Cleavage patterns, cell-lineages and cell specification are clues to phyletic lineages in Spiralia

Embryos of molluscs, annelids, nemerteans and platyhelminthes show remarkable intra- and interphyletic resemblances and differences in mesentoblast, dorso-ventral axis and trochoblast specification. These variations have been used to investigate their evolutionary relationship. In molluscs and annelids a heterochronic shift parallels evolutionary relations based on adult characters. Nemerteans and platyhelminthes lack trochal cells and differ in the specification of the mesodermal precursor cell. Nemerteans also differ fundamentally with respect to axis specification related to the first cleavage. Therefore, close phylogenetic relations exist between molluscs and annelids, whereas nemerteans and platyhelminthes are only remotely related with each other and with molluscs and annelids.     

Comparative biology of calcium signaling during fertilization and egg activation in animals.

During animal fertilizations, each oocyte or egg must produce a proper intracellular calcium signal for development to proceed normally. As a supplement to recent synopses of fertilization-induced calcium responses in mammals, this paper reviews the spatiotemporal properties of calcium signaling during fertilization and egg activation in marine invertebrates and compares these patterns with what has been reported for other animals. Based on the current database, fertilization causes most oocytes or eggs to generate multiple wavelike calcium oscillations that arise at least in part from the release of internal calcium stores sensitive to inositol 1,4,5-trisphosphate (IP3). Such calcium waves are modulated by upstream pathways involving oolemmal receptors and/or soluble sperm factors and in turn regulate calcium-sensitive targets required for subsequent development. Both "protostome" animals (e.g., mollusks, annelids, and arthropods) and "deuterostomes" (e.g., echinoderms and chordates) display fertilization-induced calcium waves, IP3-mediated calcium signaling, and the ability to use a combination of external calcium influx and internal calcium release. Such findings fail to support the dichotomy in calcium signaling modes that had previously been proposed for protostomes vs deuterostomes and instead suggest that various features of fertilization-induced calcium signals are widely shared throughout the animal kingdom.     

The repetitive calcium waves in the fertilized ascidian egg are initiated near the vegetal pole by a cortical pacemaker.

Ascidian eggs respond to fertilization with a series of repetitive calcium waves that originate mostly from the vegetal/contraction pole region (J. E. Speksnijder, C. Sardet, and L. F. Jaffe, 1990, Dev. Biol. 142, 246-249), where the myoplasm is concentrated during the first phase of ooplasmic segregation. This suggests that the myoplasm may be involved in initiating these calcium waves. To test this possibility, the starting position of the calcium waves was determined in eggs that had the subcortical, mitochondria-rich part of the myoplasm displaced by centrifugation. Such centrifuged eggs display four cytoplasmic layers: a large centrifugal yolk zone, a narrow clear zone, a mitochondria-rich layer, and a small clear zone at the centripetal pole. Imaging of the cytosolic calcium in centrifuged eggs that were injected with the calcium-specific photoprotein aequorin reveals a series of repetitive calcium waves after fertilization. About 70% of these waves start in the vegetal/contraction pole area, which is similar to the number of waves previously found to start in this area in uncentrifuged eggs. In contrast, only about 25% of the waves start close to the displaced mitochondria-rich layer. From this result it is concluded that the main wave initiation site is not displaced by the centrifugal forces that displace the subcortical, mitochondria-rich part of the myoplasm. Moreover, the observation that the animal-vegetal polarity of cortical components such as actin filaments and the endoplasmic reticulum has been retained after centrifugation further suggests that a cortical component located in the vegetal hemisphere--most likely the endoplasmic reticulum network in the cortical region of the myoplasm--is involved in initiating the repetitive calcium waves in the fertilized ascidian egg.     

