PLCzeta(zeta): a sperm protein that triggers Ca2+ oscillations and egg activation in mammals

At fertilization in mammals, the sperm activates development by causing a prolonged series of intracellular Ca(2+) oscillations that are generated by increased production of inositol trisphosphate (InsP(3)). It appears that the sperm initiates InsP(3) generation via the introduction of a sperm factor into the egg after gamete membrane fusion. We recently identified a sperm-specific form of phospholipase C (PLC), referred to as PLCzeta(zeta). We review the evidence that PLCzeta represents the sperm factor that activates development of the egg and discuss the characteristics of PLCzeta that distinguish it from the somatic forms of PLC.     

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

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.     

Mechanism of Ca2+ release at fertilization in mammals.

At fertilization in mammals the sperm triggers a series of oscillations in intracellular Ca2+ within the egg. These Ca2+ oscillations activate the development of the egg into an embryo. It is not known how the sperm triggers these Ca2+ oscillations. There are currently three different theories for Ca2+ signaling in eggs at fertilization. One idea is that the sperm acts as a conduit for Ca2+ entry into the egg after membrane fusion. Another idea is that the sperm acts upon plasma membrane receptors to stimulate a phospholipase C (PLC) within the egg which generates inositol 1,4, 5-trisphosphate (InsP(3)). We present a third idea that the sperm causes Ca2+ release by introducing a soluble protein factor into the egg after gamete membrane fusion. In mammals this sperm factor is also referred to as an oscillogen because, after microinjection, the factor causes sustained Ca2+ oscillations in eggs. Our recent data in sea urchin egg homogenates and intact eggs suggests that this sperm factor has phospholipase C activity that leads to the generation of InsP(3). We then present a new version of the soluble sperm factor theory of signaling at fertilization. J. Exp. Zool. (Mol. Dev. Evol.) 285:267-275, 1999.     

Measurement of intracellular IP3 during Ca2+ oscillations in mouse eggs with GFP-based FRET probe

Intracellular Ca2+ oscillations in fertilized mammalian eggs, the key signal that stimulates egg activation and early embryonic development, are regulated by inositol 1,4,5-trisphosphate (IP3) signaling pathway. We investigated temporal changes in intracellular IP3 concentration ([IP3]i) in mouse eggs, using a fluorescent probe based on fluorescence resonance energy transfer between two green fluorescent protein variants, during Ca2+ oscillations induced by fertilization or expression of phospholipase Czeta (PLCzeta), an egg-activating sperm factor candidate. Fluorescence measurements suggested the elevation of [IP3]i in fertilized eggs, and the enhancement of PLCzeta-mediated IP3 production by cytoplasmic Ca2+ was observed during Ca2+ oscillations or in response to CaCl2 microinjection. The results supported the view that PLCzeta is the sperm factor to stimulate IP3 pathway, and suggested that high Ca2+ sensitivity of PLCzeta activity and positive feedback from released Ca2+ are important for triggering and maintaining Ca2+ oscillations.     

Calcium oscillations and mammalian egg activation

Fertilization in all species studied to date induces an increase in the intracellular concentration of free calcium ions ([Ca2+]i) within the egg. In mammals, this [Ca2+]i signal is delivered in the form of long-lasting [Ca2+]i oscillations that begin shortly after fusion of the gametes and persist beyond the time of completion of meiosis. While not fully elucidated, recent evidence supports the notion that the sperm delivers into the ooplasm a trigger of oscillations, the so-called sperm factor (SF). The recent discovery that mammalian sperm harbor a specific phospholipase C (PLC), PLCzeta has consolidated this view. The fertilizing sperm, and presumably PLCzeta promote Ca2+ release in eggs via the production of inositol 1,4,5-trisphosphate (IP3), which binds and gates its receptor, the type-1 IP3 receptor, located on the endoplasmic reticulum, the Ca2+ store of the cell. Repetitive Ca2+ release in this manner results in a positive cumulative effect on downstream signaling molecules that are responsible for the completion of all the events comprising egg activation. This review will discuss recent advances in our understanding of how [Ca2+]i oscillations are initiated and regulated in mammals, highlight areas of discrepancies, and emphasize the need to better characterize the downstream molecular cascades that are dependent on [Ca2+]i oscillations and that may impact embryo development.     

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

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, PLCzeta, 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.     

