Oscillations In Eggs Research Articles

The eggstraordinary story of how life begins.

More than 15 years have elapsed since the identification of phospholipase C ζ1 (PLCζ) from a genomic search for mouse testis/sperm-specific PLCs. This molecule was proposed to represent the sperm factor responsible for the initiation of calcium (Ca2+ ) oscillations required for egg activation and embryo development in mammals. Supporting evidence for this role emerged from studies documenting its expression in all mammals and other vertebrate species, the physiological Ca2+ rises induced by injection of its messenger RNA into mammalian and nonmammalian eggs, and the lack of expression in infertile males that fail intracytoplasmic sperm injection. In the last year, genetic animal models have added support to its role as the long sought-after sperm factor. In this review, we highlight the findings that demonstrated the role of Ca2+ as the universal signal of egg activation and the experimental buildup that culminated with the identification of PLCζ as the soluble sperm factor. We also discuss the structural-functional properties that make PLCζ especially suited to evoke oscillations in eggs. Lastly, we examine unresolved aspects of the function and regulation of PLCζ and whether or not it is the only sperm factor in mammalian sperm.

PLCζ Induced Ca2+ Oscillations in Mouse Eggs Involve a Positive Feedback Cycle of Ca2+ Induced InsP3 Formation From Cytoplasmic PIP2.

Egg activation at fertilization in mammalian eggs is caused by a series of transient increases in the cytosolic free Ca2+ concentration, referred to as Ca2+ oscillations. It is widely accepted that these Ca2+ oscillations are initiated by a sperm derived phospholipase C isoform, PLCζ that hydrolyses its substrate PIP2 to produce the Ca2+ releasing messenger InsP3. However, it is not clear whether PLCζ induced InsP3 formation is periodic or monotonic, and whether the PIP2 source for generating InsP3 from PLCζ is in the plasma membrane or the cytoplasm. In this study we have uncaged InsP3 at different points of the Ca2+ oscillation cycle to show that PLCζ causes Ca2+ oscillations by a mechanism which requires Ca2+ induced InsP3 formation. In contrast, incubation in Sr2+ media, which also induces Ca2+ oscillations in mouse eggs, sensitizes InsP3-induced Ca2+ release. We also show that the cytosolic level Ca2+ is a key factor in setting the frequency of Ca2+ oscillations since low concentrations of the Ca2+ pump inhibitor, thapsigargin, accelerates the frequency of PLCζ induced Ca2+ oscillations in eggs, even in Ca2+ free media. Given that Ca2+ induced InsP3 formation causes a rapid wave during each Ca2+ rise, we use a mathematical model to show that InsP3 generation, and hence PLCζ's substate PIP2, has to be finely distributed throughout the egg cytoplasm. Evidence for PIP2 distribution in vesicles throughout the egg cytoplasm is provided with a rhodamine-peptide probe, PBP10. The apparent level of PIP2 in such vesicles could be reduced by incubating eggs in the drug propranolol which also reversibly inhibited PLCζ induced, but not Sr2+ induced, Ca2+ oscillations. These data suggest that the cytosolic Ca2+ level, rather than Ca2+ store content, is a key variable in setting the pace of PLCζ induced Ca2+ oscillations in eggs, and they imply that InsP3 oscillates in synchrony with Ca2+ oscillations. Furthermore, they support the hypothesis that PLCζ and sperm induced Ca2+ oscillations in eggs requires the hydrolysis of PIP2 from finely spaced cytoplasmic vesicles.

The role and mechanism of action of sperm PLC-zeta in mammalian fertilisation.

