The cytosolic sperm factor that triggers Ca2+ oscillations and egg activation in mammals is a novel phospholipase C: PLCζ

When sperm activate eggs at fertilization the signal for activation involves increases in the intracellular free Ca2+concentration. In mammals the Ca2+changes at fertilization consist of intracellular Ca2+oscillations that are driven by the generation of inositol 1,4,5-trisphosphate (InsP3). It is not established how sperm trigger the increases in InsP3and Ca2+at fertilization. One theory suggests that sperm initiate signals to activate the egg by introducing a specific factor into the egg cytoplasm after membrane fusion. This theory has been mainly based upon the observation that injecting a cytosolic sperm protein factor into eggs can trigger the same pattern of Ca2+oscillations induced by the sperm. We have recently shown that this soluble sperm factor protein is a novel form of phospholipase C (PLC), and it is referred to as PLCζ(zeta). We describe the evidence that led to the identification of PLCζ and discuss the issues relating to its potential role in fertilization.

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Potential role of a sperm-derived phospholipase C in triggering the egg-activating Ca2+ signal at fertilization

Reproduction ◽

10.1530/rep.0.1220839 ◽

2001 ◽

pp. 839-846 ◽

Cited By ~ 14

Author(s):

K Swann ◽

J Parrington ◽

KT Jones

Keyword(s):

Membrane Fusion ◽

Phospholipase C ◽

Potential Role ◽

Egg Activation ◽

Gamete Fusion ◽

Inositol 1,4,5 Trisphosphate ◽

Sperm Factor

An increase in intracellular Ca2+ at fertilization is the trigger for egg activation in all species that have been studied. Exactly how sperm-egg interaction leads to this Ca2+ increase has not been established. There is increasing support for the hypothesis that the spermatozoon introduces a Ca2+-releasing protein into the egg cytoplasm after gamete membrane fusion. This review discusses the merits of this 'sperm factor' hypothesis and presents evidence indicating that the sperm factor, at least in mammals, consists of a phospholipase C with distinctive properties. This evidence leads us to propose that, after gamete fusion, a sperm-derived phospholipase C causes production of inositol 1,4,5- trisphosphate, which then generates Ca2+ waves from within the egg cytoplasm.

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Phospholipase C isoforms in mammalian spermatozoa: potential components of the sperm factor that causes Ca2+ release in eggs

Reproduction ◽

10.1530/rep.0.1230031 ◽

2002 ◽

pp. 31-39 ◽

Cited By ~ 43

Author(s):

J Parrington ◽

ML Jones ◽

R Tunwell ◽

C Devader ◽

M Katan ◽

...

Keyword(s):

Phospholipase C ◽

Sea Urchin ◽

Soluble Protein ◽

Gel Filtration ◽

Protein Factor ◽

Sea Urchin Egg ◽

Inositol 1,4,5 Trisphosphate ◽

Sperm Factor ◽

Mammalian Eggs ◽

Mammalian Spermatozoa

Injection of a soluble protein factor from mammalian spermatozoa triggers Ca2+ oscillations in mammalian eggs similar to those seen at fertilization. This sperm factor also generates inositol 1,4,5-trisphosphate and causes Ca2+ release in sea urchin egg homogenates and frog eggs. Recent studies have indicated that the sperm factor may be an inositol-specific phospholipase C (PLC) activity. This study investigated whether any of the commonly known PLC isoforms are components of the sperm factor. PLCbeta, PLCgamma and PLCdelta isoforms were shown to be present in boar sperm extracts. However, upon column fractionation of sperm extracts, none of the PLC isoforms detected correlated with the ability to cause Ca2+ release in eggs. In addition to our previous work on recombinant PLCs, it was also shown that PLCdelta3, PLCdelta4 and its splice variant PLCdelta4 Alt1 fail to cause Ca2+ release. The recently discovered 255 kDa PLCepsilon isoform also appears unlikely to be a component of the sperm factor, as fractionation of sperm extracts on a gel filtration column demonstrated that the peak of Ca2+-releasing activity was associated with fractions of 30-70 kDa. These findings indicate that the sperm factor that triggers Ca2+ release in eggs does not appear to have a known PLC isoform as one of its components.

