Egg Cytoplasm Research Articles

Induction of androgenetic development of the brook charr (Salvelinus fontinalis) × Arctic charr (Salvelinus alpinus) hybrids in eggs derived from the parental species

Failure of interspecific androgenesis between brook charr (Salvelinus fontinalis, Mitchill 1814) and Arctic charr (Salvelinus alpinus, L.) has been attributed to the conflict between the egg cytoplasm of one species and the sperm nucleus of the other species. To overcome this incompatibility, sperm derived from the brook charr×Arctic charr hybrid male was used to induce androgenetic development in eggs originating from the parental species as well as their hybrids. The eggs were subjected to 420Gy of X-radiation to damage the maternal nuclear DNA and inseminated with untreated sperm. Haploid zygotes were exposed to high hydrostatic pressure shock (7000psi for 4min), which was applied 420min after insemination to inhibit the first cell cleavage and recover the diploid state of the zygote. The androgenetic diploid offspring that hatched from the brook charr, the Arctic charr and the hybrids eggs had survival rates of 4.7±0.6%, 1.2±0.4% and 16.8±0.5%, respectively. Drastic mortality among the hatched androgenetic individuals was observed within the first five months of rearing. Cytogenetic analysis of the androgenetic progenies exhibited residues of the irradiated maternal nuclear genome in the form of radiation-induced chromosome fragments in 47% of the specimens that were examined. Interactions between the egg cytoplasm and the sperm nucleus, the low quality of the gametes, the expression of homozygous paternal lethal alleles and the incomplete inactivation of the maternal chromosomes were identified as factors responsible for the large mortality among androgenetic embryos and hatchlings.

Authors’ response: polarization microscopy and rescue ICSI

1 h We thank Dr Coticchio for his commentary on our article ‘Spindle examination in unfertilized eggs using the polarization microscope can assist rescue ICS’. His view on some key points in our study harmonize with ideas and issues we have discussed and debated over and over. Therefore, we are pleased to be able to highlight answers for issues he has raised. First, we know the issue of whether to perform rescue intracytoplasmic sperm injection (ICSI) or to cancel the current cycle is still under debate. The goal of assisted reproductive technology is to help infertile couples have a healthy baby, not increase the number of transferable embryos of compromised quality. The biological and clinical implications of oocyte ageing are well reported in numerous articles, including cytogenetic abnormalities in the resulting embryo following rescue ICSI (Morton et al., 1997; Pehlivan et al., 2004) as well as congenital abnormalities (Tarin et al., 2002). However, we should also consider that routine ICSI and IVF might also cause an increased risk of major congenital anomalies compared with natural conception (Wen et al., 2012). To our knowledge, there have been reports of successful births following rescue ICSI (Yuzpe et al., 2000; Shalom-PAZ et al., 2011). As previously shown from data in our centre (Shalom-PAZ et al., 2011), out of 20 pregnancies, only one was diagnosed with trisomy 21 and was terminated, while the other children were found to be

The ascidian numb gene involves in the formation of neural tissues.

Notch signaling plays fundamental roles in various animal development. It has been suggested that Hr-Notch, a Notch homologue in the ascidian Halocynthia roretzi, is involved in the formation of peripheral neurons by suppressing the neural fates and promoting the epidermal differentiation. However, roles of Notch signaling remain controversial in the formation of nervous system in ascidian embryos. To precisely investigate functions of Notch signaling, we have isolated and characterized Hr-Numb, a Numb homologue which is a negative regulator of Notch signaling, in H. roretzi. Maternal expression of Hr-Numb mRNAs was detected in egg cytoplasm and the transcripts were inherited by the animal blastomeres. Its zygotic expression became evident by the early neurula stage and the transcripts were detected in dorsal neural precursor cells. Suppression of Hr-Numb function by an antisense morpholino oligonucleotide resulted in larvae with defect in brain vesicle and palps formation. Similar results have been obtained by overexpression of the constitutively activated Hr-Notch forms. Therefore, these results suggest that Hr-Numb is involved in Notch signaling during ascidian embryogenesis.

