Mammalian Eggs Research Articles

Signaling through protein kinases during egg activation

Following penetration of the zona pellucida, the mammalian spermatozoon binds and fuses with the egg plasma membrane, thereby fertilizing the egg that is still arrested at the second metaphase. Fertilization initiates in the egg a sequence of events referred to as ‘egg activation’. An initial increase in intracellular Ca 2+ concentration ([Ca 2+] i ) appears to be the very early cellular event observed which leads to the cortical granules exocytosis and resumption of meiosis. Various treatments can induce parthenogenetic activation mimicking at least part of the fertilization events. Similar to somatic cells, studies in mammalian eggs suggest that signal transduction pathways mediate egg activation. The initial increase in [Ca 2+] i appears to be critical for egg activation. However, other messengers such as protein kinase C (PKC) and protein tyrosine kinases (PTKs), were suggested as possible inducers of some aspects of egg activation. In the present work, studies concerning the involvement of protein kinases during egg activation in our laboratory and in others are summarized.

Signalling in mammalian egg activation: role of protein kinases

Embryonic development is initiated after the fertilizing spermatozoon enters the egg and triggers a process known as ‘egg activation’. Activation results in an increase in intracellular calcium concentration, cortical granule exocytosis (CGE), cell cycle resumption and recruitment of maternal mRNA. Various treatments can induce parthenogenetic activation characterized by the same manifestations. Signal transduction pathways similar to those known for somatic cells mediate the mammalian egg activation. This review focuses on the signal transduction pathways that occur during mammalian fertilization and during parthenogenetic egg activation. We discuss the possibility that members of the protein tyrosine kinase (PTKs) families, the Src family PTKs in particular, operate during egg activation and that protein kinase C can induce CGE.

Flowering Plant Sperm Contains a Cytosolic Soluble Protein Factor Which Can Trigger Calcium Oscillations in Mouse Eggs

There is evidence showing that the sperm-induced Ca2+ oscillations in mammalian eggs at fertilization are triggered by a sperm-derived protein factor. It was established recently that the activity of the putative sperm protein in causing Ca2+ oscillations in mammalian eggs is not species-specific in vertebrates (1, 16). Here we report that cytosolic soluble extracts derived from flowering plant sperms in Brassica campestris can also induce fertilization-like Ca2+ oscillations when microinjected into mouse eggs. The factor responsible for inducing Ca2+ oscillations in the plant sperm was sperm-specific and heat- or trypsin-labile. Eight to ten sperm equivalents of the plant sperm extracts had enough activity to trigger Ca2+ oscillations in mouse eggs. Our study suggests that, although plant and mammal are evolutionary divergent species, the activity of the putative sperm protein factor in triggering Ca2+ signaling in mammalian eggs is not specific to the animal kingdom.

Evidence That Phospholipase C from the Sperm Is Not Responsible for Initiating Ca2+ Release at Fertilization in Mouse Eggs

Release of Ca2+ from intracellular stores at fertilization of mammalian eggs is mediated by inositol 1,4,5-trisphosphate (IP3), but the mechanism by which the sperm initiates IP3 production is not yet understood. We tested the hypothesis that phospholipase C (PLC) activity introduced into the mouse egg as a consequence of sperm–egg fusion is responsible for causing Ca2+ release. We demonstrated that microinjecting purified, recombinant PLCγ1 protein into mouse eggs caused Ca2+ oscillations like those seen at fertilization. However, the PLC activity in the minimum amount of purified PLCγ1 protein needed to elicit Ca2+ release when injected into eggs was ∼500–900 times the PLC activity contained in a single sperm. This indicates that a single mouse sperm does not contain enough PLC activity to be responsible for causing Ca2+ release at fertilization. We also examined whether phosphatidylinositol 3-kinase (PI3K) could have a role in this process, and found that several inhibitors of PI3K-mediated signaling had no effect on Ca2+ release at fertilization.

Positive Darwinian Selection Drives the Evolution of Several Female Reproductive Proteins in Mammals

Rapid evolution driven by positive Darwinian selection is a recurrent theme in male reproductive protein evolution. In contrast, positive selection has never been demonstrated for female reproductive proteins. Here, we perform phylogeny-based tests on three female mammalian fertilization proteins and demonstrate positive selection promoting their divergence. Two of these female fertilization proteins, the zona pellucida glycoproteins ZP2 and ZP3, are part of the mammalian egg coat. Several sites identified in ZP3 as likely to be under positive selection are located in a region previously demonstrated to be involved in species-specific sperm-egg interaction, suggesting the selective pressure is related to male-female interaction. The results provide long-sought evidence for two evolutionary hypotheses: sperm competition and sexual conflict.