Recombinant phospholipase Czeta has high Ca2+ sensitivity and induces Ca2+ oscillations in mouse eggs

Sperm-specific phospholipase Czeta (PLCzeta) is known to induce intracellular Ca(2+) oscillations and subsequent early embryonic development when expressed in mouse eggs by injection of RNA encoding PLCzeta (Saunders, C. M., Larman, M. G., Parrington, J., Cox, L. J., Royse, J., Blayney, L. M., Swann, K., and Lai, F. A. (2002) Development 129, 3533-3544). The present study addressed characteristics of purified mouse PLCzeta protein that was synthesized using the baculovirus/Sf9 cell expression system. Microinjection of recombinant PLCzeta protein into mouse eggs induced serial Ca(2+) spikes quite similar to those produced by the injection of sperm extract, probably because of repetitive Ca(2+) release from the endoplasmic reticulum caused by continuously produced inositol 1,4,5-trisphosphate. Recombinant PLCdelta1 also induced Ca(2+) oscillations, but a 20-fold higher concentration was required compared with PLCzeta. In the enzymatic assay of phosphatidylinositol 4,5-bisphosphate hydrolyzing activity in vitro at various calcium ion concentrations ([Ca(2+)]), PLCzeta exhibited a significant activity at [Ca(2+)] as low as 10 nm and had 70% maximal activity at 100 nm [Ca(2+)] that is usually the basal intracellular calcium ion concentration level of cells. On the other hand, the activity of PLCdelta1 increased at a [Ca(2+)] between 1 and 30 microm. EC(50) was 52 nm for PLCzeta and 5.7 microm for PLCdelta1. Thus, PLCzeta has an approximately 100-fold higher Ca(2+) sensitivity than PLCdelta1. The ability of purified PLCzeta protein to induce Ca(2+) oscillations qualifies PLCzeta as a proper candidate of the mammalian egg-activating sperm factor. Furthermore, such a high Ca(2+) sensitivity of PLC activity as PLCzeta that can be active in cells at the resting state is thought to be an appropriate characteristic of the sperm factor, which is introduced into the ooplasm upon sperm-egg fusion, triggers Ca(2+) release first, and maintains Ca(2+) oscillations.     

Functional, biochemical, and chromatographic characterization of the complete [Ca2+]i oscillation-inducing activity of porcine sperm

A cytosolic sperm protein(s), referred to as sperm factor (SF), is delivered into eggs by the sperm during mammalian fertilization to induce repetitive increases in the intracellular concentration of free Ca2+ ([Ca2+]i) that are referred to as [Ca2+]i oscillations. [Ca2+]i oscillations are essential for egg activation and early embryonic development. Recent evidence shows that the novel sperm-specific phospholipase C (PLC), PLCzeta, may be the long sought after [Ca2+]i oscillation-inducing SF. Here, we demonstrate the complete extraction of SF from porcine sperm and show that regardless of the method of extraction a single molecule/complex appears to be responsible for the [Ca2+]i oscillation-inducing activity of these extracts. Consistent with this notion, all sperm fractions that induced [Ca2+]i oscillations, including FPLC-purified fractions, exhibited high in vitro PLC activity at basal Ca2+ levels (0.1-5 microM), a hallmark of PLCzeta. Notably, we detected immunoreactive 72-kDa PLCzeta in an inactive fraction, and several fractions capable of inducing oscillations were devoid of 72-kDa PLCzeta. Nonetheless, in the latter fractions, proteolytic fragments, presumably corresponding to cleaved forms of PLCzeta, were detected by immunoblotting. Therefore, our findings corroborate the hypothesis that a sperm-specific PLC is the main component of the [Ca2+]i oscillation-inducing activity of sperm but provide evidence that the presence of 72-kDa PLCzeta does not precisely correspond with the Ca2+ releasing activity of porcine sperm fractions.     

Phospholipase C-dependent Ca2+ release by worm and mammal sperm factors

Egg activation in all animals evidently requires the synthesis of inositol 1,4,5-trisphosphate (InsP(3)) from phosphatidylinositol 4,5-bisphosphate (PIP(2)) by phospholipase C (PLC). Depending on the organism, InsP(3) elicits either calcium oscillations or a single wave, which in turn initiates development. A soluble component in boar sperm that activates mammalian eggs has been suggested to be a PLC isoform. We tested this hypothesis in vitro using egg microsomes of Chaetopterus. Boar sperm factor elicited Ca(2+) release from the microsomes by an InsP(3)-dependent mechanism. The PLC inhibitor U-73122, but not its inactive analog U-73343, blocked the response to sperm factor but not to InsP(3). U-73122 also inhibited the activation of fertilized and parthenogenetic eggs. Chaetopterus sperm also contained a similar activity. These results strongly support the hypothesis that sperm PLCs are ubiquitous mediators of egg activation at fertilization.     