At mammalian fertilisation, the fundamental stimulus that triggers oocyte (egg) activation and initiation of early embryonic development is an acute rise of the intracellular-free calcium (Ca2+) concentration inside the egg cytoplasm. This essential Ca2+ increase comprises a characteristic series of repetitive Ca2+ oscillations, starting soon after sperm-egg fusion. Over the last 15 years, accumulating scientific and clinical evidence supports the notion that the physiological stimulus that precedes the cytosolic Ca2+ oscillations is a novel, testis-specific phospholipase C (PLC) isoform, known as PLC-zeta (PLCζ). Sperm PLCζ catalyses the hydrolysis of phosphatidylinositol 4,5-bisphosphate triggering cytosolic Ca2+ oscillations through the inositol 1,4,5-trisphosphate signalling pathway. PLCζ is the smallest known mammalian PLC isoform with the most elementary domain organisation. However, relative to somatic PLCs, the PLCζ isoform possesses a unique potency in stimulating Ca2+ oscillations in eggs that is attributed to its novel biochemical characteristics. In this review, we discuss the latest developments that have begun to unravel the vital role of PLCζ at mammalian fertilisation and decipher its unique mechanism of action within the fertilising egg. We also postulate the significant potential diagnostic and therapeutic capacity of PLCζ in alleviating certain types of male infertility.

PLCζ is the physiological trigger of the Ca2+ oscillations that induce embryogenesis in mammals but conception can occur in its absence.

Activation of the egg by the sperm is the first, vital stage of embryogenesis. The sperm protein PLCζ has been proposed as the physiological agent that triggers the Ca2+ oscillations that normally initiate embryogenesis. Consistent with this, recombinant PLCζ induces Ca2+ oscillations in eggs and debilitating mutations in the PLCZ1 gene are associated with infertility in men. However, there has been no evidence that knockout of the gene encoding PLCζ abolishes the ability of sperm to induce Ca2+ oscillations in eggs. Here, we show that sperm derived from Plcz1-/- male mice fail to trigger Ca2+ oscillations in eggs, cause polyspermy and thus demonstrate that PLCζ is the physiological trigger of these Ca2+ oscillations. Remarkably, some eggs fertilized by PLCζ-null sperm can develop, albeit at greatly reduced efficiency, and after a significant time-delay. In addition, Plcz1-/- males are subfertile but not sterile, suggesting that in the absence of PLCζ, spontaneous egg activation can eventually occur via an alternative route. This is the first demonstration that in vivo fertilization without the normal physiological trigger of egg activation can result in offspring. PLCζ-null sperm now make it possible to resolve long-standing questions in fertilization biology, and to test the efficacy and safety of procedures used to treat human infertility.

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

A series of intracellular oscillations in the free cytosolic Ca(2+) concentration is responsible for activating mammalian eggs at fertilization, thus initiating embryo development. It has been proposed that the sperm causes these Ca(2+) 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 Ca(2+) 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 invitro 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 Ca(2+) 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.

Monitoring Calcium Oscillations in Fertilized Mouse Eggs.

In mammalian species, including human, fertilization is characterized by the triggering of long-lasting calcium (Ca(2+)) oscillations in the egg cytoplasm. The monitoring of these Ca(2+) oscillations is a valuable technique to demonstrate that fertilization has occurred, to study egg activation events elicited downstream of the Ca(2+) signal, as well as to evaluate sperm quality. This chapter describes our protocol to monitor sperm-induced Ca(2+) oscillations in mouse eggs, using fluorescence microscopy techniques and the Fura-2-AM ratiometric Ca(2+) indicator.

Effect of M-phase kinase phosphorylations on type 1 inositol 1,4,5-trisphosphate receptor-mediated Ca2+ responses in mouse eggs

The type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) mediates increases in the intracellular concentration of Ca2+ ([Ca2+]i) during fertilization in mammalian eggs. The activity of IP3R1 is enhanced during oocyte maturation, and phosphorylations by M-phase kinases are thought to positively regulate the activity of IP3R1. Accordingly, we and others have found that IP3R1 is phosphorylated at S421, T799 (by Cdk1) and at S436 (by ERK). Nevertheless, the effects of these phosphorylations on the function of the receptor and their impact on [Ca2+]i oscillations in eggs have not been clearly examined. To address this, we expressed in mouse oocytes an IP3R1 variant with the three indicated phosphorylation sites replaced by acidic residues, IIIE-IP3R1, such that it would act like a constitutively phosphorylated IP3R1, and examined [Ca2+]i parameters in response to stimuli. We found that overexpression of wild type (wt-IP3R1) or IIIE-IP3R1 in oocytes containing endogenous receptors caused dominant negative-like effects on Ca2+ release and oscillations. Therefore, we first selectively removed the endogenous IP3R1, and subsequently expressed the exogenous receptors. We found that in response to injection of PLCζ cRNA, eggs without endogenous IP3R1 failed to mount persistent Ca2+ oscillations, although expression of wt-IP3R1 restored their [Ca2+]i oscillatory activity. We also observed that the Ca2+ oscillatory ability and the sensitivity to IP3 in eggs expressing IIIE-IP3R1 were greater than in those expressing wt-IP3R1. Lastly, we found that exogenous IP3R1s are resistant to downregulation and support longer oscillations and of higher amplitude. Altogether, our results show that phosphorylations by Cdk1 and MAPK enhance the activity of IP3R1, which is consistent with its maximal activity observed at the time of fertilization and the role of Ca2+ release in egg activation.