Sperm factor induces intracellular free calcium oscillations by stimulating the phosphoinositide pathway.

Injection of a porcine cytosolic sperm factor (SF) or of a porcine testicular extract into mammalian eggs triggers oscillations of intracellular free calcium ([Ca(2+)](i)) similar to those initiated by fertilization. To elucidate whether SF activates the phosphoinositide (PI) pathway, mouse eggs or SF were incubated with U73122, an inhibitor of events leading to phospholipase C (PLC) activation and/or of PLC itself. In both cases, U73122 blocked the ability of SF to induce [Ca(2+)](i) oscillations, although it did not inhibit Ca(2+) release caused by injection of inositol 1,4,5-triphosphate (IP(3)). The inactive analogue, U73343, had no effect on SF-induced Ca(2+) responses. To determine at the single cell level whether SF triggers IP(3) production concomitantly with a [Ca(2+)](i) rise, SF was injected into Xenopus oocytes and IP(3) concentration was determined using a biological detector cell combined with capillary electrophoresis. Injection of SF induced a significant increase in [Ca(2+)](i) and IP(3) production in these oocytes. Using ammonium sulfate precipitation, chromatographic fractionation, and Western blotting, we determined whether PLCgamma1, PLCgamma2, or PLCdelta4 and/or its splice variants, which are present in sperm and testis, are responsible for the Ca(2+) activity in the extracts. Our results revealed that active fractions do not contain PLCgamma1, PLCgamma2, or PLCdelta4 and/or its splice variants, which were present in inactive fractions. We also tested whether IP(3) could be the sensitizing stimulus of the Ca(2+)-induced Ca(2+) release mechanism, which is an important feature of fertilized and SF-injected eggs. Eggs injected with adenophostin A, an IP(3) receptor agonist, showed enhanced Ca(2+) responses to CaCl(2) injections. Thus, SF, and probably sperm, induces [Ca(2+)](i) rises by persistently stimulating IP(3) production, which in turn results in long-lasting sensitization of Ca(2+)-induced Ca(2+) release. Whether SF is itself a PLC or whether it acts upstream of the egg's PLCs remains to be elucidated.

Analysis of Mn2+/Ca2+influx and release during Ca2+oscillations in mouse eggs injected with sperm extract

Repetitive Ca2+release from the endoplasmic reticulum (ER) is necessary for activation of mammalian eggs. Influx and release of Mn2+and Ca2+during Ca2+oscillations induced by injection of sperm extract (SE) into mouse eggs were investigated by Mn2+-quenching of intracellular Fura-2 after adding Mn2+to external medium. Mn2+/Ca2+influx was detected at the resting state. A marked Mn2+/Ca2+influx occurred during the first Ca2+release upon SE injection, and persistently facilitated Mn2+/Ca2+influx was observed during steady Ca2+oscillations. As intracellular Mn2+concentration ([Mn2+]i) increased progressively, periodic [Mn2+]irises appeared, corresponding to each Ca2+transient but taking a slower time course. A numerical simulation based on continuous Mn2+/Ca2+influx-extrusion across the plasma membrane and release-uptake across the ER membrane in a competitive manner mimicked well the Mn2+oscillations calculated from experimental data, strongly suggesting that repetitive Mn2+release develops after Mn2+entry and uptake into the ER. In other experiments, a marked Mn2+influx occurred upon Mn2+addition to Ca2+-free medium after depletion of the ER using an ER Ca2+pump inhibitor plus repeated injection of inositol 1,4,5-trisphosphate (InsP3). No significant increase in Mn2+influx was induced by injection of SE, InsP3, or Ca2+, when Ca2+release was prevented by pre-injection of an antibody against the InsP3receptor. We concluded that Ca2+influx is activated during the initial large Ca2+release possibly by a capacitative mechanism and kept facilitated during steady Ca2+oscillations. The finding that repetitive Mn2+release is caused by continuous Mn2+entry suggests that continuous Ca2+influx may play a critical role in refilling the ER and, thereby, maintaining Ca2+oscillations in mammalian fertilization.

