Proceedings of the International Symposium on Fertilization and Development of Sea Urchin and Marine Invertebrates
Obituary
Professor Ikuo Yasumasu
- Hiraku Shimada
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- 16 July 2018, pp. S1-S2
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Special Lecture
Regulation of mitochondrial respiration in eggs and embryos of sea urchin
- Ikuo Yasumasu
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- 16 July 2018, pp. S3-S4
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It is well known that sea urchin eggs, which exhibit quite a low rate of respiration before fertilisation, undergo a sudden increase in the rate of respiration followed by its gradual decrease in about a 15 min period after fertilisation (Ohnishi & Sugiyama, 1963; Epel, 1969), in which the respiration is mediated mainly by Ca2+-activated non-mitochondrial respiratory systems (Foerder et al., 1978; Perry & Epel, 1985a,b). During this short period the rate of mitochondrial respiration gradually increases (Yasumasu et al., 1988) and stabilises at a higher rate than before fertilisation (Warburg, 1908, 1910; Whitaker, 1933; Yasumasu & Nakano, 1963), when the respiration due to non-mitochondrial respiratory systems is turned off. The rate of mitochondrial respiration, once enhanced upon fertilisation, increases further in the period between hatching and the gastrula stage, without any changes in the number of mitochondria or the capacity of electron transport in the mitochondrial respiratory chain (Fujiwara & Yasumasu, 1997; Fujiwara et al., 2000). It is likely that the respiratory rate is reduced by regulation of electron transport in the mitochondrial respiratory chain and increases due to the release of electron transport from the regulation upon fertilisation and after hatching.
A marked increase in the respiratory rate after hatching is accompanied by an evident decrease in the ATP level without any change in the levels of ADP and AMP (Mita & Yasumasu, 1984). In isolated mitochondria, the rate of respiration, estimated in the presence of ADP at the same concentration as in embryos, is reduced by a high concentration of ATP as found in embryos before hatching but is not affected at a concentration as low as in gastrulae (Fujiwara & Yasumasu, 1997; Fujiwara et al., 2000) ATP at a high concentration probably blocks ATP release from mitochondria and consequently inhibits ADP uptake coupled to ATP release in the ATP/ADP translocation reaction in the mitochondrial membrane, causing a shortage of intra-mitochondrial ADP.
A Comment on the Special Lecture
‘A century homework,’: how fertilisation causes elevation of respiration in the sea urchin egg
- Kouichi Asami
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- 16 July 2018, pp. S5-S6
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The lecture given by Dr Yasumasu should be considered from two points of view, namely its significance in the field of developmental biology and his own personal history as a developmental biologist.
In 1908 Otto Warburg published a paper entitled ‘Beobachtungen üeber die Oxydationsprozesse im Seeigelei’ (Warburg, 1908). This is one of his earliest works, where he measured respiration of eggs with Winkler's method not with his manometer. This was the first paper describing the fact that the respiration in unfertilised eggs was considerably lower than that in fertilised ones. Many researchers confirmed his experiments and extended them. Borei (1948) measured oxygen consumption of oocytes and unfertilised and fertilised eggs and compared his results with those of other researchers. He observed that the respiration of eggs declined after they were removed from the ovary and placed into seawater and that it increased at fertilisation. He observed an exponential increase in respiration of fertilised eggs or embryos from the cleavage stage to the hatching blastula. He also observed an initial burst of respiration but failed to record it exactly. Ohnishi & Sugiyama (1963) measured the initial burst of respiration quantitatively with the oxygen electrode method. Thus, respiration of sea urchin eggs and early embryos was divided into three phases: respiration of unfertilised eggs, the initial burst of respiration at fertilisation and the respiration of fertilised eggs. The respiration of fertilised eggs increased exponentially with progression of development until hatching.
Special Lecture for Citizens
How the sperm triggers development of the egg: what have we learned and what can we expect in the next millennium?
