Structure of acrosome reaction-inducing substance in the jelly coat of starfish eggs: a mini review.

Our knowledge at present on the structure of acrosome-reaction inducing substance (ARIS) in the jelly coat of starfish eggs is summarized. ARIS ia a proteoglycan-like molecule consisting of very long, linear, and highly sulfated glycans and three ARIS proteins, ARIS1-3. Detailed structures of the major glycan of ARIS and of ARIS1-3 are discussed. 3D-models of ARIS glycans are also presented. Phylogenetic distribution of ARIS proteins and/or genes indicates that ARIS genes are well preserved from the Ctenophore to Cephalochordata. In the Echinodermata, ARIS1-3 and ARIS genes were detected in all classes except for sea urchins.

Stem cells from innate sexual but not acquired sexual planarians have the capability to form a sexual individual.

Planarian species may harbor as many as three populations with different reproductive strategies. Animals from innate asexual (AS) and innate sexual (InS) populations reproduce only by fission and cross-fertilization, respectively, whereas the third population switches seasonally between the two reproductive modes. AS worms can be experimentally sexualized by feeding them with minced InS worms; we termed the resulting animals "acquired sexual" (AqS) worms. Both AqS and InS worms exhibit sexualizing activity when used as feed, suggesting that they maintain their sexual state via endogenous sexualizing substances, although the mechanisms underlying determination of reproductive strategy and sexual switching in these metazoans remain enigmatic. Therefore, we compared the endogenous sexualizing activity of InS worms and AqS worms. First, we amputated mature worms and assessed if they could re-enter a sexual state. Regenerants of InS worms, but not AqS worms, were only sexual, indicating that sexual state regulation comprises two steps: (1) autonomous initiation of sexualizing substance production and (2) maintenance of the sexual state by continuous production of sexualizing substances. Next, InS neoblasts were characterized by transplantation, finding that they successfully engrafted, proliferated, and replaced all recipient cells. Under such conditions, the AS recipients of InS worm neoblasts, but not those of AqS worms, became sexual. These results clearly show that there is a neoblast-autonomous determination of reproductive strategy in planarians.

Existence of two sexual races in the planarian species switching between asexual and sexual reproduction.

In certain planarian species that are able to switch between asexual and sexual reproduction, determining whether a sexual has the ability to switch to the asexual state is problematic, which renders the definition of sexuals controversial. We experimentally show the existence of two sexual races, acquired and innate, in the planarian Dugesia ryukyuensis. Acquired sexuals used in this study were experimentally switched from asexuals. Inbreeding of acquired sexuals produced both innate sexuals and asexuals, but inbreeding of innate sexuals produced innate sexuals only and no asexuals. Acquired sexuals, but not innate sexuals, were forced to become asexuals by ablation and regeneration (asexual induction). This suggests that acquired sexuals somehow retain asexual potential, while innate sexuals do not. We also found that acquired sexuals have the potential to develop hyperplastic and supernumerary ovaries, while innate sexuals do not. In this regard, acquired sexuals were more prolific than innate sexuals. The differences between acquired and innate sexuals will provide a structure for examining the mechanism underlying asexual and sexual reproduction in planarians.

Acrosome reaction-related steroidal saponin, Co-ARIS, from the starfish induces structural changes in microdomains.

Cofactor for acrosome reaction-inducing substance (Co-ARIS) is a steroidal saponin from the starfish Asterias amurensis. Saponins exist in many plants and few animals as self-defensive chemicals, but Co-ARIS has been identified as a cofactor for inducing the acrosome reaction (AR). In A. amurensis, the AR is induced by the cooperative action of egg coat components (ARIS, Co-ARIS, and asterosap); however, the mechanism of action of Co-ARIS is obscure. In this study we elucidated the membrane dynamics involved in the action of Co-ARIS. We found that cholesterol specifically inhibited the Co-ARIS activity for AR induction and detected the binding of labeled compounds with sperm using radioisotope-labeled Co-ARIS. Co-ARIS treatment did not reduce the content of sperm sterols, however, the condition was changed and localization of GM1 ganglioside on the periacrosomal region disappeared. We then developed a caveola-breaking assay, a novel method to detect the effect of chemicals on microdomains of culture cell, and confirmed the disturbance of somatic cell caveolae in the presence of Co-ARIS. Finally, by atomic force microscopy observations and surface plasmon resonance measurements using an artificial membrane, we revealed that Co-ARIS colocalized with GM1 clusters on the microdomains. Through this study, we revealed a capacitation-like event for AR in starfish sperm.