Activation of rabbit oocytes: the impact of the Ca2+ signal regime on development.

Postfertilization manipulation of mammalian embryos results in various developmental alterations. To determine whether the manipulation of the Ca2+ regime causing oocyte activation is a valuable experimental means in helping understand the biological process by which embryos integrate signals from outside and later regulate gene expression, we linked Ca2+ signal parameters i.e. amplitude, number and frequency, with the efficiency and quality of postimplantation development. Freshly ovulated rabbit oocytes were subjected to repetitive and modulated Ca2+ influx. The results provide three major pieces of information. Firstly, the Ca2+ stimulus is the most efficient signal activating mammalian eggs when it is applied in a repetitive manner, the amplitude being the crucial factor. Secondly, the dynamics of early cleavage does not appear to be determined by either the frequency or the amplitude of modulation of the Ca2+ signal that activates the oocyte. Thirdly, amplitude and temporal modulation of the Ca2+ signal in the early minutes influences the developmental performance and the morphology of the rabbit parthenogenetic conceptus at day 11.5 of pregnancy. The results demonstrate the importance of epigenetic events during postfertilization as well as the possible uses of Ca2+ modulation in studying long term developmental effects.

Cell surface beta-1,4-galactosyltransferase-I activates G protein-dependent exocytotic signaling.

ZP3 is a protein in the mammalian egg coat (zona pellucida) that binds sperm and stimulates acrosomal exocytosis, enabling sperm to penetrate the zona pellucida. The nature of the ZP3 receptor/s on sperm is a matter of considerable debate, but most evidence suggests that ZP3 binds to beta-1,4-galactosyltransferase-I (GalTase) on the sperm surface. It has been suggested that ZP3 induces the acrosome reaction by crosslinking GalTase, activating a heterotrimeric G protein. In this regard, acrosomal exocytosis is sensitive to pertussis toxin and the GalTase cytoplasmic domain can precipitate G(i) from sperm lysates. Sperm from mice that overexpress GalTase bind more soluble ZP3 and show accelerated G protein activation, whereas sperm from mice with a targeted deletion in GalTase have markedly less ability to bind soluble ZP3, undergo the ZP3-induced acrosome reaction, and penetrate the zona pellucida. We have examined the ability of GalTase to function as a ZP3 receptor and to activate heterotrimeric G proteins using Xenopus laevis oocytes as a heterologous expression system. Oocytes that express GalTase bound ZP3 but did not bind other zona pellucida glycoproteins. After oocyte maturation, ZP3 or GalTase antibodies were able to trigger cortical granule exocytosis and activation of GalTase-expressing eggs. Pertussis toxin inhibited GalTase-induced egg activation. Consistent with G protein activation, both ZP3 and anti-GalTase antibodies increased GTP-gamma[(35)S] binding as well as GTPase activity in membranes from eggs expressing GalTase. Finally, mutagenesis of a putative G protein activation motif within the GalTase cytoplasmic domain eliminated G protein activation in response to ZP3 or anti-GalTase antibodies. These results demonstrate directly that GalTase functions as a ZP3 receptor and following aggregation, is capable of activating pertussis toxin-sensitive G proteins leading to exocytosis.

A profile of fertilization in mammals.

Fertilization is defined as the process of union of two gametes, eggs and sperm. When mammalian eggs and sperm come into contact in the female oviduct, a series of steps is set in motion that can lead to fertilization and ultimately to development of new individuals. The pathway begins with species-specific binding of sperm to eggs and ends a relatively short time later with fusion of a single sperm with each egg. Although this process has been investigated extensively, only recently have the molecular components of egg and sperm that participate in the mammalian fertilization pathway been identified. Some of these components may participate in gamete adhesion and exocytosis, whereas others may be involved in gamete fusion. Here we describe selected aspects of mammalian fertilization and address some of the latest experimental evidence that bears on this important area of research.

