Masaaki Yamaguchi

The Participation of Speract in the Acrosome Reaction of Hemicentrotus pulcherrimus

A speract‐free macromolecular fraction was prepared from the egg jelly of the sea urchin Hemicentrotus pulcherrimus by gel filtration and tested for ability to induce the acrosome reaction in H. pulcherrimus spermatozoa with or without exogenously added synthetic speract. The macromolecular fraction without speract showed only about half the activity of the original unfractionated jelly for induction of the acrosome reaction. The rates of the acrosome reaction induced by the fraction with speract were comparable with those induced by the unfractionated jelly at all pHs tested. Speract itself, however, did not induce the acrosome reaction in the absence of the macromolecular fraction of jelly. The acrosome reaction was associated with incerase of the cyclic AMP concentration in sperm cells, the extent of incerase depending on the concentration of the macromolecular fraction. Addition of speract to the fraction enhanced both induction of the acrosome reaction and increase in the cyclic AMP concentration induced by the fraction. These results suggest that a major factor(s) responsible for the acrosome reaction is a macromolecular component(s) of the jelly and that speract promotes the reaction as a co‐factor.

Expression patterns of Hox genes in larvae of the sea lily Metacrinus rotundus

We cloned eight Hox genes (MrHox1, MrHox2, MrHox4, MrHox5, MrHox7, MrHox8, MrHox9/10, and MrHox11/13c) from the sea lily Metacrinus rotundus, a member of the most basal group of the extant echinoderms. At the auricularia stage, before the formation of the pentaradial rudiment, four MrHox genes were expressed sequentially along the anteroposterior (AP) axis in the straightened mesodermal somatocoels in the order MrHox5, MrHox7, MrHox8, and MrHox9/10. The expression of MrHox7 and MrHox8 was detected as early as the hatching stage in the presumptive somatocoel region of the archenteral sac. MrHox5 was expressed in the anteriormost region of the somatocoels, where a stalk-related structure (the chambered organ) forms later. In addition to the mesodermal somatocoels, MrHox7 was expressed in the oral hood ectoderm, which gives rise to the adhesive pit. The expression of four other MrHox genes (MrHox1, MrHox2, MrHox4, and MrHox11/13c) was not detected in any of the larval stages we examined. In comparison with the mesodermal sea urchin Hox genes, the MrHox genes are expressed more posteriorly along the AP (oral–anal) axis than the sea urchin orthologs, implying that the evolution of the eleutherozoans was accompanied by a posteriorization of the larval body. Our study illuminates the possible body plan and Hox expression patterns of the ancestral echinoderm and sheds light on the larval body plan of the last common ancestor of the echinoderms and chordates.

Induction of the Acrosome Reaction of Hemicentrotus pulcherrimus Spermatozoa by the Egg Jelly Molecules, Fucose‐Rich Glycoconjugate and Spem‐Activating Peptide I

A fucose‐rich glycoconjugate (FRG) was isolated from egg jelly of the sea urchin Hemicentrotus pulcherrimus by gel filtration. FRG induced the acrosome reaction in H. pulcherrimus spermatozoa in a concentration‐dependent manner, although it showed about half the activity of the original unfractionated jelly. Synthetic sperm‐activating peptide I (SAP‐I: Gly‐Phe‐Asp‐Leu‐Asn‐Gly‐Gly‐Gly‐Val‐Gly) increased the rate of the acrosome reaction induced by FRG; the maximal rate of the acrosome reaction with FRG and SAP‐I being that of the unfractionated jelly. The half‐maximal increase in induction of the acrosome reaction by SAP‐I with FRG occurred at 4 × 10−10 M SAP‐I, which was almost the same concentration inducing half‐maximal stimulation of sperm respiration. Pronase digestion of FRG resulted in an 50% decrease in induction of the acrosome reaction and also in the elevation of cAMP in sperm. Some reagents (monensin and 3‐isobutyl‐1‐methylxanthine) which increase intracellular pH, Ca2+ and cyclic nucleotides also increased the rates of the acrosome reaction induced by FRG or pronase‐digested FRG. However, the rates did not reach those with FRG or pronase‐digested FRG with SAP‐I. These results indicate that SAP‐I promotes induction of the acrosome reaction by acting as a specific co‐factor of FRG.