Reconstitution of Src-dependent phospholipase Cgamma phosphorylation and transient calcium release by using membrane rafts and cell-free extracts from Xenopus eggs

We reported previously that egg membrane rafts serve as a subcellular microdomain for sperm-dependent tyrosine kinase signaling in Xenopus fertilization. Moreover, we demonstrated that raft-associated Src tyrosine kinase was activated by sperm in vitro. Here we show that egg rafts incubated with sperm or hydrogen peroxide (H2O2) can promote Src-dependent phosphorylation of phospholipase Cgamma (PLCgamma) and transient calcium release in the extracts of unfertilized Xenopus eggs. In vivo egg activation by sperm or H2O2 also promotes tyrosine phosphorylation and raft-translocalization of PLCgamma. Immunodepletion of PLCgamma from the egg extracts inhibits the raft-dependent calcium release. Rafts prepared from H2O2-activated eggs also promote Src-dependent dephosphorylation of p42 mitogen-activated protein kinase and cell cycle transition from metaphase II to interphase in egg extracts. PLCgamma phosphorylation and calcium release in egg extracts can be promoted by rafts prepared from COS-7 cells expressing the Xenopus Src gene. These results demonstrate that the signaling events elicited by fertilization in Xenopus eggs can be reconstituted in vitro. The development of such experimental platforms will allow us to dissect the molecular mechanism of sperm-dependent activation of raft-associated Src and subsequent up-regulation of PLCgamma and egg activation machinery in Xenopus eggs.     

Calcium signalling in early embryos

The onset of development in most species studied is triggered by one of the largest and longest calcium transients known to us. It is the most studied and best understood aspect of the calcium signals that accompany and control development. Its properties and mechanisms demonstrate what embryos are capable of and thus how the less-understood calcium signals later in development may be generated. The downstream targets of the fertilization calcium signal have also been identified, providing some pointers to the probable targets of calcium signals further on in the process of development.In one species or another, the fertilization calcium signal involves all the known calcium-releasing second messengers and many of the known calcium-signalling mechanisms. These calcium signals also usually take the form of a propagating calcium wave or waves. Fertilization causes the cell cycle to resume, and therefore fertilization signals are cell-cycle signals. In some early embryonic cell cycles, calcium signals also control the progress through each cell cycle, controlling mitosis.Studies of these early embryonic calcium-signalling mechanisms provide a background to the calcium-signalling events discussed in the articles in this issue.     

Polarity and reorganization of the endoplasmic reticulum during fertilization and ooplasmic segregation in the ascidian egg

During the first cell cycle of the ascidian egg, two phases of ooplasmic segregation create distinct cytoplasmic domains that are crucial for later development. We recently defined a domain enriched in ER in the vegetal region of Phallusia mammillata eggs. To explore the possible physiological and developmental function of this ER domain, we here investigate its organization and fate by labeling the ER network in vivo with DiIC16(3), and observing its distribution before and after fertilization in the living egg. In unfertilized eggs, the ER-rich vegetal cortex is overlaid by the ER-poor but mitochondria-rich subcortical myoplasm. Fertilization results in striking rearrangements of the ER network. First, ER accumulates at the vegetal-contraction pole as a thick layer between the plasma membrane and the myoplasm. This accompanies the relocation of the myoplasm toward that region during the first phase of ooplasmic segregation. In other parts of the cytoplasm, ER becomes progressively redistributed into ER-rich and ER- poor microdomains. As the sperm aster grows, ER accumulates in its centrosomal area and along its astral rays. During the second phase of ooplasmic segregation, which takes place once meiosis is completed, the concentrated ER domain at the vegetal-contraction pole moves with the sperm aster and the bulk of the myoplasm toward the future posterior side of the embryo. These results show that after fertilization, ER first accumulates in the vegetal area from which repetitive calcium waves are known to originate (Speksnijder, J. E. 1992. Dev. Biol. 153:259-271). This ER domain subsequently colocalizes with the myoplasm to the presumptive primary muscle cell region.     

A hypothesis on p34cdc2 sequestration based on the existence of Ca(2+)-coordinated changes in H+ and MPF activities during Xenopus egg activation [corrected]

The entry into, and exit from, mitosis are controlled by a universal M-phase promoting factor (MPF) composed of at least p34cdc2 and a cyclin. Embryonic systems are convenient for studying the association and dissociation of the active MPF complex because oocytes and eggs are naturally arrested at a specific point of the cell cycle until progression to the next point is triggered by a hormonal signal or sperm. In amphibians, eggs prior to fertilization are arrested at metaphase 2 of meiosis due to the presence of a stabilized MPF complex. Fertilization (egg activation) produces a transient increase in intracellular free Ca2+, a propagating Ca2+ wave, that specifically triggers the destruction of cyclin, leading to MPF inactivation and entry into the first embryonic inter-phase. We have recently shown that intracellular pH (pHi) variations in amphibian eggs, a large increase at fertilization and small oscillations during the embryonic cell cycle, were temporally and functionally related to the corresponding changes in MPF activity. In addition, the recent finding that the pHi increase at fertilization in Xenopus eggs is a propagating, Ca(2+)-dependent pH wave which closely follows the Ca2+ wave, together with the absence in the egg plasma membrane of pHi-regulating systems responsible for that pHi increase, suggest the existence of cortical or subcortical vesicles acidifying in the wake of the Ca2+ wave, thus producing the pH wave.(ABSTRACT TRUNCATED AT 250 WORDS)     

Repetitive calcium transients and the role of calcium in exocytosis and cell cycle activation in the mouse egg.