Novel signalling mechanism and clinical applications of sperm-specific PLCζ.

Egg activation is the first step of embryonic development and in mammals is triggered by a series of cytoplasmic calcium (Ca2+) oscillations. Sperm-egg fusion initiates these Ca2+ oscillations by introducing a sperm-specific protein factor into the egg cytoplasm. Substantial evidence indicates that this protein is a sperm-specific phospholipase C (PLC), termed PLC-zeta (PLCζ). PLCζ stimulates cytoplasmic Ca2+ oscillations matching those at fertilization triggering early embryonic development in several mammalian species. Structurally, PLCζ is comprised of four EF-hands, a C2 domain, and X and Y catalytic domains. PLCζ is an unusual PLC since it lacks a pleckstrin homology (PH) domain. It is also distinctive in that its X-Y linker is not involved in auto-inhibition of catalytic activity, but instead binds to phosphatidylinositol 4,5-bisphosphate (PIP2). Moreover, relative to other PLC isoforms, PLCζ possesses unique potency in stimulating Ca2+ oscillations in eggs, although it does not appear to bind to plasma membrane PIP2. In contrast, PLCζ appears to interact with intracellular vesicles in eggs that contain PIP2. I discuss the recent advances in our knowledge of the intriguing biochemical and physiological properties of sperm PLCζ andpostulate potential roles for PLCζ in terms of clinical diagnosis and therapy for certain forms of male infertility.

Chimeras of sperm PLC reveal disparate protein domain functions in the generation of intracellular Ca2+ oscillations in mammalian eggs at fertilization

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 mobilizing 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 phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and their binding to PIP2 and PI(3)P with a liposome interaction assay. Most chimeras hydrolyzed 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.

PLCζ and the initiation of Ca2+ oscillations in fertilizing mammalian eggs

Mammalian eggs undergo a prolonged series of low frequency Ca2+ oscillations at fertilization. These Ca2+ oscillations are the immediate cause of egg activation. The Ca2+ oscillations in mouse eggs have been shown to be driven by increased InsP3 production. Substantial evidence now indicates that a sperm-derived phospholipase C-zeta (PLCζ) is the key molecule that causes these Ca2+ oscillations at fertilization. The fertilizing sperm is envisaged to introduce this essential molecule into the egg following gamete fusion. This review summarizes our current knowledge of how sperm PLCζ causes these oscillations and why it is so much more effective at triggering InsP3 production and Ca2+ oscillations in eggs, than other somatic isoforms of PLC. The molecular features of PLCζ and how they relate to the pattern of Ca2+ oscillations seen at fertilization are considered. We also discuss the evidence that PLCζ does not hydrolyze the conventional source of PI(4,5)P2 in the plasma membrane to make InsP3, but instead uses a distinct pool of PI(4,5)P2 present on intracellular vesicles. This leads us to suggest that sperm PLCζ may be targeted to these cytoplasmic vesicles by directly interacting with a specific but as yet unidentified egg PLCζ-binding protein.

Male infertility-linked point mutation disrupts the Ca2+ oscillation-inducing and PIP2 hydrolysis activity of sperm PLCζ

A male infertility-linked human PLCζ (phospholipase Cζ) mutation introduced into mouse PLCζ completely abolishes both in vitro PIP2 (phosphatidylinositol 4,5-bisphosphate) hydrolysis activity and the ability to trigger in vivo Ca2+ oscillations in mouse eggs. Wild-type PLCζ initiated a normal pattern of Ca2+ oscillations in eggs in the presence of 10-fold higher mutant PLCζ, suggesting that infertility is not mediated by a dominant-negative mechanism.

