scholarly journals Establishment of the mammalian membrane block to polyspermy: evidence for calcium-dependent and -independent regulation

Reproduction ◽  
2007 ◽  
Vol 133 (2) ◽  
pp. 383-393 ◽  
Author(s):  
Allison J Gardner ◽  
Carmen J Williams ◽  
Janice P Evans
Keyword(s):  
Plasma Membrane ◽  
Zona Pellucida ◽  
Egg Activation ◽  
Membrane Function ◽  
Independent Event ◽  
Calcium Dependent ◽  
Normal Sperm ◽  
Egg Membrane ◽  
Mammalian Eggs ◽  
Mouse Eggs

One crucial result of egg activation is the establishment of blocks on the zona pellucida and the egg plasma membrane to prevent fertilization by additional sperm. The mechanism(s) by which a mammalian egg regulates the establishment of the membrane block to polyspermy is largely unknown. Since Ca2+ signaling regulates several egg activation events, this study investigates how sperm-induced Ca2+ transients affect the membrane block to polyspermy, building on our previous work (Biology of Reproduction 67:1342). We demonstrate that mouse eggs that experience only one sperm-induced Ca2+ transient establish a membrane block that is less effective, than in eggs that experience normal sperm-induced Ca2+ transients but that is more effective than in eggs with completely suppressed [Ca2+]cyt increases. Sperm-induced increases in [Ca2+]cyt regulate the timing of membrane block establishment, as this block is established more slowly in eggs that experience one or no sperm-induced Ca2+ transients. Finally, our studies produce the intriguing discovery that there is also a Ca2+-independent event that is associated with fertilization in the pathway leading to membrane block establishment. Taken together, these data indicate that Ca2+ plays a role in facilitating membrane block establishment by regulating the timing with which this change in egg membrane function occurs, and also that the membrane block differs from other post-fertilization egg activation responses as Ca2+ is not the only stimulus. The membrane block to polyspermy in mammalian eggs is likely to be the culmination of multiple post-fertilization events that together modify the egg membrane’s receptivity to sperm.

scholarly journals Conventional PKCs regulate the temporal pattern of Ca2+ oscillations at fertilization in mouse eggs

2004 ◽  
Vol 164 (7) ◽  
pp. 1033-1044 ◽  
Author(s):  
Guillaume Halet ◽  
Richard Tunwell ◽  
Scott J. Parkinson ◽  
John Carroll
Keyword(s):  
Embryonic Development ◽  
Temporal Pattern ◽  
Physiological Function ◽  
Egg Activation ◽  
Present Evidence ◽  
Downstream Effectors ◽  
Living Mouse ◽  
Egg Membrane ◽  
Mammalian Eggs ◽  
Mouse Eggs

In mammalian eggs, sperm-induced Ca2+ oscillations at fertilization are the primary trigger for egg activation and initiation of embryonic development. Identifying the downstream effectors that decode this unique Ca2+ signal is essential to understand how the transition from egg to embryo is coordinated. Here, we investigated whether conventional PKCs (cPKCs) can decode Ca2+ oscillations at fertilization. By monitoring the dynamics of GFP-labeled PKCα and PKCγ in living mouse eggs, we demonstrate that cPKCs translocate to the egg membrane at fertilization following a pattern that is shaped by the amplitude, duration, and frequency of the Ca2+ transients. In addition, we show that cPKC translocation is driven by the C2 domain when Ca2+ concentration reaches 1–3 μM. Finally, we present evidence that one physiological function of activated cPKCs in fertilized eggs is to sustain long-lasting Ca2+ oscillations, presumably via the regulation of store-operated Ca2+ entry.

PLCζ: a sperm-specific trigger of Ca2+ oscillations in eggs and embryo development

Development ◽  
2002 ◽  
Vol 129 (15) ◽  
pp. 3533-3544 ◽  
Author(s):  
Christopher M. Saunders ◽  
Mark G. Larman ◽  
John Parrington ◽  
Llewellyn J. Cox ◽  
Jillian Royse ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Embryo Development ◽  
Specific Protein ◽  
Normal Embryo ◽  
Egg Activation ◽  
Sperm Factor ◽  
Mammalian Eggs ◽  
Mouse Eggs ◽  
Cytosolic Factor ◽  
Specific Trigger

Upon fertilisation by sperm, mammalian eggs are activated by a series of intracellular Ca2+ oscillations that are essential for embryo development. The mechanism by which sperm induces this complex signalling phenomenon is unknown. One proposal is that the sperm introduces an exclusive cytosolic factor into the egg that elicits serial Ca2+ release. The ‘sperm factor’ hypothesis has not been ratified because a sperm-specific protein that generates repetitive Ca2+ transients and egg activation has not been found. We identify a novel, sperm-specific phospholipase C, PLCζ, that triggers Ca2+ oscillations in mouse eggs indistinguishable from those at fertilisation. PLCζ removal from sperm extracts abolishes Ca2+ release in eggs. Moreover, the PLCζ content of a single sperm was sufficient to produce Ca2+ oscillations as well as normal embryo development to blastocyst. Our results are consistent with sperm PLCζ as the molecular trigger for development of a fertilised egg into an embryo.

scholarly journals Autoradiographic visualization of the mouse egg's sperm receptor bound to sperm.