The role of calcium in cortical granule exocytosis and activation of the cell cycle at fertilization was examined in the mouse egg using the calcium chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) and the fluorescent calcium indicator fluo-3. BAPTA and fluo-3 were introduced into zona-free mouse eggs by a 30-min incubation with 0.01-50 microM BAPTA acetoxymethyl ester (AM) and/or 1-20 microM fluo-3 AM prior to in vitro fertilization. Incubation of eggs in greater than or equal to 5.0 microM BAPTA AM inhibited cortical granule exocytosis in all cases. Introduction of the calcium chelator into the egg blocked second polar body formation at greater than or equal to 1.0 microM BAPTA AM. Sperm entry occurred in all eggs regardless of the BAPTA AM concentration. Sperm induce a large transient increase in calcium lasting 2.3 +/- 0.6 min, followed by repetitive transients lasting 0.5 +/- 0.1 min and occurring at 3.4 +/- 1.4-min intervals. Incubation with greater than or equal to 5.0 microM BAPTA AM inhibited all calcium transients. Introduction of BAPTA also inhibited calcium transients, exocytosis, and the resumption of meiosis following application of the calcium ionophore A23187 or SrCl2, which activate eggs. These results demonstrate that the calcium increase at fertilization is required for cortical granule exocytosis and resumption of the cell cycle in a mammalian egg.     

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.     

Introduction. Calcium signals and developmental patterning

Calcium ions generate ubiquitous cellular signals. Calcium signals play an important role in development. The most obvious example is fertilization, where calcium signals and calcium waves are triggered by the sperm and are responsible for activating the egg from dormancy and cell cycle arrest. Calcium signals also appear to contribute to cell cycle progression during the rapid cell cycles of early embryos. There is increasing evidence that calcium signals are an essential component of the signalling systems that specify developmental patterning and cell fate. This issue arises from a Discussion Meeting that brought together developmental biologists studying calcium signals with those looking at other patterning signals and events. This short introduction provides some background to the papers in this issue, setting out the emerging view that calcium signals are central to dorsoventral axis formation, gastrulation movements, neural specification and neuronal cell fate.     

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.     

Ascidian eggs block polyspermy by two independent mechanisms: One at the egg plasma membrane,the other involving the follicle cells

Many ascidians live in clumps and usually release sperm before the eggs. Consequently, eggs are often spawned into dense clouds of sperm. Because fertilization by more than a single sperm is lethal, ascidians have evolved at least two successive blocks to polyspermy: the rapid release of a glycosidase that inhibits sperm binding to the vitelline coat (VC) and a subsequent change in membrane potential that prevents supernumerary sperm–egg fusion. This paper shows that (1) these two blocks can be uncoupled by the use of suramin, and (2) most of the glycosidase appears to be from the follicle cells, which are accessory cells on the outside of the egg VC. Phallusia mammillata eggs initially bind numerous sperm but, after the glycosidase is released, only a few additional sperm bind. Intact eggs in 20 μM suramin release glycosidase, but the electrical response is inhibited; sperm swim actively and bind to the VC but fail to penetrate. Suramin treatment is completely reversible; intact eggs exhibit the electrical response an average of 11 minutes after the drug is washed out. Sperm must contact the follicle cells before passing through the VC; eggs with the VC removed and fertilized in the presence of 20 μM suramin show the electrical response 35% of the time, thus VC removal enhances sperm entry. Like the intact eggs, 100% of the naked eggs respond electrically to fertilization after the drug is washed out. Follicle cells that are isolated by calcium magnesium free seawater and then returned to complete seawater release N-acetylglucosaminidase activity in response to sperm. Thus, these eggs have two blocks to polyspermy that operate in sequence: an early first block resulting from enzymatic modification of the VC by N-acetylglucosaminidase released primarily from follicle cells and a second electrical block operating at the egg plasma membrane level and requiring sperm–egg fusion. Mol. Reprod. Dev. 48:137-143, 1997. © 1997 Wiley-Liss, Inc.     