Transgenic RNA interference reveals role for mouse sperm phospholipase Czeta in triggering Ca2+ oscillations during fertilization

A sperm-specific phospholipase (PL) C, termed PLCzeta, is proposed to be the soluble sperm factor that induces Ca(2+) oscillations in mammalian eggs and, thus, initiates egg activation in vivo. We report that sperm from transgenic mice expressing short hairpin RNAs targeting PLCzeta mRNA have reduced amounts of PLCzeta protein. Sperm derived from these transgenic mice trigger patterns of Ca(2+) oscillations following fertilization in vitro that terminate prematurely. Consistent with the perturbation in patterns of Ca(2+) oscillations is the finding that mating of transgenic founder males to females results in lower rates of egg activation and no transgenic offspring. These data strongly suggest that PLCzeta is the physiological trigger of Ca(2+) oscillations required for activation of development.     

Starting a new life: sperm PLC-zeta mobilizes the Ca2+ signal that induces egg activation and embryo development: an essential phospholipase C with implications for male infertility

We have discovered that a single sperm protein, phospholipase C-zeta (PLCζ), can stimulate intracellular Ca(2+) signalling in the unfertilized oocyte ('egg') culminating in the initiation of embryonic development. Upon fertilization by a spermatozoon, the earliest observed signalling event in the dormant egg is a large, transient increase in free Ca(2+) concentration. The fertilized egg responds to the intracellular Ca(2+) rise by completing meiosis. In mammalian eggs, the Ca(2+) signal is delivered as a train of long-lasting cytoplasmic Ca(2+) oscillations that begin soon after gamete fusion and persist beyond the completion of meiosis. Sperm PLCζ effects Ca(2+) release from egg intracellular stores by hydrolyzing the membrane lipid PIP(2) and consequent stimulation of the inositol 1,4,5-trisphosphate (InsP(3) ) receptor Ca(2+) -signalling pathway, leading to egg activation and early embryogenesis. Recent advances have refined our understanding of how PLCζ induces Ca(2+) oscillations in the egg and also suggest its potential dysfunction as a cause of male infertility.     

Teleost fish spermatozoa contain a cytosolic protein factor that induces calcium release in sea urchin egg homogenates and triggers calcium oscillations when injected into mouse oocytes

Established studies in a variety of organisms including amphibians, fish, ascidians, nemerteans, echinoderms, mammals, and even a species of flowering plant, clearly demonstrate that an increase in intracellular egg calcium is crucial to the process of egg activation at fertilization. In echinoderms, egg activation appears to involve an egg phospholipase C gamma (PLCgamma). However, numerous studies in mammalian species suggest that calcium is released from internal egg stores at fertilization by a sperm-derived cytosolic protein factor. Recent studies in the mouse have identified this sperm-derived factor as being a novel sperm-specific PLC isoform with distinctive properties (PLCzeta). Homologues of PLCzeta have since been isolated from human and cynomolgus monkey sperm. In addition, sperm factor activity has been detected in non-mammalian species such as chicken, Xenopus, and a flowering plant. Here we report evidence for the existence of a similar sperm-derived factor in a commercially important species of teleost fish, the Nile tilapia Oreochromis niloticus (L). Using an established bioassay for calcium release, the sea urchin egg homogenate, we demonstrate that protein extracts obtained from tilapia spermatozoa exhibit PLC activity similar to that seen in mammalian sperm extracts, and also induce calcium release when added directly to the homogenate. Further, tilapia sperm extracts induced calcium oscillations when injected into mouse oocytes.     

Mammalian fertilization: from sperm factor to phospholipase Czeta

In mammalian eggs, the fertilizing sperm evokes intracellular Ca2+ ([Ca2+]i) oscillations that are essential for initiation of egg activation and embryonic development. Although the exact mechanism leading to initiation of [Ca2+]i oscillations still remains unclear, accumulating studies suggest that a presently unknown substance, termed sperm factor (SF), is delivered from the fertilizing sperm into the ooplasm and triggers [Ca2+]i oscillations. Based on findings showing that production of inositol 1,4,5-trisphosphate (IP3) underlies the generation of [Ca2+]i oscillations, it has been suggested that SF functions either as a phospholipase C (PLC), an enzyme that catalyzes the generation of IP3, or as an activator of a PLC(s) pre-existing in the egg. This review discusses the role of SF as the molecule responsible for the production of IP3 and the initiator of [Ca2+]i oscillations in mammalian fertilization, with particular emphasis on the possible involvement of egg- and sperm-derived PLCs, including PLCzeta, a novel sperm specific PLC.     