Calcium and The Control of Mammalian Cortical Granule Exocytosis

Abstract At fertilization, the release of intracellular calcium is necessary and sufficient for most, if not virtually all, of the major events of egg activation that are responsible for the onset of embryonic development. In mammalian eggs, repetitive calcium oscillations stimulate egg activation events through calcium-dependent effectors, such as calmodulin, protein kinases, and specific proteins involved in exocytosis. One of the earliest calcium-dependent events is the exocytosis of cortical granules (CGs), a secretory event resulting in the block to polyspermy and the prevention of triploidy. Emerging studies suggest that CG release in mature eggs is dependent upon calcium-dependent proteins similar to those in somatic cells employed to undergo calcium-regulated exocytosis. In contrast, pre-ovulatory oocytes are incompetent to undergo CG exocytosis due to deficiencies in the ability to release and respond to increases in intracellular calcium. The development of competence to release and respond to calcium is relevant to both animal and human in vitro fertilization programs that largely utilize ovarian oocytes not all of which are fully activation competent.

Composition, regulation, and function of the cytoskeleton in mammalian eggs and embryos.

The cytoskeleton of the mammalian egg and early embryo is highly unique when compared to the cytoskeleton of their somatic cell brethren. Although all three cytoskeletal systems, actin filaments, microtubules, and intermediate filaments, are present as early as the unfertilized egg; each system has adapted features that allow the egg and early embryo to meet the strict demands of the developmental process. The major demands placed upon eggs and embryos are developmental transitions (i.e., fertilization, compaction, blastocyst formation, germ layer formation and gastrulation), each of which must be traversed in order for the embryo to form a new individual. To successfully complete all of the necessary processes during early development, eggs and embryos must call upon many signal transduction mechanisms, cytoskeletal components, and genes that are both unique to embryogenesis and ubiquitous among many types of somatic cells. It is the goal of this review to provide some current details into the mechanisms that drive early development primarily focusing on the cytoskeletal components eggs and embryos have adapted to promote embryogenesis.

Composition regulation and function of the cytoskeleton in mammalian eggs and embryos

Composition regulation and function of the cytoskeleton in mammalian eggs and embryos

Calcium and the control of mammalian cortical granule exocytosis.

At fertilization, the release of intracellular calcium is necessary and sufficient for most, if not virtually all, of the major events of egg activation that are responsible for the onset of embryonic development. In mammalian eggs, repetitive calcium oscillations stimulate egg activation events through calcium-dependent effectors, such as calmodulin, protein kinases, and specific proteins involved in exocytosis. One of the earliest calcium-dependent events is the exocytosis of cortical granules (CGs), a secretory event resulting in the block to polyspermy and the prevention of triploidy. Emerging studies suggest that CG release in mature eggs is dependent upon calcium-dependent proteins similar to those in somatic cells employed to undergo calcium-regulated exocytosis. In contrast, pre-ovulatory oocytes are incompetent to undergo CG exocytosis due to deficiencies in the ability to release and respond to increases in intracellular calcium. The development of competence to release and respond to calcium is relevant to both animal and human in vitro fertilization programs that largely utilize ovarian oocytes not all of which are fully activation competent.

CDNA Cloning and Functional Analysis of Ascidian Sperm Proacrosin

cDNA cloning and functional analysis of proacrosin from the ascidian Halocynthia roretzi were undertaken. The isolated cDNA of the ascidian preproacrosin consists of 2367 nucleotides, and an open reading frame encodes 505 amino acids, which corresponds to the molecular mass of 55,003 Da. The mRNA of proacrosin was found to be specifically expressed in the gonad by Northern blotting and in the spermatocytes or spermatids by in situ hybridization. The amino acid sequences around His(76), Asp(132), and Ser(227), which make up a catalytic triad, showed high homology to those of the trypsin family. Ascidian acrosin has paired basic residues (Lys(56)-His(57)) in the N-terminal region, which is one of the most characteristic features of mammalian acrosin. This region seems to play a key role in the binding of (pro)acrosin to the vitelline coat, because the peptide containing the paired basic residues, but not the peptide substituted with Ala, was capable of binding to the vitelline coat. Unlike mammalian proacrosin, ascidian proacrosin contains two CUB domains in the C-terminal region, in which CUB domain 1 seems to be involved in its binding to the vitelline coat. Four components of the vitelline coat that are capable of binding to CUB domain 1 in proacrosin were identified. In response to sperm activation, acrosin was released from sperm into the surrounding seawater, suggesting that ascidian acrosin plays a key role in sperm penetration through the coat. These results indicate that ascidian sperm contains a mammalian acrosin homologue, a multi-functional protein working in fertilization.