Reorganization of the Endoplasmic Reticulum during Meiotic Maturation of the Mouse Oocyte

The endoplasmic reticulum (ER) of live metaphase II mouse eggs and prophase I-arrested oocytes was compared using the fluorescent, lipophilic dicarbocyanine dye, DiI. DiI, dissolved in soybean oil, was microinjected into oocytes and eggs; the dye diffused throughout the cytoplasm to label the ER, which was imaged by confocal microscopy. The mature egg had a fine reticular network of ER throughout the cell and numerous dense accumulations of membrane in the cortex. These ER accumulations, 1-2 μm in diameter, were generally absent deeper in the cytoplasm. A similar staining pattern was observed when the eggs were fixed within 1 min of injection, providing evidence that the cortical accumulations of membrane are part of a continuous ER membrane system, since membrane trafficking could not occur in a fixed egg. Cortical ER accumulations were localized to the same region of the egg as the cortical granules and were not observed in the cortical granule-free region adjacent to the meiotic spindle. In contrast, ER accumulations were rarely found in the cortex of the immature, prophase I-arrested oocyte, but larger and less well-defined membrane clusters were found throughout the deeper cytoplasm of the oocyte. The appearance of ER clusters in the egg cortex following oocyte maturation correlates with an increased ability of the mature egg to release calcium at fertilization. Since the ER is a calcium store, structural reorganization of the ER may be necessary to permit the large release of calcium and resulting cortical granule exocytosis at fertilization.     

Nicotinamide alters the calcium release pattern and the degradation of MPF activity after fertilisation in ascidian oocytes.

Fertilisation in ascidian oocytes triggers a plasma membrane current, the release of intracellular calcium and the degradation of Maturation Promoting Factor (MPF) activity leading to the completion of meiosis and the initiation of embryo development. We have previously shown that the fertilisation current in ascidians is produced through the metabolism of nicotinamide nucleotide (NN) metabolites to ADP ribose. In this study we have used nicotinamide to test whether NN metabolism plays additional roles in fertilisation in ascidians. Nicotinamide treatment blocked calcium-induced calcium release (CICR) and arrested the cell cycle prior to the completion of meiosis I. Nicotinamide further prevented the abolition of MPF activity after fertilisation. Interestingly, nicotinamide treatment caused ascidian oocytes to form interphase-like pronuclei after fertilisation, despite the high MPF activity. The data demonstrate that NN metabolism is involved in calcium signalling through CICR and further suggest that a NN metabolite acts as a messenger connecting MPF activity to the formation of the meiotic apparatus.     

Mos limits the number of meiotic divisions in urochordate eggs

Mos kinase is a universal mediator of oocyte meiotic maturation and is produced during oogenesis and destroyed after fertilization. The hallmark of maternal meiosis is that two successive M phases (meiosis I and II) drive two rounds of asymmetric cell division (ACD). However, how the egg limits the number of meioses to just two, thereby preventing gross aneuploidy, is poorly characterized. Here, in urochordate eggs, we show that loss of Mos/MAPK activity is necessary to prevent entry into meiosis III. Remarkably, maintaining the Mos/MAPK pathway active after fertilization at near physiological levels induces additional rounds of meiotic M phase (meiosis III, IV and V). During these additional rounds of meiosis, the spindle is positioned asymmetrically resulting in further rounds of ACD. In addition, inhibiting meiotic exit with Mos prevents pronuclear formation, cyclin A accumulation and maintains sperm-triggered Ca(2+) oscillations, all of which are hallmarks of the meiotic cell cycle in ascidians. It will be interesting to determine whether Mos availability in mammals can also control the number of meioses as it does in the urochordates. Our results demonstrate the power of urochordate eggs as a model to dissect the egg-to-embryo transition.     