Could modifications of signalling pathways activated after ICSI induce a potential risk of epigenetic defects?

A calcium signal during oocyte or egg activation is a conserved event in virtually all species analyzed so far. This signal, that is in the form of calcium oscillations in mammals, is spatially and temporally controlled and is mainly supported by calcium release from internal calcium stores, but how it is triggered after fertilization is far from understood. The sperm factor hypothesis of egg activation postulates that sperm delivers a calcium-releasing factor into the egg following sperm-egg fusion. Among the many potential sperm factors, PLCzeta is the strongest bona fide sperm factor candidate. However, how sperm-oocyte fusion occurs prior to PLCzeta delivery and oocyte activation is not entirely known. We propose in the first part of this review the possibility that other pathways such as those involving G-proteins, tyrosine kinases or integrins could be activated besides sperm factor injection and could be upstream mechanisms involved in later embryonic development. Among different assisted reproductive technologies (ARTs), intracytoplasmic sperm injection (ICSI) is considered as the best and easiest therapeutic technique to circumvent severe male infertility. Although most reports are reassuring, some recent data suggest a greater incidence of abnormalities in children conceived by ART compared with those conceived normally. Spatio-temporal signals may be missing or abnormal during ICSI, perhaps because membrane fusion and signalling events are bypassed. We discuss in the second part of this review the hypothesis that potential perturbations during the ICSI procedure may have repercussions on epigenetic processes, inducing not only alterations of embryonic development, but also diseases in young children and, perhaps, in adults.     

Regulation of cortical vesicle exocytosis in sea urchin eggs by inositol 1,4,5-trisphosphate and GTP-binding protein

To investigate the roles of inositol 1,4,5-trisphosphate (InsP3) and guanyl nucleotide binding proteins (G-proteins) in the transduction mechanism coupling fertilization and exocytosis of cortical vesicles in sea urchin eggs, we microinjected InsP3 and guanyl nucleotide analogs into eggs of Lytechinus variegatus. Injection of 28 nM InsP3 caused exocytosis. However, if the egg was first injected with EGTA ([Cai] less than or equal to 0.1 microM; EGTA = 1.6 mM), InsP3 injection did not cause exocytosis, supporting the hypothesis that InsP3 acts by causing a rise in intracellular free calcium. Injection of 28 microM guanosine-5'-0-(3-thiotriphosphate) (GTP-gamma-S), a hydrolysis-resistant analog of GTP, caused exocytosis, but exocytosis did not occur if the egg was pre-injected with EGTA. Injection of 3 mM guanosine-5'-0-(2-thiodiphosphate) (GDP-beta-S), a metabolically stable analog of GDP, prevented sperm from stimulating exocytosis. However, injection of GDP-beta-S did not prevent the stimulation of exocytosis by InsP3. These results suggested the following sequence of events. The sperm activates a G-protein, which stimulates production of InsP3. InsP3 elevates intracellular free calcium, which causes exocytosis.     

Identification and functional analysis of an ovarian form of the egg activation factor phospholipase C zeta (PLCζ) in pufferfish

Recent studies suggest that egg activation in mammals is triggered by a sperm-specific phospholipase C, PLCzeta. In other vertebrate species such as medaka fish, chickens, and quail, PLCzeta is also expressed as a testis-specific mRNA. Functional studies suggest that PLCzeta plays a similar role as a trigger of egg activation in these species. Here, we report the identification of PLCzeta orthologues in pufferfish species Takifugu rubripes (Fugu) and Tetraodon nigroviridis (Tetraodon). Unexpectedly in these species PLCzeta is expressed not in the testis, but in ovary and brain. Injection of pufferfish PLCzeta copy ribonucleic acid (cRNA) into mouse eggs failed to trigger calcium oscillations, unlike medaka PLCzeta cRNA. Our findings provide the first evidence that PLCzeta may be expressed in the egg, rather than the sperm, in some vertebrate species, and that its mechanism of action and physiologic role at fertilization may differ in different vertebrate species.     

