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Mammalian Phospholipase C~ Induces Oocyte Activation from the Sperm Perinuclear Matrix

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Mammalian Phospholipase C~ Induces Oocyte Activation from the Sperm Perinuclear Matrix View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Developmental Biology 274 (2004) 370–383 www.elsevier.com/locate/ydbio Mammalian phospholipase C~ induces oocyte activation from the sperm perinuclear matrix Satoko Fujimotoa, Naoko Yoshidaa, Tomoyuki Fukuia, Manami Amanaia, Toshiaki Isobeb, Chiharu Itagakib, Tomonori Izumib, Anthony C.F. Perrya,* aLaboratory of Mammalian Molecular Embryology, RIKEN Center for Developmental Biology, Chuo-ku, Kobe 650-0047, Japan bDivision of Proteomics Research (ABJ & Millipore), Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan Received for publication 22 June 2004, revised 27 July 2004, accepted 27 July 2004 Available online 25 August 2004 Abstract Mammalian sperm-borne oocyte activating factor (SOAF) induces oocyte activation from a compartment that engages the oocyte cytoplasm, but it is not known how. A SOAF-containing extract (SE) was solubilized from the submembrane perinuclear matrix, a domain that enters the egg. SE initiated activation sufficient for full development. Microinjection coupled to tandem mass spectrometry enabled functional correlation profiling of fractionated SE without a priori assumptions about its chemical nature. Phospholipase C-zeta (PLC~) correlated absolutely with activating ability. Immunoblotting confirmed this and showed that the perinuclear matrix is the major site of 72- kDa PLC~. Oocyte activation was efficiently induced by 1.25 fg of sperm PLC~, corresponding to a fraction of one sperm equivalent (~0.03). Immunofluorescence microscopy localized sperm head PLC~ to a post-acrosomal region that becomes rapidly exposed to the ooplasm following gamete fusion. This multifaceted approach suggests a mechanism by which PLC~ originates from an oocyte-penetrating assembly—the sperm perinuclear matrix—to induce mammalian oocyte activation at fertilization. D 2004 Published by Elsevier Inc. Keywords: Sperm; Oocyte; Activation; SOAF; Perinuclear matrix; Mouse; Protein correlation profiling 2+ Introduction available calcium ([Ca ]i)(Whittingham and Siracusa, 1978) and presumably modulates the oscillatory activity Mammalian oocytes are released from metaphase II (mII) of the calmodulin-dependent kinase, CaMKII (Markoulaki arrest by one or more signals from a fertilizing spermato- et al., 2004). zoon. This results in asymmetric cytokinesis and the The general importance of Ca2+ as a second messenger exudation of a second polar body to restore diploidy as a has focused attention on the hypothesis that the sperm- prelude to embryogenesis. Collectively, early events during borne activating factor (SOAF) responsible for inducing 2+ and downstream of sperm-mediated signaling at fertilization activation does so via [Ca ]i oscillations (Swann, 1990). are termed oocyte activation. Following this rationale, several SOAF candidates have Entry of a spermatozoon into an oocyte rapidly (within been proposed. One, glucosamine 6-phosphate isomerase 1–3 min in the mouse; Lawrence et al., 1997) induces a oscillin, was purified from the cytosolic fraction of golden series of oscillations in the concentration of intracellular hamster spermatozoa (Parrington et al., 1996), but sub- sequent experiments failed to activate mII oocytes when enzymatically functional recombinant glucosamine 6-phos- * Corresponding author. Laboratory of Mammalian Molecular Embryo- logy, RIKEN Center for Developmental Biology, 2-2-3 Minatojima Minami- phate isomerase was injected into them (Wolosker et al., machi, Chuo-ku, Kobe 650-0047, Japan. Fax: +81 78 306 3144. 1998; Wolny et al., 1999). Other cytosol-derived candi- E-mail address: [email protected] (A.C.F. Perry). dates have included a truncated form of the c-kit ligand, tr- 0012-1606/$ - see front matter D 2004 Published by Elsevier Inc. doi:10.1016/j.ydbio.2004.07.025 S. Fujimoto et al. / Developmental Biology 274 (2004) 370–383 371 kit (Sette et al., 1997) and NO synthase (Kuo et al., 2000). Second, it is unclear whether any of the cytosolic However, these have not been validated (Hyslop et al., components that remain after acrosomal exocytosis ever 2001; Mehlmann et al., 1998). become exposed to oocyte cytoplasm at fertilization; most is Several enzymes of the phospholipase family have also topologically extracellular with respect to the fused gametes been suggested to correspond to SOAF (Jones et al., 1998; (Fig. 1). During and following gamete fusion, sperm reviewed in Runft et al., 2002). This family is attractive, membranes integrate with the oocyte plasma membrane at because its members generate the second messenger, fertilization and sperm surface markers can be shown