Studies on fertilization of the teleost. II. Nuclear behavior and changes in histone H1 kinase

In order to understand the dynamic responses of gamete nuclei upon fertilization in the fish, Oryzias latipes, the relationship between changes in the activity of histone H1 kinase and nuclear behavior was examined during fertilization. Kinase activity rapidly decreased concomitant with the initiation of the propagative exocytosis of cortical alveoli following sperm attachment to the egg plasma membrane post-insemination (PI). Activity again increased 30 min PI. Similar changes in kinase activity, migration and syngamy of pronuclei, and subsequent cleavage were observed with aphidicolin or actinomycin D treatment, except that formation of abnormal metaphase chromosomes was retarded in aphidicolin-treated zygotes. Pretreatment of unfertilized eggs with cycloheximide or 6-dimethylaminopurine (6-DMAP) caused no nuclear changes. The activity of histone H1 kinase in these eggs rapidly declined following sperm penetration and exocytosis, but did not undergo subsequent increase in the presence of these inhibitors. In these eggs with low histone H1 kinase activity, the fertilization process from sperm penetration to syngamy occurred normally, but the pronuclear membrane did not break down and the chromosomes did not condense. The present data suggest that in fish eggs, DNA replication as well as the synthesis and phosphorylation of proteins, especially cyclin B, are required for normal formation of metaphase chromosomes at the first cleavage, but not for fertilization events from sperm penetration through to nuclear migration resulting in syngamy.     

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.     

Bi-polarized translation of ascidian maternal mRNA determinant pem-1 associated with regulators of the translation machinery on cortical Endoplasmic Reticulum (cER)

Polarized cortical mRNA determinants such as maternal macho-1 and pem-1 in ascidians, like budding yeast mating factor ASH1 reside on the cER-mRNA domain a subdomain of cortical Endoplasmic Reticulum(ER) and are translated in its vicinity. Using high resolution imaging and isolated cortical fragments prepared from eggs and embryos we now find that macho-1 and pem-1 RNAs co-localize with phospho-protein regulators of translation initiation (MnK/4EBP/S6K).Translation of cortical pem-1 RNA follows its bi-polarized relocalization. About 10 min after fertilization or artificial activation with a calcium ionophore, PEM1 protein is detected in the vegetal cortex in the vicinity of pem-1 RNA. About 40 min after fertilization—when pem-1 RNA and P-MnK move to the posterior pole—PEM1 protein remains in place forming a network of cortical patches anchored at the level of the zygote plasma membrane before disappearing. Cortical PEM1 protein is detected again at the 4 cell stage in the posterior centrosome attracting body (CAB) region where the cER-mRNA domain harboring pem-1/P-MnK/P-4EBP/P-S6K is concentrated. Bi-polarized PEM1 protein signals are not detected when pem-1 morpholinos are injected into eggs or zygotes or when MnK is inhibited.We propose that localized translation of the pem-1 RNA determinant is triggered by the fertilization/calcium wave and that the process is controlled by phospho-protein regulators of translation initiation co-localized with the RNA determinant on a sub-domain of the cortical Endoplasmic Reticulum.     

The endoplasmic reticulum network in the ascidian egg: localization and calcium content

In contrast with most other eggs, where the endoplasmic reticulum is mixed with many other organelles, in ascidians, continuous sheets and tubes of endoplasmic reticulum constitute the only prominent organelle in the immediate layer (0.5-1μm) beneath the plasma membrane, and occupies 16–20% of the cortical volume. We took advantage of this unusual stratification of the organelles in the ascidian egg, to carry X-ray microanalysis. Our measurements provide the first estimate of the calcium content of the endoplasmic reticulum network in an egg, and show it is the main calcium store.     

Modulation of cell cycle control during oocyte‐to‐embryo transitions

Ex ovo omnia—all animals come from eggs—this statement made in 1651 by the English physician William Harvey marks a seminal break with the doctrine that all essential characteristics of offspring are contributed by their fathers, while mothers contribute only a material substrate. More than 360 years later, we now have a comprehensive understanding of how haploid gametes are generated during meiosis to allow the formation of diploid offspring when sperm and egg cells fuse. In most species, immature oocytes are arrested in prophase I and this arrest is maintained for few days (fruit flies) or for decades (humans). After completion of the first meiotic division, most vertebrate eggs arrest again at metaphase of meiosis II. Upon fertilization, this second meiotic arrest point is released and embryos enter highly specialized early embryonic divisions. In this review, we discuss how the standard somatic cell cycle is modulated to meet the specific requirements of different developmental stages. Specifically, we focus on cell cycle regulation in mature vertebrate eggs arrested at metaphase II (MII‐arrest), the first mitotic cell cycle, and early embryonic divisions.