A cytosolic sperm factor stimulates repetitive calcium increases and mimics fertilization in hamster eggs

Microinjection of cytosolic sperm extracts into unfertilized golden hamster eggs caused a series of increases in cytoplasmic free calcium, Ca2+i, and membrane hyperpolarizing responses, HRs. These HRs and Ca2+i transients are similar to those seen during in vitro fertilization of hamster eggs. The sperm factor that is responsible for causing these effects appears to be of high molecular weight and protein based. Injection of sperm factor activated eggs and mimicked fertilization in causing repetitive HRs in the presence of phorbol esters and in sensitizing the egg to calcium-induced calcium release. Since these effects cannot be mimicked by injecting G-protein agonists or calcium-containing solutions, it seems unlikely that a receptor-G-protein signalling system is involved at fertilization. These data instead suggest a novel signal transduction system operates during mammalian fertilization in which a protein factor is transferred from the sperm into the egg cytoplasm after gamete membrane fusion.     

Development of calcium release mechanisms during starfish oocyte maturation

In response to the maturation-inducing hormone 1-methyladenine, starfish oocytes acquire increased sensitivity to sperm and inositol trisphosphate (InsP3), stimuli that cause a release of calcium from intracellular stores and a rise in intracellular free calcium. In the immature oocyte, the calcium release in response to 10 sperm entries is less than that seen with a single sperm entry in the mature egg. Likewise, the sensitivity to injected InsP3 is less in the immature oocyte. Approximately 100 times as much InsP3 is required to obtain the same calcium release in an immature oocyte as in a mature egg. However, with saturating amounts of InsP3, immature oocytes and mature eggs release comparable amounts of calcium. These results indicate that although calcium stores are well-developed in the immature oocyte, mechanisms for releasing the calcium develop fully only during oocyte maturation.     

Nuclear translocation of phospholipase C-zeta, an egg-activating factor, during early embryonic development

Phospholipase C-zeta (PLCzeta), a strong candidate of the egg-activating sperm factor, causes intracellular Ca2+ oscillations and egg activation, and is subsequently accumulated into the pronucleus (PN), when expressed in mouse eggs by injection of RNA encoding PLCzeta. Changes in the localization of expressed PLCzeta were investigated by tagging with a fluorescent protein. PLCzeta began to translocate into the PN formed at 5-6 h after RNA injection and increased there. Observation in the same embryo revealed that PLCzeta in the PN dispersed to the cytoplasm upon nuclear envelope breakdown and translocated again into the nucleus after cleavage. The dynamics was found in the second mitosis as well. When RNA was injected into fertilization-originated 1-cell embryos or blastomere(s) of 2-8-cell embryos, the nuclear localization of expressed PLCzeta was recognized in every embryo up to blastocyst. Thus, PLCzeta exhibited alternative cytoplasm/nucleus localization during development. This supports the view that the sperm factor could control cell cycle-dependent generation of Ca2+ oscillations in early embryogenesis.     

Role of phospholipase Cgamma at fertilization and during mitosis in sea urchin eggs and embryos

It is well known that stimulation of egg metabolism after fertilization is due to a rise in intracellular free calcium concentration. In sea urchin eggs, this first calcium signal is followed by other calcium transients that allow progression through mitotic control points of the cell cycle of the early embryo. How sperm induces these calcium transients is still far from being understood. In sea urchin eggs, both InsP3 and ryanodine receptors contribute to generate the fertilization calcium transient, while the InsP3 receptor generates the subsequent mitotic calcium transients. The identity of the mechanisms that generate InsP3 after fertilization remains an enigma. In order to determine whether PLCgamma might be the origin of the peaks of InsP3 production that punctuate the first mitotic cell cycles of the fertilized sea urchin egg, we have amplified by RT-PCR several fragments of sea urchin PLCgamma containing the two SH2 domains. The sequence shares similarities with SH2 domains of PLCgamma from mammals. One fragment was subcloned into a bacterial expression plasmid and a GST-fusion protein was produced and purified. Antibodies raised to the GST fusion protein demonstrate the presence of PLCgamma protein in eggs. Microinjection of the fragment into embryos interferes with mitosis. A related construct made from bovine PLCgamma also delayed or prevented entry into mitosis and blocked or prolonged metaphase. The bovine construct also blocked the calcium transient at fertilization, in contrast to a tandem SH2 control construct which did not inhibit either fertilization or mitosis. Our data indicate that PLCgamma plays a key role during fertilization and early development.