Mammalian Sperm Contain a Ca2+-Sensitive Phospholipase C Activity That Can Generate InsP3 from PIP2 Associated with Intracellular Organelles

We have previously described a phospholipase C (PLC) activity in mammalian sperm cytosolic extracts. Here we have examined the Ca2+ dependency of the enzyme, whether there is enough in a single sperm to account for Ca2+ release at fertilization, and finally where in the egg is the phosphatidyl 4,5-bisphosphate, the substrate for the enzyme. As for all PLCs examined so far in vitro, we found that the boar sperm PLC activity was Ca2+ dependent. Specific activity increased when free Ca2+ levels were micromolar. However, even at nanomolar free Ca2+ concentration the boar sperm PLC activity was considerable, being two orders of magnitude greater than PLC activities in other tissues. We calculated that PLC activity of a single boar sperm in a mammalian egg is enough to generate 400 nM inositol 1,4,5-trisphosphate (InsP3) in 1 min, which may be sufficient to account for the observed Ca2+ changes in an egg at fertilization. We fractionated sea urchin egg homogenate and examined the ability of boar sperm extract to generate InsP3 from these fractions. The sperm PLC activity triggered InsP3 production from a PIP2-enriched nonmicrosomal egg compartment that contained yolk platelets. We propose that this sperm PLC activity, which is active at nanomolar Ca2+ levels and hydrolyzes PIP2 from intracellular membranes, could be involved in the Ca2+ changes observed at fertilization.

Selective and Continuous Elimination of Mitochondria Microinjected Into Mouse Eggs From Spermatids, but Not From Liver Cells, Occurs Throughout Embryogenesis

Exclusion of paternal mitochondria in fertilized mammalian eggs is very stringent and ensures strictly maternal mtDNA inheritance. In this study, to examine whether elimination was specific to sperm mitochondria, we microinjected spermatid or liver mitochondria into mouse embryos. Congenic B6-mt(spr) strain mice, which are different from C57BL/6J (B6) strain mice (Mus musculus domesticus) only in possessing M. spretus mtDNA, were used as mitochondrial donors. B6-mt(spr) mice and a quantitative PCR method enabled selective estimation of the amount of M. spretus mtDNA introduced even in the presence of host M. m. domesticus mtDNA and monitoring subsequent changes of its amount during embryogenesis. Results showed that M. spretus mtDNA in spermatid mitochondria was not eliminated by the blastocyst stage, probably due to the introduction of a larger amount of spermatid mtDNA than of sperm mtDNA into embryos on fertilization. However, spermatid-derived M. spretus mtDNA was eliminated by the time of birth, whereas liver-derived M. spretus mtDNA was still present in most newborn mice, even though its amount introduced was significantly less than that of spermatid mtDNA. These observations suggest that mitochondria from spermatids but not from liver have specific factors that ensure their selective elimination and resultant elimination of mtDNA in them, and that the occurrence of elimination is not limited to early stage embryos, but continues throughout embryogenesis.

Rat Spermatogenic Cell β-[formula omitted]-Galactosidase: Characterization, Biosynthesis, and Immunolocalization

In this study we have demonstrated that the rat sperm acrosomal β-d-galactosidase is expressed in late spermatocytes and spermatids (round, elongated/condensed) during spermatogenesis. The enzyme is an exoglycohydrolase which, along with other exoglycohydrolases and proteases, is thought to aid in penetration of the zona pellucida, the extracellular glycocalyx that surrounds the mammalian egg. The presence of the enzyme in spermatocytes was confirmed by multiple approaches using biochemical, biosynthetic, and immunohistochemical protocols. The germ cells (spermatocytes, round spermatids, and elongated/condensed spermatids), purified from rat testis, were found to contain β-galactosidase and four other glycohydrolases (β-d-glucuronidase, α-d-mannosidase, α-l-fucosidase, and β-N-acetylglucosaminidase). With the exception of α-l-fucosidase, the other enzymes assayed demonstrated a two- to threefold higher activity per cell in spermatocytes than in round spermatids. Immunoblotting approaches of affinity-purified germ cell extracts demonstrated several molecular forms of β-galactosidase in spermatocytes and round spermatids; one of these forms (62 kDa) was seen only in round spermatids. The biosynthetic approach demonstrated that the enzyme is synthesized in spermatocytes and round spermatids in culture in high-molecular-weight precursor forms (90/88 kDa) which undergo processing to lower molecular weight mature forms in a cell-specific manner. The net result is the formation of predominantly 64- and 62-kDa forms in spermatocytes and round spermatids, respectively. The conversion of precursor forms to mature forms in the diploid and haploid cells in culture is rapid with t1/2 of 6.5 and 9.0 h, respectively. Immunohistochemical approaches revealed an immunopositive reaction in the Golgi membranes, Golgi-associated vesicles, and lysosome-like structures in the late spermatocytes and early round spermatids. The forming/formed acrosome in round and elongated spermatids was also immunoreactive.