Novel conserved structural domains of acrosome reaction-inducing substance are widespread in invertebrates.

In the starfish Asterias amurensis, acrosome reaction inducing substance (ARIS) is the main factor responsible for allowing sperm to recognize the egg jelly and begin the acrosome reaction (AR). ARIS is a large proteoglycan-like molecule, and its pentasaccharide repeat, Fragment 1 (Fr. 1), is responsible for inducing AR. Here, we investigated the primary structure of ARIS for the first time in order to improve our understanding of its functionality. Electrophoretic analysis revealed that ARIS is a complex of three proteins, all of which are modified by the Fr. 1 sugar chain. Sequencing indicated that there are two novel, conserved domains in all three ARIS proteins: ARIS N-terminus (AR-N) and ARIS C-terminus (AR-C) domains. We also found that other echinoderms possess ARIS proteins that are capable of inducing the AR for homologous sperm, indicating that ARIS proteins may be a ubiquitous component for echinoderm fertilization. Moreover, we identified ARIS-like genes from Ctenophora to Protochordata.

Sex-inducing effect of a hydrophilic fraction on reproductive switching in the planarian Dugesia ryukyuensis (Seriata, Tricladida).

BACKGROUND: The mechanisms underlying the switching from an asexual to a sexual mode of reproduction, and vice versa, remain unknown in metazoans. In planarians, asexual worms acquire cryptic sexuality when fed with sexual worms, indicating that sexual worms contain a sex-inducing substance. Although such a chemical compound will provide clues about the mechanisms underlying the switching, information on the sex-inducing substance is poor. In order to identify this substance, we have established an assay system for sexual induction in asexual worms of Dugesia ryukyuensis by feeding them with sexual worms. Here, we carried out an isolation study on the sex-inducing substance using this assay system. RESULTS: After centrifugation of sexual worms homogenised in saline solution, we found that not only did the precipitate have a sex-inducing effect on the asexual worms, which has been shown previously, but the cytosolic fraction did as well. We confirmed that the sex-inducing activity in the cytosolic fraction was recovered in a hydrophilic fraction separated on an octadecylsilane (ODS) column. We showed that the sex-inducing substance in the hydrophilic fraction is papain-resistant and a putative low-molecular-weight compound of less than 500. We also suggest the presence of an enhancer of sexual induction with a molecular weight (MW) of more than 5 K in the hydrophilic fraction. CONCLUSION: Our experiments showed the existence of a sex-inducing substance and an enhancer of sex-induction in a hydrophilic fraction, and a putative hydrophobic sex-inducing substance in the precipitate. Sexual induction in the asexual worms might be triggered by additive or synergistic effects of these chemical compounds.

Regulation of the starfish sperm acrosome reaction by cGMP, pH, cAMP and Ca2+.

In the starfish, Asterias amurensis, three components in the jelly coat of eggs, namely acrosome reaction-inducing substance (ARIS), Co-ARIS and asterosap, act in concert on homologous spermatozoa to induce the acrosome reaction (AR). Molecular recognition between the sperm surface molecules and the egg jelly molecules must underlie signal transduction events triggering the AR. Asterosap is a sperm-activating molecule, which stimulates rapid synthesis of intracellular cGMP, pH and Ca2+. This transient elevation of Ca2+ level is caused by a K+-dependent Na+/Ca2+ exchanger, and the increase of intracellular pH is sufficient for ARIS to induce the AR. The concerted action of ARIS and asterosap could induce elevate intracellular cAMP levels in starfish sperm and the sustained increase in [Ca2+], which is essential for the AR. The signaling pathway induced by these factors seems to be synergistically regulated to trigger the AR in starfish sperm.

Neoblast-enriched fraction rescues eye formation in eye-defective planarian 'menashi' Dugesia ryukyuensis.