Towards germline gene therapy of inherited mitochondrial diseases

Mutations in mitochondrial DNA (mtDNA) are associated with serious human diseases and inherited from mother's eggs. Here we investigated the feasibility of mtDNA replacement in human oocytes by spindle transfer (ST). Of 106 human oocytes donated for research, 65 were subjected to reciprocal ST and 33 served as controls. Fertilization rate in ST oocytes (73%) was similar to controls (75%). However, a significant portion of ST zygotes (52%) displayed abnormal fertilization as determined by irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell (ESC) isolation (38%) rates were comparable to controls. All ESC lines derived from ST zygotes displayed normal euploid karyotypes and contained exclusively donor mtDNA. The mtDNA can be efficiently replaced in human oocytes. Although some ST oocytes displayed abnormal fertilization, remaining embryos were capable of developing to blastocysts and producing ESCs similar to controls.

Histone H1 compacts DNA under force and during chromatin assembly

Histone H1 binds to linker DNA between nucleosomes, but the dynamics and biological ramifications of this interaction remain poorly understood. We performed single-molecule experiments using magnetic tweezers to determine the effects of H1 on naked DNA in buffer or during chromatin assembly in Xenopus egg extracts. In buffer, nanomolar concentrations of H1 induce bending and looping of naked DNA at stretching forces below 0.6 pN, effects that can be reversed with 2.7-pN force or in 200 mM monovalent salt concentrations. Consecutive tens-of-nanometer bending events suggest that H1 binds to naked DNA in buffer at high stoichiometries. In egg extracts, single DNA molecules assemble into nucleosomes and undergo rapid compaction. Histone H1 at endogenous physiological concentrations increases the DNA compaction rate during chromatin assembly under 2-pN force and decreases it during disassembly under 5-pN force. In egg cytoplasm, histone H1 protects sperm nuclei undergoing genome-wide decondensation and chromatin assembly from becoming abnormally stretched or fragmented due to astral microtubule pulling forces. These results reveal functional ramifications of H1 binding to DNA at the single-molecule level and suggest an important physiological role for H1 in compacting DNA under force and during chromatin assembly.

Mitochondrial GCD1 Dysfunction Reveals Reciprocal Cell-to-Cell Signaling during the Maturation of Arabidopsis Female Gametes

Cell-to-cell communication in embryo sacs is thought to regulate the development of female gametes in flowering plants, but the details remain poorly understood. Here, we report a mitochondrial protein, GAMETE CELL DEFECTIVE 1 (GCD1), enriched in gametophytes that is essential for final maturation of female gametes. Using Arabidopsis gcd1 mutants, we found that final maturation of the egg and central cells is not required for double fertilization but is necessary for embryogenesis initiation and endosperm development. Furthermore, nonautonomous effects, observed when GCD1 or AAC2 function is disrupted, suggest that mitochondrial function influences reciprocal signaling between central and egg cells to regulate maturation of the partner (egg or central) cell. Our findings confirm that cell-to-cell communication is important in functional maturation of female gametic cells and suggest that both egg and central cells sense and transmit their mitochondrial metabolic status as animportant cue that regulates the coordination of gamete maturation.

Egg activation in physiological polyspermy

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 Ca(2+) concentration induced by the fertilizing sperm is both necessary and sufficient for egg activation in polyspermy. Multiple small Ca(2+) waves induced by several fertilizing sperm result in a long-lasting Ca(2+) 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 Ca(2+) waves. Citrate synthase may perform dual functions: as an enzyme in mitochondria and as a Ca(2+)-inducing factor in egg cytoplasm. We also discuss the close relationship between the mode of fertilization and the Ca(2+) rise at egg activation and consider changes in this process through evolution in vertebrates.

Exploring the effect of the Cardinium endosymbiont on spiders

Spiders have recently emerged as important diversity hot spots for endosymbiotic bacteria, but the consequences of these symbiotic interactions are largely unknown. In this article, we examined the evolutionary history and effect of the intracellular bacterium Cardinium hertigii in the marbled cellar spider Holocnemus pluchei. We showed that Cardinium infection is primarily transmitted in spider populations maternally via egg cytoplasm, with 100% of the progeny from infected mothers being also infected. Examination of a co-inherited marker, mitochondrial DNA (mtDNA), revealed that Cardinium infection is associated with a wide diversity of mtDNA haplotypes, showing that the interaction between Cardinium and H.pluchei has a long-term evolutionary dimension and that horizontal transmission among individuals could also occur. Although Cardinium is well known to exert sex ratio distortion or cytoplasmic incompatibility in various arthropod hosts, we show, however, that Cardinium does not interact with the reproductive biology of H.pluchei. A field survey shows a clear geographical structuring of Cardinium infection, with a marked gradual variation of infection frequencies from ca. 0.80 to 0. We discuss different mechanistic and evolutionary explanations for these results as well as their consequences for spider phenotypes. Notably, we suggest that Cardinium can either behave as a neutral cytoplasmic element within H.pluchei or exhibit a context-dependent effect, depending on the environmental conditions.

