Extracts from both the vitelline envelope (VE) and fertilisation envelopes (FE) of rainbow trout eggs have the ability to exert a bactericidal effect on Gram-positive and -negative bacteria. The effect may be due to the presence of phospholipase D (PLD), lysozyme, proteinase and DNases, as the extracts contain these enzyme activities. The intensity of chorionic PLD and lysozyme activities in the VE extract was maintained in the FE without any alteration in activity even after transformation in the course of the cortical reaction, as components of a fundamental architecture of the envelope. Both extracts also contain different types of proteinase activities. Treatment with VE or FE extract seriously damaged the outer membrane of Gram-negative bacteria and the plasma membrane of Gram-positive and -negative bacteria at the ultrastructural level. Chorionic DNases probably degrade DNA of bacterial cells killed by virtue of the action of PLD and/or lysozyme and contribute to the transmigration of nucleosides and/or nucleotides produced by degrading bacterial DNA after degradation of bacterial components by the actions of the chorionic PLD, lysozyme and proteinase. These results suggest that the bactericidal process manifested by the VE or FE extract may start with the action of PLD and/or lysozyme against bacteria and be completed by subsequent degradation of constitutive proteins and DNA by the action of proteinases and DNases, respectively. Thus the VE and FE are able to protect the egg itself and the embryo, respectively, from bacterial infection in the internal or external environments.

A transient increase in cytosolic free calcium ion concentration ([Ca2+]i) (Ca2+-transient) takes place in the early stages of fertilisation of sea urchin eggs as well as in other animal eggs (Miyazaki et al., 1993). This transient increase in [Ca2+]i propagates across the egg as a Ca2+ wave, which is thought to be a necessary and sufficient event for egg activation (Whitaker & Swarm, 1993). In sea urchin eggs, the rise in [Ca2+], is caused by release of Ca2+ from the endoplasmic reticulum (ER) via one or both of two pathways: (a) inositol 1,4,5-trisphosphate (IP3) and the inositol 1,4,5-trisphosphate receptor/channel (IP3R) or (b) cADP-ribose (cADPR) and/or cGMP and the ryanodine receptor/channel (RyR) (Berridge, 1993). The signalling pathways from sperm to ER of eggs are not yet fully explained. Recent evidence from two lines of experiments has excited more controversy. First, intracellular injection of SH2 domain of phospholipase Cγ, which produced IP3, completely inhibited the increase in [Ca2+]i (Carroll et al., 1999). Another series of experiments showed that nitric oxide (NO) gas was produced in sperm during their acrosome reaction and in eggs during fertilisation, and that the intracellular injection of NO synthase caused egg activation (Epel, this supplement). NO gas is expected to stimulate the production of cGMP by activating soluble guanylyl cyclase (Garthewaite, 1991). Thus, it seems that direct measurements of the second messenger candidates during sea urchin fertilisation are essential to an understanding of the calcium signalling pathway. We previously measured the IP3, cGMP and cADPR contents of sea urchin eggs, and compared the time courses of their changes with that of the [Ca2+]i change (Kuroda et al., 1997). We now examine further the involvement of guanylyl cyclase in the Ca2+ signalling pathway at fertilisation of sea urchin eggs.

The aim of the present study was to determine the presence of the connexins Cx43, Cx32 and Cx26 in Bufo arenarum ovarian follicles during the breeding season as well as to analyse the possible alterations in the meiotic process when connexins are blocked by specific antibodies. Western blot analysis revealed that the Cx43 and Cx32 proteins were present but not Cx26. We demonstrated that the anti-Cx43 and anti-Cx32 antibodies produced the uncoupling of the gap junctions. When these junctions are blocked the maturation process is triggered in the oocytes. We determined that dbcAMP exerts an inhibitory effect on the maturation induced by the uncoupling of the gap junctions when the oocytes are injected or pretreated with this metabolite. We propose the idea that cAMP is the regulatory molecule in meiotic arrest in this amphibian species.