1986 ◽  
Vol 102 (4) ◽  
pp. 1363-1371 ◽  
Author(s):  
J D Bleil ◽  
P M Wassarman
Keyword(s):  
Plasma Membrane ◽  
Zona Pellucida ◽  
Present Report ◽  
Fusion Event ◽  
Acrosomal Membrane ◽  
Specific Receptors ◽  
Species Specific ◽  
Mammalian Eggs ◽  
Silver Grains ◽  
Sperm Receptor

The extracellular coat, or zona pellucida, of mammalian eggs contains species-specific receptors to which sperm bind as a prelude to fertilization. In mice, ZP3, one of only three zona pellucida glycoproteins, serves as sperm receptor. Acrosome-intact, but not acrosome-reacted, mouse sperm recognize and interact with specific O-linked oligosaccharides of ZP3 resulting in sperm-egg binding. Binding, in turn, causes sperm to undergo the acrosome reaction; a membrane fusion event that results in loss of plasma membrane at the anterior region of the head and exposure of inner acrosomal membrane with its associated acrosomal contents. Bound, acrosome-reacted sperm are able to penetrate the zona pellucida and fuse with the egg's plasma membrane (fertilization). In the present report, we examined binding of radioiodinated, purified, egg ZP3 to both acrosome intact and acrosome reacted sperm by whole-mount autoradiography. Silver grains due to bound 125I-ZP3 were found localized to the acrosomal cap region of heads of acrosome-reacted sperm. Under the same conditions, 125I-fetuin bound at only bacKground levels to heads of both acrosome-intact and -reacted sperm, and 125I-ZP2, another zona pellucida glycoprotein, bound preferentially to acrosome-reacted sperm. These results provide visual evidence that ZP3 binds preferentially and specifically to heads of acrosome intact sperm; properties expected of the mouse egg's sperm receptor.

Sperm entry into fertilised mouse eggs

Zygote ◽  
1994 ◽  
Vol 2 (2) ◽  
pp. 129-131 ◽  
Author(s):  
Darek Maluchnik ◽  
Ewa Borsuk
Keyword(s):  
Plasma Membrane ◽  
Zona Pellucida ◽  
Motile Sperm ◽  
Perivitelline Space ◽  
Sperm Entry ◽  
Mouse Eggs

SummaryWe have investigated the oolemma block to polyspermy in the mouse. Zona-free and zona-intact eggs were fertilised and subsequently re-inseminated (the latter following zona pellucida removal). The ‘perivitelline’ block to polyspermy in zona-intact eggs renders motile sperm in the perivitelline space unable to bind to the oolemma. This is not connected with irreversible changes in the egg plasma membrane, because freshly added sperm can still fuse with such eggs freed from the zona. Fertilised eggs eventually lose the ability to fuse with sperm within 1 h, while still being able to bind many sperm.

scholarly journals Mammalian membrane block to polyspermy: new insights into how mammalian eggs prevent fertilisation by multiple sperm

2006 ◽  
Vol 18 (2) ◽  
pp. 53 ◽  
Author(s):  
Allison J. Gardner ◽  
Janice P. Evans
Keyword(s):  
Plasma Membrane ◽  
Zona Pellucida ◽  
Animal Species ◽  
Mammalian Species ◽  
Experimental Studies ◽  
Present Review ◽  
Cellular Signalling ◽  
Classical Studies ◽  
Vitelline Envelope ◽  
Mammalian Eggs

To inhibit fertilisation by more than one sperm (a condition known as polyspermy), eggs have developed preventative mechanisms known as blocks to polyspermy. The block at the level of the egg extracellular coat (the zona pellucida in mammals, the vitelline envelope in non-mammals) has been well characterised in many different animal species and the block at the level of the egg plasma membrane is understood in some non-mammalian species. However, virtually nothing is known about the membrane block to polyspermy in mammalian eggs, despite data dating back 50–90 years that provide evidence for its existence. In the present review, we will discuss the background on blocks to polyspermy used by animal eggs and then focus on the membrane block to polyspermy in mammalian eggs. This will include a summary of classical studies that provide evidence for this block in mammalian eggs, assays used to study the mammalian membrane block and what has been elucidated from recent experimental studies about the cellular signalling events that lead to membrane block establishment and the mechanism of how the membrane block may prevent additional fertilisation.