The dynamics of calcium oscillations that activate mammalian eggs

It has been known for some time that mammalian eggs are activated by a series of intracellular calcium oscillations that occur shortly after sperm egg membrane fusion. Recent work has identified a novel sperm specific phospholipase C zeta as the likely agent that stimulates the calcium oscillations in eggs after sperm-egg membrane fusion. PLCzeta is stimulated by low intracellular calcium levels in a manner which suggests that there is a regenerative feedback of calcium release and PLCzeta induced inositol 1,4,5-trisphophate (InsP3) production in eggs. This implies calcium oscillations in fertilizing mammalian eggs are driven by underlying oscillations of InsP3. This model of oscillations is supported by the response of mouse eggs to sudden increases in InsP3. The cellular targets of calcium oscillations include calmodulin-dependent protein kinases, protein kinase C and mitochondria. There is evidence that eggs might be best activated by multiple calcium increases rather than a single calcium rise. As yet we do not fully understand how the target of calcium in a mammalian egg might decode the patterns of calcium changes that can occur during egg activation.

Intracellular calcium oscillations in mammalian eggs at fertilization

Intracellular calcium oscillations in mammalian eggs at fertilization

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.

PLC zeta: a sperm-specific trigger of Ca(2+) oscillations in eggs and embryo development.

Upon fertilisation by sperm, mammalian eggs are activated by a series of intracellular Ca(2+) 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 Ca(2+) release. The 'sperm factor' hypothesis has not been ratified because a sperm-specific protein that generates repetitive Ca(2+) transients and egg activation has not been found. We identify a novel, sperm-specific phospholipase C, PLC zeta, that triggers Ca(2+) oscillations in mouse eggs indistinguishable from those at fertilisation. PLC zeta removal from sperm extracts abolishes Ca(2+) release in eggs. Moreover, the PLC zeta content of a single sperm was sufficient to produce Ca(2+) oscillations as well as normal embryo development to blastocyst. Our results are consistent with sperm PLC zeta as the molecular trigger for development of a fertilised egg into an embryo.

How a Sperm Turns On An Egg

When a sperm joins with an egg, development is begun by a signal encoded in oscillations in the concentration of intracellular free calcium ([Ca2+]i) in the cytoplasm of the egg. Precisely how the interaction of egg and sperm triggers these oscillations in [Ca2+]i has been a matter of debate, but soluble extracts of sperm cells can reproduce the effect. The Ca2+ is released from intracellular stores in response to increased concentrations of inositol 1,4,5-trisphosphate (IP3), suggesting that a phosphatidylinositol-specific phospholipase C, which generates IP3 from phosphoinositides could be the culprit. But known PLCs can't substitute for sperm in activation of the egg. Saunders et al. now report that mouse sperm produce a previously uncharacterized isoform of PLC that they call PLCζ. Eggs microinjected with complimentary RNA encoding PLCζ showed oscillations in [Ca2+]i with temporal characteristics similar to those in normally fertilized eggs. Furthermore, the amount of PLCζ from just one sperm cell (about 50 fg) mimicked activation by a sperm, and depletion of PLCζ from sperm extracts by immunoprecipitation removed egg-activating activity. Thus, PLCζ from the sperm cytoplasm appears to be the key molecular determinant of egg activation. The authors note that their findings may be applied to treatment of male infertility or to egg activation for production of stem cells.C. M. Saunders, M. G. Larman, J. Parrington, L. J. Cox, J. Royse, L. M. Blayney, K. Swann, F. A. Lai, PLCζ: A sperm-specific trigger of Ca2+ oscillations in eggs and embryo development. Development 129, 3533-3544 [Abstract] [Full Text]