Experiments were conducted to examine the effects of (a) different activation methods, (b) incubation time in calcium-free medium and (c) bisbenzimide staining on the activation and subsequent development of pig oocytes. Oocytes were matured in vitro and activated by one of the following methods: combined thimerosal/dithiothreitol (DTT) treatment, calcium ionophore A23187 treatment followed by incubation in the presence of 6-dimethylaminopurine (6-DMAP), electroporation, and electroporation followed by incubation with cytochalasin B. There were no significant differences in the activation rate (ranging from 70.0% to 88.3%) and the percentage of cleaved embryos after activation (ranging between 48.8% and 58.8%) among the four treatment groups (p < 0.05). The rate of development to the blastocyst stage in oocytes activated by thimerosal/DTT (10.0%) or electroporation followed by cytochalasin B treatment (12.3%) was significantly higher (p < 0.05) than in the group activated with A23187/6-DMAP (2.5%). Both the activation rate and the rate of blastocyst formation in oocytes that were incubated in Ca2+-free medium for 8 h before thimerosal/DTT activation were significantly lower (p < 0.05) than in those incubated for 0, 1 or 4 h. Intracellular Ca2+ measurements revealed that the Ca2+ homeostasis in these oocytes were severely altered. Staining of oocytes with 5 μg/ml bisbenzimide for 2 h decreased the quality of blastocysts and increased the rate of degenerated embryos at day 6. Two activation protocols (thimerosal/DTT and electroproation) were used for activation after nuclear transfer; the rate of nuclear formation did not differ in the oocytes activated by the two different methods.

We are studying the regulation of ion transport in sperm physiology. Sperm ion permeability is modulated by components from the outer layer of the egg which, depending on the species, regulate sperm motility, Chemotaxis and the acrosome reaction (AR). This reaction is required for sperm to fertilise the egg in many species from sea urchins to man (Darszon et al., 1999).

Speract, a decapeptide from the external layer of Strongylocentrotus purpuratus sea urchin eggs, influences sperm respiration, motility and possibly the AR. Signal transduction starts when speract binds to a protein of 77 kDa closely coupled to sperm guanylyl cyclase (Garbers, 1989). Our recent receptor binding experiments using fluorescent-labelled speract (fluorescein and rhodamine) have allowed estimates of the association (kon 2.4 × 107 M−1s−1) and dissociation rate constants (koff 1.3 × 10−4 s−1). Furthermore, studies with fluorescent speract analogues indicate that the receptor undergoes conformational changes that depend on intracellular pH (pHi). The overall results are consistent with the possibility that speract may induce in sea urchin sperm a hyperactivated-like flagellar movement inside the jelly coat to accelerate sperm penetration through this layer.

Ultrastructural and morphometric analyses of centromeric regions by scanning and transmission electron microscopy have been performed in chromosomes from male pronuclei obtained by heterologous fertilisation of hamster oocytes with human spermatozoa. In 1308 of 1323 chromosomes analysed, the primary constriction showed a defined biconcave constriction of variable length (0.56–1.34 μm) and constant width (0.64–0.7 μm). A positive correlation was observed between centromeric length and chromosome length. In some chromosomes, the primary constriction appears as decondensed regions of variable length (1.6–2.51 μm) composed of chromatin fibres with a minimum diameter of 30 nm.

This study was conducted to investigate the possibility of using bovine oocytes for a heterologous fertility test by intracytoplasmic sperm injection (ICSI) and to compare the pronuclear formation of ram, bull and minke whale spermatozoa after injection into bovine oocytes. Bovine oocytes were cultured in vitro for 24 h and those with a polar body were selected for ICSI. Frozen-thawed semen from the three species were treated with 5 mM dithiothreitol for 1 h and spermatozoa were killed by storing them in a -20 °C refrigerator before use. ICSI was performed using a Piezo system. Three experiments were designed. In experiment 1, a higher (p < 0.05) male pronuclear formation rate was found in the oocytes injected with ram (52.6%) or bull (53.4%) spermatozoa than with minke whale spermatozoa (39.1%). In experiment 2, sperm head decondensation was detected at 2 h after ICSI in the oocytes injected with a spermatozoon of each species. Male pronuclei were first observed at 4 h in the oocytes injected with ram or bull spermatozoa and at 6 h in oocytes injected with minke whale spermatozoa. The mean diameters of male pronuclei derived from both whale and bull spermatozoa were larger than those from ram spermatozoa (30.4 μm and 28.3 μm vs 22.4 μm, p < 0.005). The mean diameter of female pronuclei in the oocytes injected with whale spermatozoa was also larger than with ram spermatozoa (29.3 μm vs 24.7 μm, p < 0.05). The development of male and female pronuclei was synchronous. In experiment 3, ethanol-activated oocytes injected with a spermatozoon from any of the three species achieved significantly higher (p < 0.05-0.001) cleavage rates than control oocytes. Blastocyst formation was only observed when bull spermatozoa were used. The results of this study indicate that dead foreign spermatozoa can participate in fertilisation activities in bovine oocytes after ICSI.