The animal body plan, the prototypic body segment, and eye evolution

Developmental genetics of Drosophila continue to have a great impact on our understanding of evolution. The specification of the body plan involves four conceptual steps: 1) Localization of maternal mRNAs in the egg cytoplasm. 2) Translation of these RNAs and formation of morphogen gradients. 3) Subdivision of the antero-posterior gradient into a repetitive pattern of body segments. 4) Assignment of a specific identity to each segment by the Hox genes. The discovery of the Hox genes has uncovered a universal principle shared by all bilaterians; they serve as master control genes specifying organization along the antero-posterior axis. The ancestral arthropods presumably consisted of a series of more or less identical segments, which may be represented by recently discovered precambrian Lobopodia which have a pair of legs and a pair of eyes in each segment. The progressive divergence of Hox genes has led to progressive cephalization and caudalization. From the amino acid sequences of the clustered homeodomains we can deduce that the mesothoracic segment represents the prototype from the more anterior and the more posterior segments evolved. Pax6 has been identified as a master control gene for eye development in all bilaterians. Since Pax6 is involved in eye development in all bilaterian phyla, this argues strongly for a monophyletic origin of the metazoan eye. With the same tool box of transcription factors all the different eye-types can be constructed.

Amphibian interorder nuclear transfer embryos reveal conserved embryonic gene transcription, but deficient DNA replication or chromosome segregation

Early interspecies nuclear transfer (iNT) experiments suggested that a foreign nucleus may become permanently damaged after a few rounds of cell division in the cytoplasm of another species. That is, in some distant species combinations, nucleocytoplasmic hybrid (cybrid) blastula nuclei can no longer support development, even if they are back-transferred into their own kind of egg cytoplasm. We monitored foreign DNA amplification and RNA production by quantitative PCR (qPCR) and RT-qPCR in interorder amphibian hybrids and cybrids formed by the transfer of newt (Pleurodeles waltl) embryonic nuclei into intact and enucleated frog (Xenopus laevis) eggs. We found a dramatic reduction in the expansion of foreign DNA and cell numbers in developing cybrid embryos that correlated with reduced gene transcription. Interestingly, expansion in cell numbers was rescued by the recipient species (Xenopus) maternal genome in iNT hybrids, but it did not improve P. waltl DNA expansion or gene transcription. Also, foreign gene transcripts, normalized to DNA copy numbers, were mostly normal in both iNT hybrids and cybrids. Thus, incomplete foreign DNA replication and/or chromosome segregation during cell division may be the major form of nuclear damage occurring as a result of nuclear replication in a foreign cytoplasmic environment. It also shows that the mechanisms of embryonic gene transcription are highly conserved across amphibians and may not be a major cause of cybrid lethality.

The maternal muscle determinant in the ascidian egg

Muscle formation in ascidian embryos has been investigated for more than a century as a representative example of cell fate specification by localized maternal factors within the egg cytoplasm. Observations of colored cytoplasm in combination with micromanipulation techniques have suggested the presence of a muscle-forming factor. The molecular basis has been elucidated with the discovery of macho-1. macho-1 mRNA is already present in the unfertilized egg, and translocates to the posterior region of the egg during ooplasmic movements. It encodes a zinc-finger transcription factor that positively regulates the expression of target genes. macho-1-binding cis-elements have been identified in muscle-specific zygotic genes. Maternally localized macho-1 appears to have originated in the ascidian lineage, but it activates a muscle-forming developmental program that is shared by the vertebrates. macho-1 is also involved in establishment of the anterior-posterior axis as a competence factor in mesenchyme induction in the posterior region. It is suggested that translation of the macho-1 protein is initiated at the eight-cell stage, and that the protein is inherited by all descendant blastomeres of the posterior-vegetal region. The macho-1 activities in nonmuscle descendants are suppressed or modified by cell interactions during the cleavage stages. In addition to the primary muscle specified by maternal macho-1, ascidian embryos develop secondary muscle, whose fate is determined by cell interactions. Dozens of maternal mRNAs show similar localization to macho-1, and these are known as postplasmic/PEM RNAs, being also involved in various posterior-specific developmental events. Evolutionary aspects relevant to macho-1 and tail muscle formation are also discussed in this article.