Transient activation of the micro1 homeobox gene family in the sea urchin (Hemicentrotus pulcherrimus) micromere

Abstract. The animal-vegetal (A-V) axis of sea urchin embryos is morphologically evident at the 16-cell stage of development. Mesomeres, macromeres, and micromeres are arrayed along the A-V axis. The vegetal micromere differentiates into the skeletogenic mesenchyme and functions as a signaling center. To date, no zygotic or maternally specified gene with restricted expression in the micromere at the 16-cell stage has been reported. We performed subtraction PCR and dot blot hybridization using poly(A)+ RNA extracted from the micromere (tester) and the mesomere (driver) in order to identify micromere-specific genes. Using a cDNA fragment identified in this screen, we isolated four similar but distinct cDNA clones from a library, which corresponded to a group of genes that we refer to as the micro1 family. The micro1 family encoded putative transcription factors with a homeodomain which had 87–95% identity between family members. The most highly conserved protein was encoded by PlHbox12 from Paracentrotus lividus (71–76% identity among family members). Northern blot hybridization and in situ hybridization demonstrated that micro1 was transiently activated during the early cleavage stages and that the transcript was restricted to the micromere. Thus, the expression domain was complementary to that of PlHbox12 along the A-V axis. The micro1 gene family has at least six loci, including polymorphic alleles, which are probably clustered in the genome. PlHbox12 and micro1 constitute a novel family of paired-like class homeobox genes. Phylogenetic analyses suggest that PlHbox12/micro1 evolved exceptionally rapidly.

Purification and Characterization of the Egg Jelly Macromolecules, Sialoglycoprotein and Fucose Sulfate Glycoconjugate, of the Sea Urchin Hemicentrotus Pulcherrimus

A sialoglycoprotein and a fucose sulfate glycoconjugate (FSG) were purified from egg jelly of the sea urchin Hemicentrotus pulcherrimus. Sialoglycoprotein which consisted of sialic acid (90%, w/w) and protein (10%, w/w) did not cause induction of the acrosome reaction and sperm isoagglutination. FSG which contained one mol sulfate/mol fucose possessed 2.0 times protein to fucose by weight. The proteins in intact FSG were separated to two major (258 kDa and 237 kDa) and one minor (120 kDa) proteins by SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) in the presence of 2‐mercaptoethanol (2‐ME) while the proteins could not be separated by HPLC in the presence of 0.1% SDS or SDS‐PAGE without 2‐ME. However, after carboxymethylation of FSG, two major (260 kDa and 240 kDa) proteins and two minor (140 kDa and 135 kDa) proteins were separated from the fucose sulfate moiety by HPLC in the presence of 0.1% SDS or SDS‐PAGE without 2‐ME. When FSG was first carboxymethylated with non‐radioactive iodoacetic acid and then reduced with 2‐ME and finally carboxymethylated with 14C‐iodoacetic acid, the most of radioactivity was detected in 140 kDa and 135 kDa proteins. Carboxymethylted‐FSG was less potent than intact FSG in induction of the acrosome reaction. Fucoidan, a fucose sulfate polymer, did not induce the acrosome reaction.

The Development of the Enteropneust Hemichordate Balanoglossus misakiensis Kuwano

Abstract We describe development from fertilization to metamorphosis of the enteropneust hemichor-date Balanoglossus misakiensis. This is the first report to describe the complete development of an indirect-developing hemichordate under laboratory conditions. Mature adults were induced to spawn by shifting the temperature of seawater from 23 to 28°C. Eggs (200 μm diameter) were enclosed within a nonmucilaginous membrane, and dispersed readily in seawater. After artificial insemination, a fertilization envelope was elevated from the egg surface beneath the egg membrane; this was followed by the formation of the first and second polar bodies within the envelope. Zygotes cleaved at 20-min intervals to form blastulae, and gastrulation started 9 h after fertilization. Embryos hatched 1 day after fertilization to become typical feeding tornaria larvae. The larvae metamorphosed 7–10 days after fertilization without undergoing the first (Müller) or forth (Krohn) stage of indirect-developing hemichordate development. Larvae that were not fed failed to metamorphose. Juveniles completed adult body formation within a week of settling in sand at the bottom of the culture tube. We discuss heterochronical modifications of B. misakiensis development, and make the case for this species as a potential model organism for the investigation of indirect-developing hemichordates.