Four porcine sperm plasma membrane proteins were previously identified as putative ligands for the oocyte plasma membrane. The present study examined the binding of these proteins and two additional porcine sperm membrane proteins to oocytes from sheep, mice and hamsters as a first step in assessing potential conservation of these putative sperm ligands across species and across mammalian orders. Plasma membrane vesicles were isolated from porcine sperm, solubilised, and the proteins separated by one-dimensional gel electrophoresis. The 7, 27, 39 and 62 kDa porcine sperm protein bands demonstrating predominant binding of the porcine oocyte plasma membrane on ligand blots, a 90 kDa protein band demonstrating minor binding, and a 97 kDa protein band that did not bind the oocyte plasma membrane probe were electroeluted. Proteins were biotinylated, and incubated with zona-free oocytes. Bound biotinylated protein was labelled with fluorescent avidin and the oocytes examined with a confocal microscope. The 7 kDa, 27 kDa and the 39 kDa proteins bound to the sheep oocytes but not to a majority of the hamster or mouse oocytes. The 62 kDa protein bound to sheep oocytes and mouse oocytes but not to a majority of the hamster oocytes. The 90 kDa protein bound to oocytes from all three species. The 97 kDa protein, which did not recognise the porcine oocyte probe on a Western ligand blot, did not bind to oocytes from any species and served as a negative control. These observations are consistent with significant conservation of molecule and function among species within the same mammalian order. Hence, one species may be a good model for other species from the same order. Only limited conservation of binding activity of porcine sperm plasma membrane proteins to rodent oocytes was observed, suggesting a greater divergence either in molecular structure or in function among species from different orders.

The aim of the present study was to investigate whether growth hormone (GH) has any effect on the development of cultured rat pre-antral follicles. Pre-antral follicles with a diameter between 120 μm and 160 μm were mechanically isolated from 10-day-old rat ovaries and cultured in groups for 6 days in serum-free medium without GH or with GH supplemented at concentrations of 1, 10 and 100 ng/ml, respectively. DNA content of the follicles before and after culture was measured to determine whether possible growth is due to proliferation of follicular cells. To investigate the quality of follicles cultured under different conditions, the ultrastructure of the cultured follicles was studied with transmission electron microscopy. Furthermore, reverse transcriptase polymerase chain reaction (RT-PCR) was used to assess the expression of growth hormone receptor (GHR) in pre-antral follicles. GH, regardless of the concentration, stimulated the growth of pre-antral follicles. However, follicles cultured in medium supplemented with high-dose GH (100 ng/ml) showed a significantly lower survival rate compared with the other groups. Follicles cultured in GH-containing medium showed a better ultrastructure in comparison with those cultured in medium without GH. Remarkably, scattered cortical granules were observed in oocytes of follicles cultured in the presence of GH. With RT-PCR, the presence of the mRNA of GHR was demonstrated in pre-antral follicles. It can be concluded that GH promotes rat pre-antral follicle development in vitro and better supports the morphology of cultured pre-antral follicles. The gene expression of GHR suggests that the action of GH could be mediated by its receptors present in pre-antral follicles.

Early embryonic development is largely dependent on maternal RNAs and proteins synthesised during oogenesis. Zygotic transcription is an essential event that occurs at a species-specific time after fertilisation. In the absence of zygotic transcription the embryo dies since it can no longer support requirements for successful embryo development. Molecular genetics of gene expression during early embryogenesis, especially in the bovine species, remain one of the unsolved questions in modern biology. Earlier studies suggested that embryonic transcription in cattle begins at the late 4-cell or 8-cell stage. However, more recent studies suggest that bovine zygotes and 2-cell embryos are both transcriptionally and translationally active. Moreover, changes in chromatin structure due to acetylation of core histones and DNA replication play important roles in the regulation of zygotic/embryonic gene expression. This review will summarise results of recent studies about the timing and mechanisms of zygotic/embryonic gene expression in cattle. In addition, terminology in the literature regarding gene expression during early embryogenesis will be clarified. These terminologies include: ‘zygotic/embryonic gene expression’, ‘maternal to embryonic transition in control of development (MET)’ and ‘zygotic/embryonic genome activation (ZEGA)’.