Structural Analysis of the Asparagine-Linked Glycan Units of the ZP2 and ZP3 Glycoproteins from Mouse Zona Pellucida

Zona pellucida (ZP), the extracellular glycocalyx that surrounds the mammalian egg plasma membrane, is a relatively simple structure consisting of three to four glycoproteins. In the mouse, the ZP is composed of three glycoproteins, namely ZP1 (200 kDa), ZP2 (120 kDa), and ZP3 (83 kDa). Extensive studies in this species have resulted in the identification of primary (mZP3) and secondary (mZP2) binding sites for spermatozoa. The two zona components are highly glycosylated containing N-linked and O-linked glycan units. In an attempt to characterize N-linked glycan units, mZP2 and mZP3 were purified and the N-linked carbohydrate chains were released by exhaustive digestion with N-glycanase. The released oligosaccharides (OSs) were radiolabeled by reduction with NaB3H4 and resolved by gel filtration on a column of Bio-Gel P-4. The OSs separated into several peaks indicating the presence of a variety of N-linked glycans. Interestingly, the radioactive peaks resolved from mZP2 and mZP3 were quite different, a result suggesting qualitative and quantitative differences in the glycans. The [3H]-labeled glycans present in mZP2 and mZP3 were pooled separately and fractionated by serial lectin chromatography. Experimental evidence included in this report strongly suggests that mZP3 (but not mZP2) contains polylactosaminyl glycan with terminal, nonreducing α-galactosyl residues. The mZP3 glycans eluted from the immobilized lectin columns were further characterized by lectin and sizing column chromatography before or after digestion with endo-/exo-glycohydrolases. Data revealed the presence of a variety of OSs, including poly-N-acetyllactosaminyl, bi-, tri-, and tetraantennary complex-type, and high-mannose-type glycans. Taken together, these results provide additional evidence on the complex nature of the glycan chains present on mZP glycoconjugates.

Sperm-Induced Ca2+ Oscillations in Mouse Oocytes and Eggs Can Be Mimicked by Photolysis of Caged Inositol 1,4,5-Trisphosphate: Evidence to Support a Continuous Low Level Production of Inositol 1,4,5-Trisphosphate during Mammalian Fertilization

During mouse fertilization the spermatozoon induces a series of low-frequency long-lasting Ca2+ oscillations. It is generally accepted that these oscillations are due to Ca2+ release through the inositol 1,4,5-trisphosphate (InsP3) receptor. However, InsP3 microinjection does not mimic sperm-induced Ca2+ oscillations, leading to the suggestion that the spermatozoon causes Ca2+ release by sensitizing the InsP3 receptor to basal levels of InsP3. This contradicts recent evidence that the spermatozoon triggers Ca2+ oscillations by introducing a phospholipase C or else an activator of phospholipase C. Here we show for the first time that sperm-induced Ca2+ oscillations may be mimicked by the photolysis of caged InsP3 in both mouse metaphase II eggs and germinal vesicle stage oocytes. Eggs, and also oocytes that had displayed spontaneous Ca2+ oscillations, gave long-lasting Ca2+ oscillations when fertilized or when caged InsP3 was photolyzed. In contrast, oocytes that had shown no spontaneous Ca2+ oscillations did not generate many oscillations when fertilized or following photolysis of caged InsP3. Fertilization in eggs was most closely mimicked when InsP3 was uncaged at relatively low amounts for extended periods. Here we observed an initial Ca2+ transient with superimposed spikes, followed by a series of single transients with a low frequency; all characteristics of the Ca2+ changes at fertilization. We therefore show that InsP3 can mimic the distinctive pattern of Ca2+ release in mammalian eggs at fertilization. It is proposed that a sperm Ca2+-releasing factor operates by generating a continuous small amount of InsP3 over an extended period of time, consistent with the evidence for the involvement of a phospholipase C.