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PLCζ, a sperm-specific PLC and its potential role in fertilization

Biochemical Society Symposium ◽

10.1042/bss2007c03 ◽

2007 ◽

Vol 74 ◽

pp. 23-36 ◽

Cited By ~ 36

Author(s):

Christopher M. Saunders ◽

Karl Swann ◽

F. Anthony Lai

Keyword(s):

Intracellular Calcium ◽

Membrane Fusion ◽

Causative Agent ◽

Calcium Concentration ◽

Potential Role ◽

Vital Role ◽

The Other ◽

Sperm Protein ◽

Dramatic Rise ◽

The Moment

A dramatic rise in intracellular calcium plays a vital role at the moment of fertilization, eliciting the resumption of meiosis and the initiation of embryo development. In mammals, the rise takes the form of oscillations in calcium concentration within the egg, driven by an elevation in inositol trisphosphate. The causative agent of these oscillations is proposed to be a recently described phosphoinositide-specific phospholipase C, PLCζ, a soluble sperm protein that is delivered into the egg following membrane fusion. In the present review, we examine some of the distinctive structural and functional characteristics of this crucial enzyme that sets it apart from the other known forms of mammalian PLC.

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The sperm phospholipase C-ζ and Ca2+ signalling at fertilization in mammals

Biochemical Society Transactions ◽

10.1042/bst20150221 ◽

2016 ◽

Vol 44 (1) ◽

pp. 267-272 ◽

Cited By ~ 20

Author(s):

Karl Swann ◽

F. Anthony Lai

Keyword(s):

Phospholipase C ◽

Embryo Development ◽

Early Embryo ◽

Egg Activation ◽

Stable Form ◽

Ph Domain ◽

Vitro Fertilization ◽

Sperm Factor ◽

Internal Sources

A series of intracellular oscillations in the free cytosolic Ca2+ concentration is responsible for activating mammalian eggs at fertilization, thus initiating embryo development. It has been proposed that the sperm causes these Ca2+ oscillations after membrane fusion by delivering a soluble protein into the egg cytoplasm. We previously identified sperm-specific phospholipase C (PLC)-ζ as a protein that can trigger the same pattern of Ca2+ oscillations in eggs seen at fertilization. PLCζ appears to be the elusive sperm factor mediating egg activation in mammals. It has potential therapeutic use in infertility treatments to improve the rate of egg activation and early embryo development after intra-cytoplasmic sperm injection. A stable form of recombinant human PLCζ could be a prototype for use in such in vitro fertilization (IVF) treatments. We do not yet understand exactly how PLCζ causes inositol 1,4,5-trisphosphate (InsP3) production in eggs. Sperm PLCζ is distinct among mammalian PI-specific PLCs in that it is far more potent in triggering Ca2+ oscillations in eggs than other PLCs, but it lacks a PH domain that would otherwise be considered essential for binding to the phosphatidylinositol 4,5-bisphosphate (PIP2) substrate. PLCζ is also unusual in that it does not appear to interact with or hydrolyse plasma membrane PIP2. We consider how other regions of PLCζ may mediate its binding to PIP2 in eggs and how interaction of PLCζ with egg-specific factors could enable the hydrolysis of internal sources of PIP2.

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Stretching releases Ca2+ from intracellular storage sites in canine cerebral arteries

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y94-004 ◽

1994 ◽

Vol 72 (1) ◽

pp. 19-24 ◽

Cited By ~ 30

Author(s):

Yoshio Tanaka ◽

Shinzo Hata ◽

Hiromi Ishiro ◽

Kunio Ishii ◽

Koichi Nakayama

Keyword(s):

Phospholipase C ◽

Cerebral Artery ◽

Basilar Artery ◽

Cerebral Arteries ◽

Mechanical Stretch ◽

Inositol 1,4,5 Trisphosphate ◽

Intracellular Ca ◽

Canine Basilar Artery ◽

Intracellular Storage

Mechanical stretch applied to canine cerebral artery produced myogenic contraction. The contraction of the artery in response to quick stretch was dependent on not only the transmembrane influx of Ca2+ through 1,4-dihydropyridine-sensitive Ca2+ channels but also the release of Ca2+ from intracellular storage sites: the stretch-produced contractile component that was resistant to 0.1 μM nicardipine, a Ca2+-channel antagonist, was inhibited by about 50% after treatment with ryanodine, and was almost completely suppressed by 0.1 mM 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate, a putative phospholipase C inhibitor, or by lowering the temperature from 35 to 20 °C. The results suggest that in addition to transmembrane influx of Ca2+ through L-type Ca2+ channels, the release of Ca2+ from both ryanodine-sensitive and -insensitive intracellular storage sites, which increases intracellular Ca2+, accounts for the stretch-induced contraction of canine basilar artery. It seems also possible that inositol 1,4,5-trisphosphate is a common mediator for the release of Ca2+ from both types of intracellular storage sites.Key words: stretch-induced contraction, cerebral artery, phospholipase C, ryanodine, Ca2+ storage sites, inositol 1,4,5-trisphosphate, Ca2+ release, Ca2+-channel antagonist.