For over 20 years it has been known that cGMP concentrations are increased by a wide variety of agents. The formation of cGMP from GTP is catalysed by guanylyl cyclase. Guanylyl cyclase is found in various cellular compartments of most organisms including animals, plants and bacteria, in soluble and/or membrane-bound forms (Drewett & Garbers, 1994). Membrane-bound guanylyl cyclase (mGC) is a single polypeptide which was first established by cloning and sequencing of the cDNA encoding a sea urchin sperm protein crosslinked to a sperm-activating peptide (SAP) IIA (Chinkers & Garbers, 1991). Soluble guanylyl cyclase (sGC) consists of two different subunits (alpha and beta). mGC is composed of an extracellular, a single transmembrane and an intracellular domain that is further divided into a protein-kinase-like domain and a cyclase catalytic domain. The primary structure of the catalytic domain of both mGC and sGC is highly conserved among vertebrates and invertebrates (Suzuki et al., 1999).

In the early 1980s both our group (Hansbrough & Garbers, 1981; Garbers et al., 1982) and that of Norio Suzuki (Suzuki et al., 1981) identified the active material in sea urchin egg conditioned media that could stimulate sperm motility and metabolism. In the sea urchins Hemicentrotus pulcherrimus or Strongylocentrotus purpuratus, the active material was a small peptide that we named speract, and the Suzuki group named this and subsequent peptides SAPs, for sperm activating peptides. Subsequently, both groups identified other peptides (see Suzuki & Yoshino, 1992 for review), one of the most interesting being one named resact, the active material in Arbacia punctulata egg conditioned media. This peptide turned out to be the first animal sperm chemoattractant identified (Ward et al., 1985a). A peptide also turned out to be the active principle that explained previous observations of Ward and Vacquier (Ward et al., 1985b; Suzuki et al., 1984) that egg conditioned media could cause the rapid dephosphorylation of a major membrane protein of spermatozoa. The apparent receptor for resact was later identified as a guanylyl cyclase, establishing a new paradigm for low-molecular-weight second messenger signalling, and the major phosphoprotein regulated by resact was also the receptor itself.

Animal eggs are generally encased in one or more extra-cellular coats that protect the egg from biological, chemical and mechanical hazards. These coats contain some essential molecules for sperm to fertilise an appropriate egg, such as the specific ligand for sperm binding and the specific signal for induction of the acrosome reaction. In starfish, the outermost egg coat is a relatively thick gelatinous layer called the jelly coat. When starfish sperm encounter the jelly coat of homologous eggs, they undergo the acrosome reaction within a second or less (Dale et al., 1981; Ikadai & Hoshi, 1981; Sase et al., 1995). We have thus searched the jelly coat for the signal molecule(s) that triggers the acrosome reaction in the starfish, Asterias amurensis. It is known that three components in the jelly coat, namely acrosome reaction-inducing substance (ARIS), Co-ARIS and asterosap, act in concert on homologous spermatozoa to elicit the acrosome reaction immediately and efficiently (Hoshi et al., 1994,1999).

ARIS alone induces the acrosome reaction only in high calcium or high pH seawater. In normal seawater, besides ARIS, either Co-ARIS or asterosap is required for the induction. Without ARIS, no combination of Co-ARIS and asterosap can induce the acrosome reaction in normal, high calcium or high pH seawater. A mixture of ARIS and Co-ARIS increases the intracellular Ca2+ level, whereas asterosap increases the intra-cellular pH (Matsui et al., 1986a, b; Nishigaki et al., 1996). These events are prerequisites for the induction of the acrosome reaction. Indeed, the triad of ARIS, CoARIS and asterosap provides the best conditions for the induction of the acrosome reaction in normal sea-water (Hoshi et al., 1994, 1999).