Planarians are well known for their remarkable regenerative capacity. This capacity to regenerate is thought to be due to the presence of totipotent somatic stem cells known as 'neoblasts', which have particular morphological characteristics. The totipotency of neoblasts was supported by Baguñà's experiment, which involved the introduction of donor cells into irradiated hosts. However, since Baguñà's experiment did not include the use of a phenotypic marker, the donor cells could not be traced. In the current study, a genetic mutant planarian, menashi, an eye-defective mutant that lacks the pigmented area in the eyes, was established. This planarian is excellent for tracing the fate of cells after their introduction into irradiated hosts. To investigate the differentiation potency more directly, a neoblast-rich fraction obtained from normal worms was transplanted into an X-ray-irradiated menashi strain. Planarians that survive X-ray irradiation were developed, and we observed the pigment of the area in the eyes of the regenerating planarians. This result suggests that the neoblast-rich fraction contains cells that can proliferate and differentiate. These cells can replace the cells and structures lost by X-ray irradiation and ablation, and they can also differentiate into eye pigment cells.

Egg and sperm recognition systems during fertilization.

Fertilization is a programmed process that has many molecules and sequential events amenable to study. The biochemistry of fertilization has identified cellular and acellular components fundamental to the interactions between sperm and egg. Recent studies highlight the molecular details of the species-specificity of fertilization that involve protein-protein and protein-carbohydrate interactions. Although the diversity of structure and mechanism may imply rapid evolution of fertilization proteins, understanding the structure-function relationships has become important. Here, we introduce the molecules controlling the sperm AR, sperm attachment to, and penetration through, the egg investments.

A chloride ion channel in Halocynthia roretzi hemocytes is associated with PO activity but not pigmentation during the contact reaction.

When hemocytes of two different individuals of the solitary ascidian Halocynthia roretzi come into contact (allogeneic recognition), they devacuolate in several seconds following contact, release phenoloxidase (PO) into the supernatant, and form coagulates. These coagulates show brown pigmentation. This reaction is referred to as the contact reaction (CR). In this study, the CR-inhibitory monoclonal antibody ku-4-96, which inhibits devacuolation, increase in PO activity, coagulation, and pigmentation, was constructed. This antibody is thought to exert its inhibitory action at an early stage in the CR. A differential display analysis was conducted by using ku-4-96 to search exhaustively for differentially expressed genes involved in the CR. One of the genes cloned was downregulated in the presence of ku-4-96 and upregulated during the CR. This gene showed very high similarity to the Cl(-) channel gene ClC-2 and was named HrClC-2. We examined the effects of Cl(-) channel inhibitors on the CR to examine whether the Cl(-) channel was involved in the CR signal cascade. Devacuolation, coagulation, and pigmentation were not affected by different concentrations of these inhibitors, which inhibited PO activity. This suggests that the PO activity is independent of these other phenomena occurring during the CR.

Characterization of novel genes expressed specifically in the sexual organs of the planarian Dugesia ryukyuensis.

The planarian Dugesia ryukyuensis reproduces both asexually (fissiparous) and sexually (oviparous) and can switch from the asexual mode to the sexual mode. By feeding with mature Bdellocephala brunnea oviparous worms, the fissiparous worms, which do not possess sexual organs, can be converted to fully sexualized worms in a process termed sexualization. As sexualization proceeds, the sexual organs are formed uniformly and five stages (stages 15) of the process have been identified histologically. In order to clarify the sexualization process, we attempted to isolate the genes expressed specifically at stage 5 by the differential display method. We isolated five genes expressed in the testis and two genes expressed in the yolk gland, which is an organ specific to sexualized worms. By BLAST search, one of the testis-specific genes was coded as testis-specific alpha-tubulin and two yolk gland-specific genes are similar to ribose-phosphate pyrophosphokinase I and F-box/SPRY-domain protein 1. Drs1, Drs2 and Drs3 were expressed in spermatocytes and spermatids from the early stage of spermatogenesis and Drs4 and Drs5 were expressed in spermatogonia, spermatocytes and spermatids. These genes are useful markers for elucidating the sexualization process.

The Dugesia ryukyuensis database as a molecular resource for studying switching of the reproductive system.