- David Epel
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- 16 July 2018, pp. S7-S8
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The problem of how the sperm activates the egg has captivated the attention of cell and developmental biologists since the turn of the century. An early focus concerned species-specific fertilisation and the pioneering work of Lilly and Tyler (Tyler & Tyler, 1966) used immunological analogies to provide explanations of species-specific fertilisation. Contemporary work has focused on the identity of unique receptors on the sperm and the egg as exemplified in the work of Lennarz (Ohlendieck & Lennarz, 1996), Vacquier (Vacquier, et al., 1995) and Wasserman (1999). Lately, this approach has provided unexpected insights on how speciation might occur. Speciation requires reproductive isolation and creative research from the Vacquier laboratory has demonstrated how reproductive barriers might occur through rapid evolution of sperm/egg recognition systems (Lee et al., 1995).
Studies on the cell biology of activation received a major impetus in the 1930s with Mazia's observation of a calcium increase in eggs of the sea urchin following fertilisation (Mazia, 1937). His discovery, however, was a premature one in that there was no satisfactory model at that time for explaining how a calcium increase could affect cell activity. It took almost 40 years to develop a paradigm, and this came from studies on muscle and nerve which revealed how calcium increases could somehow control cell activity. Work in the 1970s rapidly established a similar role for calcium in activation of the egg at fertilisation. The first break-through was the direct demonstration by Steinhardt & Epel (1974) that calcium was involved in egg activation, through manipulation of calcium levels in sea urchin oocytes by use of calcium ionophores.
1-Methyladenine: a starfish oocyte maturation-inducing substance
- Masatoshi Mita
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- 16 July 2018, pp. S9-S11
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1-Methyladenine (1-MeAde) in starfish was the first compound to be identified as an oocyte maturation-inducing substance (MIS) among invertebrates in 1969 by Kanatani and co-workers. In starfish, the ripe ovary contains a huge number of fully grown oocytes of almost equal size. Each oocyte possesses a large nucleus (germinal vesicle, GV), which is arrested in late prophase of the first maturation division. The oocyte is surrounded by a single follicle layer. Such immature oocytes are not fertilisable. Resumption of meiosis in immature oocytes can be induced by 1-MeAde, and the mature oocytes thus become fertilisable (Kanatani et al., 1969; Kanatani, 1985). 1-MeAde is produced by ovarian follicle cells upon stimulation with a gonad-stimulating substance (GSS) released from the radial nerves (Fig. 1).
It has been demonstrated that GSS is a peptide hormone (Kanatani et al., 1971). The action of GSS on 1-MeAde production in follicle cells appears to be mediated by its receptor, G-proteins and adenylyl cyclase (Mita & Nagahama, 1991). These findings suggest that a G-protein coupled (seven transmembrane type) receptor is involved in GSS signal transduction, similarly to the pituitary-gonadal axis in vertebrates.
Thus, using degenerate probes derived from consensus sequences of the mammalian glycoprotein hormone (GTH and TSH) receptors, cDNA was cloned from mRNA of ovaries of Asterina pectinifera. The cDNA showed striking structural homology with members of the glycoprotein hormone receptor family in the transmembrane region, and contained a very large extracellular region. Expression was observed in isolated ovarian follicle cells. Thus, it seems likely that the glycoprotein hormone receptor (GTHR) family gene is related to GSS receptor in ovarian follicle cells. The phylogenic relatedness of the starfish GTHR was also assessed in relation to other vertebrate GTHRs. The analysis showed that the starfish gene diverged before differentiation of the gonadotropin (LH and FSH) and TSH receptors in vertebrates.
Centrosome inheritance in starfish zygotes: behaviour and duplicating capacity of the meiotic centrosomes in maturation division
- Miwa Tamura, Shin-ichi Nemoto
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- 16 July 2018, pp. S12-S13
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In zygotes of almost all animals, it has been believed that only the sperm centrosome acts as the mitotic spindle poles. As first proposed a century ago by Boveri (1887), this uniparental (paternal) inheritance of the centrosome must depend on the selective loss of the maternal centrosomes. To trace the fate and duplicating capacity of all the maternal centrosomes/centrioles, including those cast off into polar bodies, we used two kinds of procedures: (1) suppression of polar body (PB) extrusion and (2) transplantation of PB centro-somes into artificially activated eggs.