to inositol 1,4,5-trisphosphate (IP3), which might in turn integrate into the oocyte surface (Gaunt, 1983). activate IP3 receptors to trigger calcium-induced calcium Third, cytosolic sperm components may be involved in release at fertilization. Most recently, a reverse genetic the induction of a distinct Ca2+-mobilizing event integral to approach identified a phospholipase C (PLC), designated sperm at fertilization: the acrosome reaction (Arnoult et al., PLC-zeta (PLC~), whose corresponding cRNA induced 1996; Fukami et al., 2003). Indeed, a distinct PLC, PLCy4, 2+ [Ca ]i oscillations following injection into mII oocytes was recently shown to play an important role in the (Saunders et al., 2002). Recombinant, baculovirus-derived acrosome reaction, although sperm from gene-targeted mice 2+ PLC~ also induced [Ca ]i oscillations when its longer lacking the enzyme still underwent ZP3-induced acrosomal isoform was injected into oocytes at 60 fg/pl (Kouchi et exocytosis at a rate of 20% of controls (Fukami et al., 2003), al., 2004) and cRNA-encoded PLC~ associates with indicating that a redundant mechanism exists. The intro- pronuclei in zygotes (Larman et al., 2004; Yoda et al., duction of factor(s) involved in acrosome reaction into an 2004). ectopic site—an mII oocyte—might be capable of inducing Aside from its oscillogenic nature, little is known of the Ca2+ transients although they play no physiological role in biological properties of PLC~ that might account for any oocyte activation. role in oocyte activation. Only a single report addresses Clearly, the demonstration of a sperm-derived factor that PLC~ derived from sperm (Saunders et al., 2002) and this stimulates oocyte activation is insufficient absent evidence uses cytosolic material derived from whole sperm by that it derives from a compartment that enters the oocyte at cyclical freeze–thaw as a starting point for its character- fertilization. ization. This is problematic for reasons that have dogged Alternative SOAF candidates have thus been proposed previous attempts to isolate SOAF. based on their residence in noncytosolic, submembrane First, the cytosol (defined as the extranuclear part of the sperm head components that enter the oocyte at fertilization. cell, including organelles, whose components are in These include SOAF-I, MN13 antigen and PT32 (Mana- solution) is greatly depleted in acrosome-reacted sperm ndhar and Toshimori, 2003; Perry et al., 1999; Sutovsky et (Yanagimachi, 1994) such that material derived from whole al., 2003). Each is a component of the perinuclear matrix sperm may not be extant at the time of gamete fusion (Fig. (PNM), a macromolecular assembly comprised of several 1). The acrosome reaction is an absolute prerequisite for hundred protein species that enshrouds the nuclear mem- sperm binding and fusion (Yanagimachi, 1994). brane (Bellve´ et al., 1993). We previously reported that Fig. 1. Simplified diagram showing the membrane topology of the sperm head during mammalian fertilization. Acrosomal exocytosis triggered by the zona pellucida glycoprotein, ZP3 (B), results in the discharge of many soluble and membrane-associated components (C). Acrosome-reacted spermatozoa contain little water due to the tight juxtaposition of membranes and the high packing ratio of chromatin. Most of what little cytosol is extant at the time of membrane fusion (D) is on the exterior of the oocyte (E). 372 S. Fujimoto et al. / Developmental Biology 274 (2004) 370–383 SOAF-I protein is solubilized from membrane-free sperm Preparation of mouse spermatozoa and sperm extracts 2+ heads in reducing environments and is able to induce [Ca ]i oscillations and activation when injected into mII oocytes Motile mouse spermatozoa were obtained by chopping (Perry et al., 1999, 2000). caudae epididymides acutely isolated from 12–30-week- It is now possible to identify proteins present within old male B6D2F1 mice in Nuclear Isolation Medium complex mixtures in femtomolar amounts by tandem mass (NIM: 125 mM KCl, 2.6 mM NaCl, 7.8 mM Na2HPO4, spectrometry (Aebersold and Mann, 2003). This approach 1.4 mM KH2PO4, 3.0 mM EDTA; pH6.9) as described has been applied to generate protein profiles for yeast previously (Perry et al., 1999). Sperm were demembra- (Washburn et al., 2001) and Deinococcus radiodurans nated according to the Core Protocol (Perry et al., 1999). (Lipton et al., 2002). Moreover, correlative nano-liquid In brief, sperm were sonicated in detergent (typically 0.05– chromatography (nano-LC) tandem mass spectrometric 1% [v/v] TX-100) and washed 3Â in NIM. To obtain analysis permits the analysis of any multiprotein complex mouse SOAF extract (SE), the final pellet was resus- that can be enriched by fractionation, even where its pended to give a density of 2–5 Â 106 sperm/100 Alin components cannot be purified to homogeneity.
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