The role of the zona pellucida in the development of mouse eggs in vivo

Development ◽  
1970 ◽  
Vol 23 (3) ◽  
pp. 539-547
Author(s):  
Jacek A. Modliński
Keyword(s):  
Zona Pellucida ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Development In Vitro ◽  
Single Blastomeres ◽  
Mammalian Eggs ◽  
Mouse Eggs

Up to the present time the function and significance of the zona pellucida in the development of mammalian eggs has not been fully explained. Zona-free mouse eggs will develop in vitro from the 2-cell stage, or later, up to the blastocyst stage (Tarkowski, 1961; Mintz, 1962; Gwatkin, 1963). Single blastomeres isolated at the 2-cell (Mulnard, 1965), 4- and 8-cell stage (Tarkowski & Wróblewska, 1967) will also develop in vitro up to the blastocyst stage. Similar experiments on development in vitro of 1- and 2-cell rabbit eggs (Edwards, 1964) showed that in this species also cleavage can occur when the zona pellucida is absent, although the blastomeres exhibit a tendency to fall away from each other. Tarkowski's observations (unpublished) would appear to show, however, that naked 1-, 2- and 4-cell mouse eggs do not develop when transferred to the oviduct. A few hours after transplanting the naked eggs none could be recovered by flushing the oviduct, whereas eggs surrounded by zonae which were transplanted simultaneously were recovered.

scholarly journals Conserved Furin Cleavage Site Not Essential for Secretion and Integration of ZP3 into the Extracellular Egg Coat of Transgenic Mice

2002 ◽  
Vol 22 (9) ◽  
pp. 3111-3120 ◽  
Author(s):  
Ming Zhao ◽  
Lyn Gold ◽  
Ann M. Ginsberg ◽  
Li-Fang Liang ◽  
Jurrien Dean
Keyword(s):  
Transgenic Mice ◽  
Zona Pellucida ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Furin Cleavage ◽  
Furin Cleavage Site ◽  
Green Fluorescent ◽  
Mammalian Eggs ◽  
Mouse Eggs

ABSTRACT The extracellular zona pellucida surrounding mammalian eggs is formed by interactions of the ZP1, ZP2, and ZP3 glycoproteins. Female mice lacking ZP2 or ZP3 do not form a stable zona matrix and are sterile. The three zona proteins are synthesized in growing oocytes and secreted prior to incorporation into the zona pellucida. A well-conserved furin site upstream of a transmembrane domain near the carboxyl terminus of each has been implicated in the release of the zona ectodomains from oocytes. However, mutation of the furin site (RNRR → ANAA) does not affect the intracellular trafficking or secretion of an enhanced green fluorescent protein (EGFP)-ZP3 fusion protein in heterologous somatic cells. After transient expression in growing oocytes, normal EGFP-ZP3 and mutant EGFP-ZP3 associate with the inner aspect of the zona pellucida, which is distinct from the plasma membrane. These in vitro results are confirmed in transgenic mice expressing EGFP-ZP3 with or without the mutant furin site. In each case, EGFP-ZP3 is incorporated throughout the width of the zona pellucida and the transgenic mice are fertile. These results indicate that the zona matrix accrues from the inside out and, unexpectedly, suggest that cleavage at the furin site is not required for formation of the extracellular zona pellucida surrounding mouse eggs.

scholarly journals Roles of heterotrimeric and monomeric G proteins in sperm-induced activation of mouse eggs

Development ◽  
1994 ◽  
Vol 120 (11) ◽  
pp. 3313-3323 ◽  
Author(s):  
G.D. Moore ◽  
T. Ayabe ◽  
P.E. Visconti ◽  
R.M. Schultz ◽  
G.S. Kopf
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Zona Pellucida ◽  
Polar Body ◽  
Active Form ◽  
Dominant Negative ◽  
Egg Activation ◽  
Cell Stage ◽  
Second Polar Body ◽  
Mouse Eggs

Results of several lines of experimentation suggest that sperm-induced egg activation has several features in common with G protein-coupled receptor signal transduction mechanisms. We report that microinjection of GDP beta S into metaphase II-arrested mouse eggs blocks sperm-induced egg activation. Since GDP beta S inactivates both heterotrimeric and monomeric classes of G proteins, the involvement of members of each of these families in sperm-induced egg activation was evaluated. Neither pertussis toxin treatment of eggs nor microinjection of eggs with inhibitory antibodies toward G alpha q blocked sperm-induced egg activation. Nevertheless, microinjection of phosducin, a protein that binds tightly to free G protein beta gamma subunits, specifically inhibited second polar body emission, the fertilization evoked decrease of H1 kinase activity and pronucleus formation. Microinjection of phosducin, however, did not inhibit the fertilization-induced modifications of the zona pellucida and microinjection of beta gamma t did not result in egg activation in the absence of sperm. Inactivation of the monomeric Rho family of G proteins with C3 transferase from Clostridium botulinum inhibited emission of the second polar body and cleavage to the 2-cell stage, but did not affect the modifications of the zona pellucida or pronucleus formation. Microinjection of Rasval12, which is a constitutively active form of Ras, did not result in egg activation in the absence of sperm. Moreover, microinjection of either an anti-Ras neutralizing antibody (Y13-259) or a dominant negative form of Ras (RasT) did not affect events of sperm-induced egg activation. In contrast, microinjection of RasT inhibited embryo cleavage to the 2-cell stage. These results suggest that both heterotrimeric and monomeric G proteins are involved in various aspects of sperm-induced egg activation.