Gametes used in this study were from the starfish, Asterina pectinifera. Oocyte maturation was induced with 1-methyladenine (Kanatani, 1969). Suppression of PB extrusion and artificial activation were done according to Washitani-Nemoto et al. (1994). Micromanipulation was performed by the method of Saiki & Hamaguchi (1993). Behaviour of the centrosomes was examined by staining with an antibody against α-tubulin, polarisation and differential interference-contrast microscopy and transmission electron microscopy.
In starfish oocytes, no centriole duplication occurs in meiosis II, hence each pole of a meiosis II spindle is formed by the splitting of paired centrioles in the inner centrosome of a meiosis I spindle into singles. Eventually, each of a second PB (PB2) and a mature egg inherits only one centriole from a meiosis II spindle (a PB1 inherits a pair of centrioles). So, either PB2 and the mature egg inherit a single centriole (Fig. 1; cf. Sluder et al., 1989; Kato et al., 1990). When mature eggs were artificially activated with the Ca2+-ionophore A23187, a single monaster was formed.
Ascidian sperm receptor attached to the vitelline coat during oocyte maturation
- Hitoshi Sawada, Etsuko Tanaka, Yukichi Abe, Satoshi Takizawa, Youko Takahashi, Junko Fujino, Hideyoshi Yokosawa
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- 16 July 2018, pp. S14-S15
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While all ascidians (Urochordata) are hermaphroditic, some, including Halocynthia roretzi (Fuke, 1983) and Ciona intestinalis (Rosati & De Santis, 1978) are strictly self-sterile because of a self–nonself recognition system in the interaction between the sperm and the vitelline coat of the eggs. However, immature oocytes (Fuke & Numakunai, 1996) and acidic-seawater-treated mature oocytes (Morgan, 1939; Kawamura et al., 1991) are self-fertile. It is thought that a putative self–nonself recognition molecule, which is detached or modified by treatment with acidic seawater, may be attached to the vitelline coat during oocyte maturation. Although the existence of a self–nonself recognition system in the fertilisation process is well known, the molecular entity has yet to be conclusively identified. However, there have been several attempts to identify such a molecule in Ciona (Marino et al., 1999). In the present study, we have isolated and analysed a molecule which appears to be responsible for allorecognition in the interaction between sperm and eggs of the ascidian Halocynthia roretzi.
Biologicals. A solitary ascidian Halocynthia roretzi Type C was used in this study. The fertilisation experiment was carried out as described previously (Sawada et al., 1982).
Isolation and N-terminal Sequencing of Hr VC70. Vitelline coats were isolated from immature and mature oocytes of the ascidian by homogenisation and repeated washing with 5× diluted artificial seawater. The isolated vitelline coats were subjected to SDS-PAGE, followed by blotting to a PVDF membrane. The N-terminal amino acid sequence of the 70 kDa main component (HrVC70) was determined by a protein sequencer.
The role of Src family kinases in starfish egg fertilisation
- Andrew F. Giusti, Kathy R. Foltz, Laurinda. A. Jaffe
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- 16 July 2018, pp. S16-S17
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A common early feature in the activation of all eggs during fertilisation is an increase in the level of intra-cellular free calcium (Ca2+) that, in most species, propagates as a wave across the egg (reviewed in Strieker, 1999). In echinoderms, this Ca2+ release is the result of a signal transduction cascade that requires phospholipase Cγ (PLCγ)-mediated production of inositol trisphosphate (IP3) (Carroll et al., 1997, 1999). PLCγ is most commonly regulated by tyrosine phosphorylation (Rhee & Bae, 1997), indicating that a tyrosine kinase is a likely upstream regulator of PLCγ enzymatic activity at fertilisation. In support of this hypothesis, an increase in tyrosine kinase activity and an increase in tyrosine-phosphorylated proteins at fertilisation has been observed in echinoderm eggs (Satoh & Garbers, 1985; Ciapa & Epel, 1991; Kinsey, 1997). Moreover, the tyrosine kinase inhibitors genistein (Shen et al., 1999) and PP1 (Abassi et al., 2000) have been used to show that in sea urchin eggs a tyrosine kinase activity is required for normal Ca2+ release in response to fertilisation.