How a Sperm Turns On An Egg

When a sperm joins with an egg, development is begun by a signal encoded in oscillations in the concentration of intracellular free calcium ([Ca 2+ ] i ) in the cytoplasm of the egg. Precisely how the interaction of egg and sperm triggers these oscillations in [Ca 2+ ] i has been a matter of debate, but soluble extracts of sperm cells can reproduce the effect. The Ca 2+ is released from intracellular stores in response to increased concentrations of inositol 1,4,5-trisphosphate (IP 3 ), suggesting that a phosphatidylinositol-specific phospholipase C, which generates IP 3 from phosphoinositides could be the culprit. But known PLCs can't substitute for sperm in activation of the egg. Saunders et al. now report that mouse sperm produce a previously uncharacterized isoform of PLC that they call PLCζ. Eggs microinjected with complimentary RNA encoding PLCζ showed oscillations in [Ca 2+ ] i with temporal characteristics similar to those in normally fertilized eggs. Furthermore, the amount of PLCζ from just one sperm cell (about 50 fg) mimicked activation by a sperm, and depletion of PLCζ from sperm extracts by immunoprecipitation removed egg-activating activity. Thus, PLCζ from the sperm cytoplasm appears to be the key molecular determinant of egg activation. The authors note that their findings may be applied to treatment of male infertility or to egg activation for production of stem cells. C. M. Saunders, M. G. Larman, J. Parrington, L. J. Cox, J. Royse, L. M. Blayney, K. Swann, F. A. Lai, PLCζ: A sperm-specific trigger of Ca 2+ oscillations in eggs and embryo development. Development 129 , 3533-3544 [Abstract] [Full Text]

The soluble mammalian sperm factor protein that triggers Ca 2+ oscillations in eggs: Evidence for expression of mRNA(s) coding for sperm factor protein(s) in spermatogenic cells

At fertilisation in mammals the sperm initiates a series of Ca 2+ oscillations that activate development. One theory of signalling at fertilisation suggests that the sperm contains a soluble protein factor that causes these Ca 2+ oscillations by entering the egg after sperm–egg membrane fusion. This theory is supported by the finding that, in some species, injection of sperm protein extracts into eggs triggers a pattern of Ca 2+ oscillations similar to those seen at fertilisation. So far, all the direct evidence for a sperm factor has been based upon the injection of soluble proteins from mature sperm. Here, we demonstrate that injection of mRNA extracted from hamster spermatogenic cells also leads to generation of prolonged Ca 2+ oscillations in mouse eggs. The ability of spermatogenic cell mRNA to induce Ca 2+ oscillations is dependent upon translation into protein and also appears to be specific to spermatogenic cells since injection of mRNA isolated from somatic tissues into eggs was ineffective. These data support the hypothesis that sperm contain a soluble, cytosolic protein factor that induces Ca 2+ oscillations in eggs at fertilisation. These data are discussed in the light of our recent findings that suggest that the sperm factor possesses a phospholipase C activity.

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.

The Frequency of Calcium Oscillations in Mouse Eggs at Fertilization Is Modulated by the Number of Fused Sperm

In a variety of calcium signaling systems, the frequency of intracellular calcium oscillations is physiologically important. Probably multiple factors control the frequency of calcium oscillations in the egg after fertilization and many of these remain to be identified. In this study, we present the first rigorous set of data showing that monospermic fertilization is important for setting the physiological calcium oscillation frequency. Recordings in 152 zona-free eggs show that the general pattern of the calcium oscillations is identical in monospermic and polyspermic eggs; however, the oscillation frequency is higher in polyspermic eggs (P < 10−6). The frequency of the late oscillations increases with the number of sperm heads incorporated: 5.2 ± 0.3 spikes per hour (mean ± SEM; n = 55) in monospermic eggs, 6.6 ± 0.3 (n = 62) in dispermic eggs, 8.7 ± 0.7 (n = 23) in trispermic eggs, and 8.9 ± 0.9 (n = 12) in eggs with four or more sperm heads. The frequency of the early oscillations is also increased in polyspermic eggs. Seventy-eight additional eggs were divided into two groups and inseminated with two different sperm concentrations (“low” and “high”) to obtain one group mainly monospermic and the other mainly polyspermic. The two groups of eggs oscillated at different frequencies (P < 10−5). These data rule out the possibility of an egg effect in which some eggs would have the dual properties of oscillating faster and of being able to fuse with several sperm cells. These data instead suggest that the sperm modulates the frequency of the oscillations in a dose-dependent manner.