Structure, regulation, and function of micro1 in the sea urchin Hemicentrotus pulcherrimus

The animal-vegetal axis of sea urchin embryos is morphologically apparent at the 16-cell stage, when the mesomeres, macromeres, and micromeres align along it. At this stage, the micromere is the only autonomously specified blastomere that functions as a signaling center. We used a subtraction PCR survey to identify the homeobox gene micro1 as a micromere-specific gene. The micro1 gene is a representative of a novel family of paired-like class homeobox genes, along with PlHbox12 from Paracentrotus lividus and pmar1 from Strongylocentrotus purpuratus. In the present study, we showed that micro1 is a multicopy gene with six or more polymorphic loci, at least three of which are clustered in a 30-kb region of the genome. The micro1 gene is transiently expressed during early cleavage stages in the micromere. Recently, nuclear β-catenin was shown to be essential for the specification of vegetal cell fates, including micromeres, and the temporal and spatial coincidence of micro1 expression with the nuclear entry of β-catenin is highly suggestive. We demonstrated that micro1 is a direct target of β-catenin. In addition, we showed that micro1 is necessary and sufficient for micromere specification. These observations on the structure, regulation, and function of micro1 lead to the conclusion that micro1 and pmar1 (and potentially PlHbox12) are orthologous.

The extracellular-matrix-retaining cyanobacterium Nostoc verrucosum accumulates trehalose, but is sensitive to desiccation

The aquatic cyanobacterium Nostoc verrucosum forms macroscopic colonies, which consist of both cellular filaments and massive extracellular matrix material. In this study, the physiological features of N. verrucosum were investigated and compared with those of the anhydrobiotic cyanobacterium Nostoc commune. Nostoc verrucosum cells were sensitive to desiccation, but tolerant of freeze-thawing treatment in terms of both cell viability and photosynthetic O(2) evolution. Natural colonies of these cyanobacteria contained similar levels of chlorophyll a, carotenoids, the UV-absorbing pigments scytonemin and mycosporine-like amino acids, and uronic acid [a component of extracellular polysaccharides (EPS)]. EPS from both N. verrucosum and N. commune indicated low acidity and a high affinity for divalent cations, although their sugar compositions differed. The WspA protein, known to be a major component of the extracellular matrix of N. commune, was detected in N. verrucosum. Desiccation caused similarly high levels of trehalose accumulation in both cyanobacteria. Although previously considered relevant to anhydrobiosis in the terrestrial cyanobacterium N. commune, the data presented here suggest that extracellular matrix production and trehalose accumulation are not enough for standing extreme desiccation in N. verrucosum.

The micro1 gene is necessary and sufficient for micromere differentiation and mid/hindgut-inducing activity in the sea urchin embryo

In the sea urchin embryo, micromeres have two distinct functions: they differentiate cell autonomously into the skeletogenic mesenchyme cells and act as an organizing center that induces endomesoderm formation. We demonstrated that micro1 controls micromere specification as a transcriptional repressor. Because micro1 is a multicopy gene with at least six polymorphic loci, it has been difficult to consistently block micro1 function by morpholino-mediated knockdown. Here, to block micro1 function, we used an active activator of micro1 consisting of a fusion protein of the VP16 activation domain and the micro1 homeodomain. Embryos injected with mRNA encoding the fusion protein exhibited a phenotype similar to that of micromere-less embryos. To evaluate micro1 function in the micromere, we constructed chimeric embryos composed of animal cap mesomeres and a micromere quartet from embryos injected with the fusion protein mRNA. The chimeras developed into dauerblastulae with no vegetal structures, in which the micromere progeny constituted the blastula wall. We also analyzed the phenotype of chimeras composed of an animal cap and a mesomere expressing micro1. These chimeras developed into pluteus larvae, in which the mesomere descendants ingressed as primary mesenchyme cells and formed a complete set of skeletal rods. The hindgut and a part of the midgut were also generated from host mesomeres. However, the foregut and nonskeletogenic mesoderm were not formed in the larvae. From these observations, we conclude that micro1 is necessary and sufficient for both micromere differentiation and mid/hindgut-inducing activity, and we also suggest that micro1 may not fulfill all micromere functions.

Some more speract derivatives associated with eggs of sea urchins Pseudocentrotus depressus, Strongylocentrotus purpuratus, Hemicentrotus pulcherrimus and Anthocidaris crassispina

1. Fourteen peptides were isolated from the egg jelly of sea urchins, Pseudocentrotus depressus, Strongylocentrotus purpuratus, Hemicentrotus pulcherrimus and Anthocidaris crassispina and their amino acid sequences were determined. 2. The peptides stimulated H. pulcherrimus sperm respiration one half-maximally at about 8-60 pM. 3. Addition of speract to intact spermatozoa of P. depressus, H. pulcherrimus and A. crassispina resulted in the appearance of a newly stained protein (Mr 128,000 for P. depressus, Mr 128,000 for H. pulcherrimus and Mr 131,000 for A. crassispina) on sodium dodecyl sulfate-polyacrylamide gels.