Cytological features of oogenesis and their evolutionary significance in the fern Osmunda japonica

The development of the egg and canal cells in the fern Osmunda japonica Thunb. was studied during oogenesis by transmission electron microscopy. The mature egg possesses no fertilization pore and no typical egg envelope. In addition, an extra wall formed around the canal cells during oogenesis and apparently blocked protoplasmic connections between the egg and the canal cells. The periodic acid Schiff (PAS) reaction revealed that the extra wall was most likely composed of polysaccharides. Maturation of the egg was accompanied by the formation of a separation cavity above the egg and by some changes in the morphology of the nucleus and cytoplasmic organelles. The chromatin of the nucleus becomes condensed and the upper surface of the nucleus becomes closely associated with the plasmalemma. Amyloplasts in the egg cytoplasm were numerous and conspicuous, with most in close proximity to the nucleus. Finally, the cytoplasm on one side of the egg became vesiculated and the overlying plasmalemma was easily disrupted. These cytological features of the egg and the canal cells during oogenesis in O. japonica are markedly different from those of the leptosporangiate ferns and suggest a significant evolutionary divergence in reproductive cellular features between Osmundaceae and leptosporangiate ferns.

Deficient Induction Response in a Xenopus Nucleocytoplasmic Hybrid

Author SummaryWhen two species evolve separately for several million years, their respective genomes accumulate many small changes that together are responsible for the differences in their characters. Some of these affect the way eggs are prepared inside the germline, and/or how embryos develop, such that the egg cytoplasm of a given species can only support development promoted by its own genome or nucleus. Thus, developmental incompatibility arises between the cytoplasm and the nucleus of distant species during evolution and we don't know its mechanism. We have studied this phenomenon in an advantageous system using two evolutionarily distant frog species (Xenopus laevis and Xenopus tropicalis). We found that hybrid frog embryos with X. laevis cytoplasm and X. tropicalis nuclei are always defective in an important process that is necessary to generate morphogenetic cell movements during development. Through a series of experiments in which we dissect out and/or recombine parts of such hybrid embryos and observe their behaviour in culture, we show that this phenomenon occurs because of malfunctions in the signalling cascade that is responsible for generating these cell movements. Thus, we postulate that inefficient molecular signalling contributes to the death of such hybrids.

Rat eggs cannot wait: Spontaneous exit from meiotic metaphase‐II arrest

Mammalian eggs await fertilisation while arrested at the second metaphase stage of meiotic division. A network of signalling pathways enables the establishment and maintenance of this metaphase-II arrest. In the absence of fertilisation, mammalian eggs can spontaneously exit metaphase II when parthenogenetically stimulated, or sometimes without any obvious stimulation. Ovulated rat eggs abortively release from metaphase-II arrest once removed from egg donors. Spontaneously activated rat eggs extrude the second polar body and proceed to the so-called metaphase III-'like' stage, with clumps of condensed chromatin scattered in the egg cytoplasm. It is still unclear what makes rat eggs susceptible to spontaneous activation; however, a vague picture of the signalling pathways involved in the process of spontaneous activation is beginning to emerge. Such cell cycle instability is one of the major reasons why it is more difficult to establish nuclear transfer in the rat. This review examines the known predisposing factors and biochemical mechanisms involved in spontaneous activation. The strategies used to prevent spontaneous metaphase-II release in rat eggs will also be discussed.

Divergent effect of mammalian PLCζ in generating Ca2+ oscillations in somatic cells compared with eggs