In Strongylocentrotus purpuratus, the fucose sulphate polymer (FSP) of egg jelly induces the sperm acrosome reaction (AR; Vacquier & Moy, 1997). Protease treatment of sperm renders the cells insensitive to FSP, indicating that sperm membrane receptors mediate the signal transduction events underlying the AR. Monoclonal antibodies to a 210 kDa membrane glycoprotein induce Ca2+ influx into sperm and trigger the AR (Trimmer et al., 1986; Moy et al., 1996). Purified 210 kDa protein binds species-specifically to egg jelly and blocks AR induction by antibody (Podell & Vacquier, 1985; Moy et al., 1996). FSP binds to the 210 kDa protein attached to Sepharose (Vacquier & Moy, 1997). Monoclonal antibodies localise the 210 kDa protein on the plasma membrane over the acrosome and also on the sperm flagellum. The 210 kDa protein has the attributes of a sperm receptor for egg jelly and is henceforth named suREJ1 (Moy et al., 1996). We describe here the three REJ proteins found thus far in S. purpuratus sperm.

Disruption of plasma membranes is a widespread, common and normal event that occurs in many mechanically challenged tissues (McNeil & Steinhardt, 1997). After injury to the plasma membrane, rapid resealing of the membrane occurs with little loss of intracellular contents.

Analysis of plasma membrane repair in the sea urchin egg and early embryos revealed a new model of the mechanism for plasma membrane repair. Resealing of disrupted plasma membranes required external Ca2+ that could be antagonised by Mg2+. Block of Ca2+/calmodulin kinase II, which regulates exocytotic vesicle availability at synapses (Llinás et al., 1991), inhibited membrane resealing. Resealing was also inhibited by botulinum neurotoxins A, B, C1, and tetanus toxin, which disrupt SNARE vesicle docking/fusion proteins. Confocal microscopic observations of exocytotic events in sea urchin eggs and embryos during membrane resealing showed that inhibition of kinesin or myosin motor activity, which are believed to be required for vesicle transport (Goodson et al., 1997), also inhibited membrane resealing and delivery of vesicles to sites of membrane disruption. This pattern of inhibition indicates that membrane repair of micrometre-sized lesions requires vesicle delivery, docking and fusion, similar to the exocytosis of neurotransmitter (Steinhardt et al., 1994; Bi et al., 1995, 1997).

The mechanism of resealing in eggs and embyros was found to be a general property of all cells (Steinhardt et al., 1994; Togo et al., 1999). It is now known that elevated intracellular Ca2+ triggers exocytosis in various types of cells (Dan & Poo, 1992; Coorssen et al., 1996), and that endosomal compartments such as lysosomes can behave as Ca2+-regulated exocytotic vesicles (Rodríguez et al., 1997).

In sea urchin embryogenesis it has been suggested that the initial territories are specified by a combination of the asymmetric distribution of cytoplasmic determinants and cell-cell interactions. At the 60-cell stage blastomeres clonally originated from founder cells divide the embryo into five distinct territories: small micromeres, large micromeres, vegetal plate, oral ectoderm and aboral ectoderm. The territories are identified by the expression of specific marker genes and their cell lineages (Davidson, 1989, 1991). The large micromeres are thought to play a role as an organiser and initiate a cascade of signal transduction toward overlying cells (Davidson, 1989). In this model the large micromeres induce the overlying veg2 tier, specifying the vegetal plate (Ransick & Davidson, 1993, 1995). The veg2 tier then induces the overlying cells, which include gut cells and cells of the prospective ectodermal territories (Wikramanayake et al., 1995; Wikramanayake & Klein, 1997). Thus, the large micromeres, which are the prospective primary mesenchyme cells (PMCs), play a key role in cell fate specification and axis determination during sea urchin embryogenesis. Previous data suggested that the large micromeres are autonomously specified to become PMCs by maternally inherited determinants (Okazaki, 1975; Kitajima & Okazaki, 1980). An important question in sea urchins embryogenesis is the identity and function of the proposed maternal determinants.