Recepter-mediated signal transduction and egg activation

Zygote ◽  
1993 ◽  
Vol 1 (4) ◽  
pp. 276-279 ◽  
Author(s):  
Kathleen R Foltz ◽  
Fraser M Shilling
Keyword(s):  
Signal Transduction ◽  
Plasma Membrane ◽  
Species Differences ◽  
Second Messenger ◽  
Signalling Pathways ◽  
Egg Activation ◽  
Gamete Fusion ◽  
Gamete Recognition ◽  
Egg Membrane ◽  
Cytoplasmic Side

Egg activation at fertilisation is composed of a complex, choreographed series of events, the initiation of which still is not understood. Two major hypotheses have emerged as explanations for the mechanism of egg activation (reviewed by Nuccitelli, 1991; Whitaker & Swann, 1993). The first holds that the sperm delivers an ‘activating factor’ (e.g. inositol trisphosphate, calcium, or a protein) that diffuses into the egg cytoplasm after gamete fusion. The second hypothesis holds that sperm bind to receptors spanning the egg plasma membrane which then transduce a signal to second messenger enzymes. We present here the evidence for receptor-mediated signal transduction in egg activation at fertilisation. By ‘receptor’ we mean only molecules that bind an extra-membrane ligand and which transduce a signal to molecules residing on the cytoplasmic side of the egg membrane. It is critical to be aware that several alternative activating mechanisms are not mutually exclusive and that species differences may exist. In fact, as more has been learnt about the molecules involved in gamete recognition and binding and of the signalling pathways in the egg, it seems likely that multiple pathways exist to trigger complete egg activation.

scholarly journals PLCζ causes Ca2+ oscillations in mouse eggs by targeting intracellular and not plasma membrane PI(4,5)P2

2012 ◽  
Vol 23 (2) ◽  
pp. 371-380 ◽  
Author(s):  
Yuansong Yu ◽  
Michail Nomikos ◽  
Maria Theodoridou ◽  
George Nounesis ◽  
F. Anthony Lai ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Inositol Polyphosphate ◽  
Ph Domain ◽  
Phosphoinositide Signaling ◽  
Intracellular Ca ◽  
Mammalian Fertilization ◽  
Green Fluorescent ◽  
Mammalian Eggs ◽  
Mouse Eggs

Sperm-specific phospholipase C ζ (PLCζ) activates embryo development by triggering intracellular Ca2+ oscillations in mammalian eggs indistinguishable from those at fertilization. Somatic PLC isozymes generate inositol 1,4,5-trisphophate–mediated Ca2+ release by hydrolyzing phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in the plasma membrane. Here we examine the subcellular source of PI(4,5)P2 targeted by sperm PLCζ in mouse eggs. By monitoring egg plasma membrane PI(4,5)P2 with a green fluorescent protein–tagged PH domain, we show that PLCζ effects minimal loss of PI(4,5)P2 from the oolemma in contrast to control PLCδ1, despite the much higher potency of PLCζ in eliciting Ca2+ oscillations. Specific depletion of this PI(4,5)P2 pool by plasma membrane targeting of an inositol polyphosphate-5-phosphatase (Inp54p) blocked PLCδ1-mediated Ca2+ oscillations but not those stimulated by PLCζ or sperm. Immunolocalization of PI(4,5)P2, PLCζ, and catalytically inactive PLCζ (ciPLCζ) revealed their colocalization to distinct vesicular structures inside the egg cortex. These vesicles displayed decreased PI(4,5)P2 after PLCζ injection. Targeted depletion of vesicular PI(4,5)P2 by expression of ciPLCζ-fused Inp54p inhibited the Ca2+ oscillations triggered by PLCζ or sperm but failed to affect those mediated by PLCδ1. In contrast to somatic PLCs, our data indicate that sperm PLCζ induces Ca2+ mobilization by hydrolyzing internal PI(4,5)P2 stores, suggesting that the mechanism of mammalian fertilization comprises a novel phosphoinositide signaling pathway.

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