Sperm PLCζ (phospholipase Cζ) is a distinct phosphoinositide-specific PLC isoform that is proposed to be the physiological trigger of egg activation and embryo development at mammalian fertilization. Recombinant PLCζ has the ability to trigger Ca2+ oscillations when expressed in eggs, but it is not known how PLCζ activity is regulated in sperm or eggs. In the present study, we have transfected CHO (Chinese-hamster ovary) cells with PLCζ fused with either YFP (yellow fluorescent protein) or luciferase and found that PLCζ-transfected cells did not display cytoplasmic Ca2+ oscillations any differently from control cells. PLCζ expression was not associated with changes in CHO cell resting Ca2+ levels, nor with a significantly changed Ca2+ response to extracellular ATP compared with control cells transfected with either YFP alone, a catalytically inactive PLCζ or luciferase alone. Sperm extracts containing PLCζ also failed to cause Ca2+ oscillations in CHO cells. Despite these findings, PLCζ-transfected CHO cell extracts exhibited high recombinant protein expression and PLC activity. Furthermore, either PLCζ-transfected CHO cells or derived cell extracts could specifically cause cytoplasmic Ca2+ oscillations when microinjected into mouse eggs. These data suggest that PLCζ-mediated Ca2+ oscillations may require specific factors that are only present within the egg cytoplasm or be inhibited by factors present only in somatic cell lines.

Vertical and horizontal transmission drive bacterial invasion

A huge variety of Arthropod species is infected with endosymbiotic Wolbachia bacteria that manipulate their host's reproduction to invade populations. In addition to vertical transmission from mother to offspring through the egg cytoplasm, it has been demonstrated through phylogenetic analyses and natural transfer experiments that horizontal transmission of Wolbachia (i.e. contagion) can occur between Arthropod hosts. More recently, factors influencing horizontal transfer have also been explored. While it is clear that horizontal transmission between species plays a major role in the evolutionary history of Wolbachia infections among insects, its role in the spread of a new infection through a host population, notably through within-species transfers, remained unknown. In this issue of Molecular Ecology, Kraaijeveld etal. (2011) present the first evidence that horizontal transmission played a key role in the early spread of parthenogenesis-inducing Wolbachia through the parasitoid wasp Leptopilina clavipes. To support their finding, the authors studied genetic variation in three types of markers, including host nuclear DNA, mitochondrial DNA and Wolbachia DNA. Specifically, they examined potential associations between their diversity patterns. No diversity was detected in Wolbachia genes, indicating that a single Wolbachia strain must have infected and spread through L.clavipes. In addition, a correlation between substantial variation in mitochondrial and nuclear genotypes suggested that horizontal transmission played an important role in the current clonal genetic variation in this wasp. Such horizontal transmission could be facilitated by a specific host ecology (e.g. parasitoid wasps sharing the same host resource) and potentially impact co-evolution between host and symbiont.

Actin, more than just a housekeeping protein at the scene of fertilization

Since the first demonstration of sperm entry into the fertilized eggs of Mediterranean sea urchin Paracentrotus lividus by Hertwig (1876), enormous progress and insights have been made on this topic. However, the precise molecular mechanisms underlying fertilization are largely unknown. The two most dramatic changes taking place in the zygote immediately after fertilization are: (i) a sharp increase of intracellular Ca(2+) that initiates at the sperm interaction site and traverses the egg cytoplasm as a wave, and (ii) the concomitant dynamic rearrangement of the actin cytoskeleton. Traditionally, this has been studied most extensively in the sea urchin eggs, but another echinoderm, starfish, whose eggs are much bigger and transparent, has facilitated experimental approaches using microinjection and fluorescent imaging methodologies. Thus in starfish, it has been shown that the sperm-induced Ca(2+) increase in the fertilized egg can be recapitulated by several Ca(2+)-evoking second messengers, namely inositol 1,4,5-trisphosphate (InsP3), cyclic ADP-ribose (cADPr) and nicotinic acid adenine dinucleotide phosphate (NAADP), which may play distinct roles in the generation and propagation of the Ca(2+) waves. Interestingly, it has also been found that the dynamic rearrangement of the actin cytoskeleton in the fertilized eggs plays pivotal roles in guiding monospermic sperm entry and in the fine modulation of the intracellular Ca(2+) signaling. As it is well known that Ca(2+) regulates the structure of the actin cytoskeleton, our finding that Ca(2+) signaling can be reciprocally affected by the state of the actin cytoskeleton raises an intriguing possibility that actin and Ca(2+) signaling may form a 'positive feedback loop' that accelerates the downstream events of fertilization. Perturbation of the cortical actin networks also inhibits cortical granules exocytosis. Polymerizing actin bundles also compose the 'acrosome process,' a tubular structure protruding from the head of fertilizing sperm. Hence, actin, which is one of the most strictly conserved proteins in eukaryotes, modulates almost all major aspects of fertilization.