Understanding how the embryonic germ layers become competent to form their characteristic tissue types is a problem of fundamental importance to developmental biology. Knowledge of how the endodermal layer is first determined and then differentiates has only recently begun to accumulate. In sea urchins, several different signals have been implicated in endoderm formation, beginning as early as the fourth cleavage division and continuing until just prior to invagination of the endoderm. Recent experiments in sea urchin embryos have shown that the activity of glycogen synthase kinase 3-β and entry of β-catenin into the nucleus during cleavage stages is required for mesoderm and endoderm formation (Emily-Fenouil et al., 1998; Logan et al., 1999), implicating the Wnt signalling pathway in this process. Overexpression of β-catenin leads to an exaggeration of endoderm and mesoderm in the embryo at the expense of ectoderm (Wikramanayake et al., 1998). Since this signal is required for both mesoderm and endoderm, some other signal must be present to differentiate between these two germ layers. Micromeres formed by the fourth cleavage division have the ability to induce endoderm (Ransick & Davidson, 1995). This induction can occur independently of the entry of β-catenin into the nucleus of the cells induced to form endoderm (Logan et al., 1999), indicating micromere induction acts through a different signalling pathway. Final determination of endoderm also requires cell interactions through the late mesenchyme blastula stage, since cells from embryos dissociated prior to that stage fail to develop into endoderm autonomously (Chen & Wessel, 1996). A sea urchin member of the hedgehog family of signalling molecules has been reported to be expressed in the vegetal plate, indicating it also may play a role in endoderm formation.

Protease activities have been recognised as important elements in controlling the composition of the extracellular matrix. Regulated remodelling of the matrix is required for a number of physiological processes including embryonic development. Excessive and unregulated remodelling has been associated with a number of pathological conditions including the metastatic phenotype of malignant cancer (Kim et al., 1998). We have begun a search for protease activities which utilise components of the sea urchin extracellular matrix as substrates. We have identified and purified a 41 kDa protease which is present in the sea urchin egg and embryo. This species possesses a non-specific gelatin-cleavage activity as well as a collagen cleavage activity which appears to be specific for echinoderm collagen (Mayne & Robinson, 1996, 1999).

The 41 kDa collagenase/ gelatinase was inhibited by EGTA and reactivated by calcium. The calcium-concentration dependence for reactivation indicated an apparent kd of 3.7 mM and was coincident with the binding of 80 moles calcium/mole of protein. These results are interpretable in terms of the high concentration of calcium (10 mM) present in seawater. In addition to calcium, seawater also contains 50 mM magnesium. The substantial amounts of calcium bound to the 41 kDa protease suggest the existence of binding sites with both low affinity and specificity for binding metal ions. To determine whether high concentrations of magnesium could influence the interaction of calcium with the 41 kDa species we used both qualitative and quantitative gelatin-cleavage assays to examine protease activity in the presence of both calcium and magnesium.

The ingression of primary mesenchyme cells (PMCs) in the sea urchin embryo is initiated with local degradation of the basal lamina at the vegetal plate epithelium (e.g. Katow & Solursh, 1980). The ingressed PMCs encounter pamlin, a cell adhesion protein in the basal lamina (Katow, 1995), which guides PMC migration to a particular embryonic region to form a ring pattern (Katow & Komazaki, 1996; Katow et al, 2000). Thus extracellular matrix (ECM) provides a necessary guidance cue to the migratory cells, and this implicates the occurrence of intracellular signalling to promote not only cell locomotion but also orientation for the migration. Using embryos of the sea urchin, Hemicentrotus pulcherrimus, I report the temporal expression of P35, a PMC surface protein, during the very early stages of PMC ingression that is downregulated with SUp62 protein in the cytoplasm, and tyrosine phosphorylation of SUp62 as a consequence of PMCs encountering pamlin in light of ECM/cell signal transduction.

It has long been recognized that micromeres have special significance in early specification events in the sea urchin embryo. Micromeres have the ability to induce a secondary axis if transferred to the animal pole at the 16-cell stage of sea urchin embryos (Hörstadius, 1939). Without micromeres an isolated animal hemisphere develops into an ectodermal ball called a dauer blastula. Addition of micromeres to an animal half rescues a normal pluteus larva, including endoderm (Hörstadius, 1939). Despite these well-known experiments, however, neither the molecular basis of that induction nor the endogenous inductive role of micromeres in development was known. In recent experiments we learned that if one eliminates micromeres from the vegetal pole at the 16-cell stage the resulting embryo makes no secondary mesenchyme. Earlier it had been found that β-catenin is crucial for specification events that lead to mesoderm and endoderm (Wikra-manayake et al., 1998; Emily-Fenouil et al., 1998; Logan et al., 1999). We noticed that at the 16-cell stage β-catenin enters the nuclei of micromeres, then enters the nuclei of macromeres at the 32-cell stage (Logan et al., 1999). Since nuclear entry of β-catenin is known to be important for its signalling function in the Wnt pathway, we asked whether β-catenin functions in the micromere induction pathway.