The planarian Dugesia ryukyuensis reproduces both asexually and sexually, and can switch from one mode of reproduction to the other. We recently developed a method for experimentally switching reproduction of the planarian from the asexual to the sexual mode. We constructed a cDNA library from sexualized D. ryukyuensis and sequenced and analyzed 8,988 expressed sequence tags (ESTs). The ESTs were analyzed and grouped into 3,077 non-redundant sequences, leaving 1,929 singletons that formed the basis of unigene sets. Fifty-six percent of the cDNAs analyzed shared similarity (E-value<1E -20) with sequences deposited in NCBI. Highly redundant sequences encoded granulin and actin, which are expressed in the whole body, and other redundant sequences encoded a Vasa-like protein, which is known to be a component of germ-line cells and is expressed in the ovary, and Y-protein, which is expressed in the testis. The sexualized planarian expressed sequence tag database (http://planaria.bio.keio.ac.jp/planaria/) is an open-access, online resource providing access to sequence, classification, clustering, and annotation data. This database should constitute a powerful tool for analyzing sexualization in planarians.

The identification of á´ -tryptophan as a bioactive substance for postembryonic ovarian development in the planarian Dugesia ryukyuensis.

Many metazoans start germ cell development during embryogenesis, while some metazoans possessing pluripotent stem cells undergo postembryonic germ cell development. The latter reproduce asexually but develop germ cells from pluripotent stem cells or dormant primordial germ cells when they reproduce sexually. Sexual induction of the planarian Dugesia ryukyuensis is an important model for postembryonic germ cell development. In this experimental system, hermaphroditic reproductive organs are differentiated in presumptive gonadal regions by the administration of a crude extract from sexual planarians to asexual ones. However, the substances involved in the first event during postembryonic germ cell development, i.e., ovarian development, remain unknown. Here, we aimed to identify a bioactive compound associated with postembryonic ovarian development. Bioassay-guided fractionation identified ʟ-tryptophan (Trp) on the basis of electrospray ionization-mass spectrometry, circular dichroism, and nuclear magnetic resonance spectroscopy. Originally masked by a large amount of ʟ-Trp, ᴅ-Trp was detected by reverse-phase high-performance liquid chromatography. The ovary-inducing activity of ᴅ-Trp was 500 times more potent than that of ʟ-Trp. This is the first report describing a role for an intrinsic ᴅ-amino acid in postembryonic germ cell development. Our findings provide a novel insight into the mechanisms of germ cell development regulated by low-molecular weight bioactive compounds.

Biochemical Studies on the Acrosome Reaction of the Starfish, Asterias Amurensis II. Purification and Characterization of Acrosome Reaction-Inducing Substance.

The acrosome reaction-inducing substance (ARIS) was purified from egg jelly of the starfish, Asterias amurensis. The purification procedure included elimination of neutral glycoproteins from the ARIS fraction by isoelectric pointprecipitation and subsequent gel filtrations on Sephadex G-50 and Bio-Gel A-50m columns. The final preparation of ARIS was homogeneous as judged by cellulose acetate electrophoresis of ARIS and by ion-exchange chromatography on DEAE-Sephadex A-25 of S-carboxymethylated ARIS. ARIS is a very large, sulfated glycoprotein containing fucose, galactose, galactosamine and glucosamine as sugar components. It requires diffusible cofactor (Co-ARIS) for full biological activity. A Pronase digest of ARIS retained its capacity to induce the acrosome reaction when Co-ARIS was added to the bioassay system. The physiological significance of the carbohydrate moiety of ARIS is discussed.

Biochemical Studies on the Acrosome Reaction of the Starfish, Asterias Amurensis I. Factors Participating in the Acrosome Reaction.

In contrast with the case in sea urchin sperm, in starfish the acrosome reaction is not spontaneously induced by simply increasing the extracellular Ca2+ concentration or pH. At higher pHs, starfish sperm undergo morphological changes accompanied by exocytosis of the acrosomal vacuole, but they do not form acrosomal filaments. Nomarski-microscopic observation confirmed that spermatozoa undergo the acrosome reaction within the jelly coat. Acrosome reaction-inducing substance, a glycoprotein from the egg jelly, required a diffusible cofactor(s) present in the egg jelly for full activity. Several lines of evidence showed that this diffusible factor(s) is not merely Ca2+ .

Three Phases of Cortical Maturation during Meiosis Reinitiation in Starfish Oocytes: (starfish oocytes/fertilization envelope/calcium maturation).