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

Development ◽

10.1242/dev.129.15.3533 ◽

2002 ◽

Vol 129 (15) ◽

pp. 3533-3544 ◽

Cited By ~ 22

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.

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Egg activation in physiological polyspermy

Reproduction ◽

10.1530/rep-12-0104 ◽

2012 ◽

Vol 144 (1) ◽

pp. 11-22 ◽

Cited By ~ 40

Author(s):

Yasuhiro Iwao

Keyword(s):

Citrate Synthase ◽

Sperm Nucleus ◽

Embryonic Cell ◽

Egg Activation ◽

Cell Cycles ◽

Close Relationship ◽

Intracellular Ca ◽

Sperm Factor ◽

Necessary And Sufficient ◽

Dual Functions

Fertilization is indispensable not only for restoring diploid genomes but also for the initiation of early embryonic cell cycles in sexual reproduction. While most animals exhibit monospermy, which is ensured by polyspermy blocks to prevent the entry of extra sperm into the egg at fertilization, several animals exhibit physiological polyspermy, in which the entry of several sperm is permitted but only one sperm nucleus participates in the formation of a zygote nucleus. Polyspermy requires that the sperm transmit the egg activation signal more slowly, thus allowing the egg to accept several sperm. An increase in intracellular Ca2+ concentration induced by the fertilizing sperm is both necessary and sufficient for egg activation in polyspermy. Multiple small Ca2+ waves induced by several fertilizing sperm result in a long-lasting Ca2+ rise, which is a characteristic of polyspermic amphibian eggs. We introduced a novel soluble sperm factor for egg activation, sperm-specific citrate synthase, into polyspermic newt eggs to cause Ca2+ waves. Citrate synthase may perform dual functions: as an enzyme in mitochondria and as a Ca2+-inducing factor in egg cytoplasm. We also discuss the close relationship between the mode of fertilization and the Ca2+ rise at egg activation and consider changes in this process through evolution in vertebrates.

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Egg Activation at Fertilization by a Soluble Sperm Protein

Physiological Reviews ◽

10.1152/physrev.00012.2015 ◽

2016 ◽

Vol 96 (1) ◽

pp. 127-149 ◽

Cited By ~ 42

Author(s):

Karl Swann ◽

F. Anthony Lai

Keyword(s):

Mechanism Of Action ◽

Empirical Studies ◽

Specific Protein ◽

Cellular Level ◽

Egg Activation ◽

Soluble Factor ◽

Unresolved Issue ◽

Sperm Protein ◽

Mammalian Sperm ◽

Intracellular Ca

The most fundamental unresolved issue of fertilization is to define how the sperm activates the egg to begin embryo development. Egg activation at fertilization in all species thus far examined is caused by some form of transient increase in the cytoplasmic free Ca2+ concentration. What has not been clear, however, is precisely how the sperm triggers the large changes in Ca2+ observed within the egg cytoplasm. Here, we review the studies indicating that the fertilizing sperm stimulates a cytosolic Ca2+ increase in the egg specifically by delivering a soluble factor that diffuses into the cytosolic space of the egg upon gamete membrane fusion. Evidence is primarily considered in species of eggs where the sperm has been shown to elicit a cytosolic Ca2+ increase by initiating Ca2+ release from intracellular Ca2+ stores. We suggest that our best understanding of these signaling events is in mammals, where the sperm triggers a prolonged series of intracellular Ca2+ oscillations. The strongest empirical studies to date suggest that mammalian sperm-triggered Ca2+ oscillations are caused by the introduction of a sperm-specific protein, called phospholipase C-zeta (PLCζ) that generates inositol trisphosphate within the egg. We will discuss the role and mechanism of action of PLCζ in detail at a molecular and cellular level. We will also consider some of the evidence that a soluble sperm protein might be involved in egg activation in nonmammalian species.