Gamete membrane microdomains and their associated molecules in fertilization signaling

Fertilization is the fundamental system of biological reproduction in many organisms, including animals, plants, and algae. A growing body of knowledge has emerged to explain how fertilization and activation of development are accomplished. Studies on the molecular mechanisms of fertilization are in progress for a wide variety of multicellular organisms. In this review, we summarize recent findings and debates about the long-standing questions concerning fertilization: how egg and sperm become competent for their interaction with each other, how the binding and fusion of these gamete cells are made possible, and how the fertilized eggs initiate development to a newborn. We will focus on the structure and function of the membrane microdomains (MDs) of egg and sperm that may serve as a platform or signaling center for the aforementioned cellular functions. In particular, we provide evidence that MDs of eggs from the African clawed frog, Xenopus laevis, play a pivotal role in receiving extracellular signals from fertilizing sperm and then transmitting them to the egg cytoplasm, where the tyrosine kinase Src is present and responsible for the subsequent signaling events collectively called egg activation. The presence of a new signaling axis involving uroplakin III, an MD-associated transmembrane protein, and Src in this system will be highlighted and discussed.

The small GTPase Cdc42 promotes membrane protrusion during polar body emission via ARP2-nucleated actin polymerization

Polar body emission is a specialized cell division throughout the animal kingdom, serving to reduce chromosome ploidy while preserving the egg cytoplasm. Critical to polar body emission are the asymmetric positioning of the meiotic spindle prior to anaphase, with one pole attached to the oocyte cortex, and the simultaneous membrane protrusion during subsequent cytokinesis. We have shown that, during Xenopus oocyte maturation, the small GTPase Cdc42 promotes membrane protrusion while a classical RhoA contractile ring forms and constricts at the base of the protrusion. We report here that treating oocytes with low concentrations of nocodazole diminished the size of metaphase I spindles and prevented polar body emission, and yet an active Cdc42 cap of correspondingly diminished size still developed, on time, atop of the spindle pole. Conversely, treating oocytes with low concentrations of taxol resulted in a spindle with multiple poles attached to the cortex, but still each of these poles were associated with activated cortical Cdc42 at the appropriate time. Therefore, the asymmetric positioning of the meiotic spindle with one pole anchored to the cortex is a prerequisite for Cdc42 activation. Furthermore, we demonstrated that the Cdc42-regulated F-actin nucleator ARP2/3 complex was similarly localized at the cortex of the protruding polar body membrane, suggesting that Cdc42 promotes membrane protrusion through an F-actin meshwork mechanism. Finally, we demonstrated that Cdc42 and RhoA formed similarly complementary activity zones during egg activation and that inhibition of Cdc42 prevented second polar body emission. Therefore, Cdc42 activation likely promotes membrane protrusion during polar body emission in widespread systems.

The Ca2+ increase by the sperm factor in physiologically polyspermic newt fertilization: Its signaling mechanism in egg cytoplasm and the species-specificity

The newt, Cynops pyrrhogaster, exhibits physiological polyspermic fertilization, in which several sperm enter an egg before egg activation. An intracellular Ca2+ increase occurs as a Ca2+ wave at each sperm entry site in the polyspermic egg. Some Ca2+ waves are preceded by a transient spike-like Ca2+ increase, probably caused by a tryptic protease in the sperm acrosome at the contact of sperm on the egg surface. The following Ca2+ wave was induced by a sperm factor derived from sperm cytoplasm after sperm–egg membrane fusion. The Ca2+ increase in the isolated, cell-free cytoplasm indicates that the endoplasmic reticulum is the major Ca2+ store for the Ca2+ wave. We previously demonstrated that citrate synthase in the sperm cytoplasm is a major sperm factor for egg activation in newt fertilization. In the present study, we found that the activation by the sperm factor as well as by fertilizing sperm was prevented by an inhibitor of citrate synthase, palmitoyl CoA, and that an injection of acetyl-CoA or oxaloacetate caused egg activation, indicating that the citrate synthase activity is necessary for egg activation at fertilization. In the frog, Xenopus laevis, which exhibits monospermic fertilization, we were unable to activate the eggs with either the homologous sperm extract or the Cynops sperm extract, indicating that Xenopus sperm lack the sperm factor for egg activation and that their eggs are insensitive to the newt sperm factor. The mechanism of egg activation in the monospermy of frog eggs is quite different from that in the physiological polyspermy of newt eggs.