Oocytes of the starfish, Asterina pectinifera, respond differently to calcium ionophore A23187 depending upon their stage of maturation. Oocytes not-treated with 1-methyladenine (1-MA) formed only a partial fertilization envelope (FE) in response to A23187. Those treated with 1-MA formed no FE if the ionophore was introduced to them before germinal vesicle breakdown (GVBD), in contrast with which they did fully elevate the FE if it was introduced after GVBD. Similar stage-dependent results were obtained if the intracellular concentration of calcium was increased by microinjection of calcium-EGTA buffers. In good accordance with the FE formation, a stage-dependent protease release from oocytes by the ionophore was observed. It is concluded from these results that, in starfish oocytes, their ability to undergo the exocytosis of cortical granules in response to an increase in intracellular calcium greatly changes along the way of maturation.

Activation of Starfish Oocytes Modifies their Hormone Dependent Period for 1-Methyladenine in Meiosis Reinitiation: (starfish oocyte/maturation/hormone dependent period/fertilization/A23187).

Starfish oocytes reinitiate the meiosis when they are exposed to the hormone, 1-methyladenine (1-MA): 1-MA must be present for a period of about 10-20 min (20°C) known as the hormone dependent period (HDP). We investigated several conditions which affect the duration of the HDP in the starfish Asterina pectinifera. Fertilization lengthened the HDP about 30-80%, when spermatozoa were added to the oocytes at the same time as 1-MA was applied. When oocytes were inseminated before 1-MA treatment, HDP was reduced progressively correlating with the time of the insemination coming earlier, and eventually became shorter than the control. Similarly, A23187 and caffeine lengthened the HDP if they were added to the oocytes at the same time as 1-MA was applied, and shortened it if they are applied sufficiently in advance to 1-MA treatment. These modifications of HDP are likely to be brought by changes in the intracellular calcium concentration. A23187 did not lengthen the HDP, if it was administered 10 min after 1-MA application. Thus the effect of A23187 on the HDP occurred specifically during the first 5 or 10 min after 1-MA application. These results indicate that the HDP comprises at least two distinct phases.

Mass Isolation of Germinal Vesicles from Starfish Oocytes*: (germinal vesicle/nucleus/oocyte/starfish/mass isolation).

A rapid, simple and efficient isolation procedure for germinal vesicles was developed using fully grown oocytes from the starfish, Asterina pectinifera. It depends on removal of the vitelline coat by trypsin digestion, gentle cell lysis by hypotonic treatment and centrifugation on a discontinuous sucrose gradient. The germinal vesicles isolated by this method are not clumped, fairly uniform in size and morphology, and rimmed with a very thin layer of cytoplasmic embroidery. They appear morphologically very similar to those in the oocytes. Potential applications of this method and possible functions of the cytoplasmic embroidery are discussed.

Low-Na+ Seawater Induces the Acrosome Reaction and Histone Degradation of Starfish Sperm in the Absence of Egg Jelly: (starfish/sperm/histone degradation/acrosome reaction/low-Na+ seawater).

Egg jelly induces the degradation of histones as well as the acrosome reaction in the spermatozoa of Asterina pectinifera. Much similar degradation of histones without any apparent morphological changes such as the acrosome reaction was induced in the spermatozoa by merely dispersing them into Na+ -free seawater. It required external Ca2+ much less than the jelly-induced one in normal seawater, and was not susceptible to Ca2+ -channel antagonists, verapamil and diltiazem. Once spermatozoa were incubated with egg jelly in Ca2+ -free seawater, they did not undergo the histone degradation even after subsequent addition of Ca2+ , but Na+ -free seawater rescued such blockage. Spontaneous acrosome reaction occurred in seawater containing 10-30 mM Na+ in a Ca2+ -dependent manner. This reaction was accompanied by a rapid increase in intracellular pH (pHi) followed by a large pHi decrease. Diltiazem blocked a large decrease in pHi but scarcely inhibited the acrosome reaction induced by low-Na+ seawater. Increasing K+ inhibited both pHi changes and the acrosome reaction induced by low-Na+ seawater. Decreasing pH of seawater also inhibited the pHi changes but did not affect the acrosome reaction. Strontium was also effective to induce a rapid increase, followed by a gradual decrease, in pHi and the acrosome reaction.