLCζ/PLCδ1 chimeras. We examined their ability to cause Ca(2+) oscillations in mouse eggs, enzymatic properties using in vitro PIP2 hydrolysis and their binding to PIP2 and PI(3)P with a liposome interaction assay. Most chimeras hydrolysed PIP2 with no major differences in Ca(2+) sensitivity and enzyme kinetics. Insertion of a PH domain or replacement of the PLCζ EF hands domain had no deleterious effect on Ca(2+) oscillations. In contrast, replacement of either XY-linker or C2 domain of PLCζ completely abolished Ca(2+) releasing activity. Notably, chimeras containing the PLCζ XY-linker bound to PIP2-containing liposomes, while chimeras containing the PLCζ C2 domain exhibited PI(3)P binding. Our data suggest that the EF hands are not solely responsible for the nanomolar Ca(2+) sensitivity of PLCζ and that membrane PIP2 binding involves the C2 domain and XY-linker of PLCζ. To investigate the relationship between PLC enzymatic properties and Ca(2+) oscillations in eggs we have developed a mathematical model that incorporates Ca(2+)-dependent InsP3 generation by the PLC chimeras and their levels of intracellular expression. These numerical simulations can for the first time predict the empirical variability in onset and frequency of Ca(2+) oscillatory activity associated with specific PLC variants.
    No preview · Article · Oct 2013 · Molecular Human Reproduction
  • Source
    Jose Raul Gonzalez-Garcia · Zoltan Machaty · F Anthony Lai · Karl Swann
    [Show abstract] [Hide abstract]
    ABSTRACT: Fertilization of mammalian eggs is characterized by a series of Ca(2+) oscillations triggered by a phospholipase C activity. These Ca(2+) increases and the parallel generation of diacylglycerol (DAG) stimulate protein kinase C (PKC). However, the dynamics of PKC activity have not been directly measured in living eggs. Here, we have monitored the dynamics of PKC-induced phosphorylation in mouse eggs, alongside Ca(2+) oscillations, using fluorescent C-kinase activity reporter (CKAR) probes. Ca(2+) oscillations triggered either by sperm, phospholipase C zeta (PLCζ) or Sr(2+) all caused repetitive increases in PKC-induced phosphorylation, as detected by CKAR in the cytoplasm or plasma membrane. The CKAR responses lasted for several minutes in both the cytoplasm and plasma membrane then returned to baseline values before subsequent Ca(2+) transients. High frequency oscillations caused by PLCζ led to an integration of PKC-induced phosphorylation. The conventional PKC inhibitor, Gö6976, could inhibit CKAR increases in response to thapsigargin or ionomycin, but not the repetitive responses seen at fertilization. Repetitive increases in PKCδ activity were also detected during Ca(2+) oscillations using an isoform-specific δCKAR. However, PKCδ may already be mostly active in unfertilized eggs, since phorbol esters were effective at stimulating δCKAR only after fertilization, and the PKCδ-specific inhibitor, rottlerin, decreased the CKAR signals in unfertilized eggs. These data show that PKC-induced phosphorylation outlasts each Ca(2+) increase in mouse eggs but that signal integration only occurs at a non-physiological, high Ca(2+) oscillation frequency. The results also suggest that Ca(2+) -induced DAG formation on intracellular membranes may stimulate PKC activity oscillations at fertilization. J. Cell. Physiol. 228: 110-119, 2013. © 2012 Wiley Periodicals, Inc.
    Full-text · Article · Jan 2013 · Journal of Cellular Physiology
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The XY-linker region of somatic cell PLC (phospholipase)-beta, -gamma, -delta and -epsilon isoforms confers potent catalytic inhibition, suggesting a common auto-regulatory role. Surprisingly, the sperm PLC zeta XY-linker does not mediate auto-inhibition. Unlike for somatic PLCs, the absence of the PLC zeta XY-linker significantly diminishes both in vitro PIP2, (phosphatidylinositol 4,5-bisphosphate) hydrolysis and in vivo Ca2+ -oscillation-inducing activity, revealing evidence for a novel PLC zeta enzymatic mechanism.
    Full-text · Article · Jul 2011 · Biochemical Journal

Publication Stats

41 Citations
17.41 Total Impact Points


  • 2013-2014
    • Cardiff University
      • Institute of Molecular & Experimental Medicine
      Cardiff, Wales, United Kingdom
  • 2011
    • National Center for Scientific Research Demokritos
      • Biomolecular Physics Laboratory
      Athínai, Attica, Greece