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Ca(2+) oscillations induced by a cytosolic sperm protein factor are mediated by a maternal machinery that functions only once in mammalian eggs

Development ◽

10.1242/dev.127.5.1141 ◽

2000 ◽

Vol 127 (5) ◽

pp. 1141-1150 ◽

Cited By ~ 1

Author(s):

T.S. Tang ◽

J.B. Dong ◽

X.Y. Huang ◽

F.Z. Sun

Keyword(s):

Receptor Sensitivity ◽

Present Evidence ◽

Sperm Protein ◽

Protein Factor ◽

Bovine Sperm ◽

Sperm Factor ◽

Mammalian Eggs ◽

Mouse Eggs ◽

Fertilized Egg ◽

Stimulation Of

At fertilization in mammals, the sperm activates the egg by inducing a series of oscillations in the intracellular free Ca(2+) concentration. There is evidence showing that this oscillatory event is triggered by a sperm-derived protein factor which diffuses into egg cytoplasm after gamete membrane fusion. At present the identity of this factor and its precise mechanism of action is unknown. Here, we studied the specificity of action of the sperm factor in triggering Ca(2+) oscillations in mammalian eggs. In doing so, we examined the patterns of Ca(2+) signaling in mouse eggs, zygotes, parthenogenetic eggs and maturing oocytes following the stimulation of bovine sperm extracts which contain the sperm factor. It is observed that the sperm factor could induce Ca(2+) oscillations in metaphase eggs, maturing oocytes and parthenogenetically activated eggs but not in the zygotes. We present evidence that Ca(2+) oscillations induced by the sperm factor require a maternal machinery. This machinery functions only once in mammalian oocytes and eggs, and is inactivated by sperm-derived components but not by parthenogenetic activation. In addition, it is found that neither InsP(3) receptor sensitivity to InsP(3) nor Ca(2+) pool size are the determinants that cause the fertilized egg to lose its ability to generate sperm-factor-induced Ca(2+) oscillations at metaphase. In conclusion, our study suggests that the orderly sequence of Ca(2+) oscillations in mammalian eggs at fertilization is critically dependent upon the presence of a functional maternal machinery that determines whether the sperm-factor-induced Ca(2+) oscillations can persist.

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Release of phospholipase C ζand [Ca2+]i oscillation-inducing activity during mammalian fertilization

Reproduction ◽

10.1530/rep-07-0259 ◽

2007 ◽

Vol 134 (5) ◽

pp. 695-704 ◽

Cited By ~ 49

Author(s):

Sook-Young Yoon ◽

Rafael A Fissore

Keyword(s):

Phospholipase C ◽

Embryo Development ◽

Intracytoplasmic Sperm Injection ◽

Egg Activation ◽

Mouse Sperm ◽

Mammalian Sperm ◽

Sperm Entry ◽

Sperm Factor ◽

Mammalian Fertilization ◽

Mammalian Eggs

During fertilization of mammalian eggs a factor from the sperm, the sperm factor (SF), is released into the ooplasm and induces persistent [Ca2+]i oscillations that are required for egg activation and embryo development. A sperm-specific phospholipase C (PLC), PLCz, is thought to be the SF. Here, we investigated whether the SF activity and PLCζare simultaneously and completely released into the ooplasm soon after sperm entry. To accomplish this, we enucleated sperm heads within 90 min of intracytoplasmic sperm injection (ICSI) and monitored the persistence of the [Ca2+]i oscillations in eggs in which the sperm had been withdrawn. We also stained the enucleatedsperm heads to ascertain the presence/absence of PLCζ. Our results show that by 90 min all the SF activity had been released from the sperm, as fertilized enucleated eggs oscillated as fertilized controls, even in cases in which oscillations were prolonged by arresting eggs at metaphase. In addition, we found that the released SF activity became associated with the pronucleus (PN), as induction of PN envelope breakdown evoked comparable [Ca2+]i responses in enucleated and non-manipulated zygotes. Lastly, we found that PLCzlocalized to the equatorial area of bull sperm and to the post-acrosomal region of mouse sperm and that by 90 min after ICSI all the sperm’s PLCζimmunoreactivity was lost in both species. Altogether, our findings show that during fertilization the SF activity and PLCζimmunoreactivity are simultaneously released from the sperm, suggesting that PLCζmay be the only [Ca2+]i oscillation-inducing factor of mammalian sperm.

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