In eggs of the starfish Asterina miniata, a Src-type tyrosine kinase has been identified as a potential regulator of PLCγ activity at fertilisation (Giusti et al., 1999a). This kinase exhibits a rapid fertilisation-dependent association specifically with the Src Homology 2 (SH2) domains of PLCγ. Moreover, the timing of this association correlates with an increase in the tyrosine kinase activity bound to the PLCγ SH2 domains, and neither the Src kinase nor the associated kinase activity was observed to associate with the PLCγ SH2 domains after treating eggs with the calcium ionophore A23187 (Giusti et al., 1999a). These data identify an egg Src family kinase as a potential upstream regulator of PLCγ during starfish egg fertilisation.
Role of guanylyl cyclase in fertilisation of sea urchin eggs
- Ritsu Kuroda, Kenji Kontani, Yasunari Kanda, Toshiaki Katada, Yu-ichi Satoh, Norio Suzuki, Hideyo Kuroda
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- 16 July 2018, pp. S18-S19
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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.
Speract-receptor interaction and the modulation of ion transport in Strongylocentrotus purpuratus sea urchin sperm
- Blanca-Estela Galindo, Takuya Nishigaki, Esmeralda Rodríguez, Daniel Sánchez, Camen Beltrán, Alberto Darszon
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- 16 July 2018, pp. S20-S21
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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.
Diverse isoforms of guanylyl cyclases in the gonads of echinoderms and medaka fish
- Norio Suzuki
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- 16 July 2018, pp. S22-S23
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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).
The guanylyl cyclase receptors
- David L. Garbers
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- 16 July 2018, pp. S24-S25
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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.
Acrosome reaction in starfish: signal molecules in the jelly coat and their receptors
- Motonori Hoshi, Takuya Nishigaki, Mayu Kawamura, Masako Ikeda, Jayantha Gunaratne, Shoichi Ueno, Manabu Ogiso, Hideaki Moriyama, Midori Matsumoto
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- 16 July 2018, pp. S26-S27
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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).
suREJ proteins: new signalling molecules in sea urchin spermatozoa
- Kathryn J. Mengerink, Gary W. Moy, Victor D. Vacquier
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- 16 July 2018, pp. S28-S30
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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.
The mechanism of cell membrane repair
- Tatsuru Togo, Janet M. Alderton, Richard A. Steinhardt
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- 16 July 2018, pp. S31-S32
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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).
HpEts implicated in primary mesenchyme cell differentiation of the sea urchin (Hemicentrotus pulcherrimus) embryo
- Daisuke Kurokawa, Takashi Kitajima, Keiko Mitsunaga-Nakatsubo, Shonan Amemiya, Hiraku Shimada, Koji Akasaka
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- 16 July 2018, pp. S33-S34
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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.
Gene expression in the endoderm during sea urchin development
- Brian Livingston, Elizabeth-Sharon David, Cary Thurm
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- 16 July 2018, pp. S35-S36
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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.
Characterisation of a 41 kDa collagenase/gelatinase activity expressed in the sea urchin embryo
- John J. Robinson, Janice Mayne
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- 16 July 2018, pp. S37-S38
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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.
Delamination and tyrosine phosphorylation of SUp62 during early embryogenesis of sea urchin
- Hideki Katow
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- 16 July 2018, pp. S39-S40
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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.
Specification of endoderm and mesoderm in the sea urchin
- David R. McClay
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- 16 July 2018, p. S41
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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.