Correlation Between the Molecular Structure and the Biological Activity of Co-ARIS, a Cofactor for Acrosome Reaction-Inducing Substance

Two components in the egg jelly are required for inducing the acrosome reaction in starfish; a sulfated glycoprotein called acrosome reaction-inducing substance (ARIS) and its cofactor called Co-ARIS. Three distinct molecules were isolated as the major Co-ARIS' and designated as Co-ARIS' I, II and III. Structural analysis of Co-ARIS' revealed that they are steroidal saponins comprising a sulfated steroid and a pentasaccharide chain. Co-ARIS' I and II differ only in the steroidal side chain. In the presence of ARIS, each Co-ARIS induced the acrosome reaction with a maximal effect at 100–200 μM (Co-ARIS I) or 25–50 μM (Co-ARIS' II and III). Mixtures of Co-ARIS' I, II and III were more effective than the individuals. The activity of Co-ARIS was considerably reduced by solvolytic desulfation but was not affected at all by periodate oxidation. Reduction by NaBH₄ decreased the activity of Co-ARIS I and enhanced that of Co-ARIS II. Treatment of Co-ARIS III with NaBH₄ did not affect the activity as anticipated from its structure. These results suggest that the sulfate moiety and the side chain of steroid are important for the activity of Co-ARIS. The saccharide chain, however, seems not necessarily to be strictly specified for the activity.     

EXOGASTRULATION INDUCED BY CHILLING IN SEA URCHIN LARVAE

Exogastrulation is induced by chilling in several species of sea urchins, including Strongylocentrotus intermedius, Strongylocentrotus nudus, Pseudocentrotus depressus and Anthocidaris crassispina , but not in Hemicentrotus pulcherrimus. When early gastrulae are raised at low temperatures, no pseudopodia of secondary mesenchyme cells are formed, but the invagination of the endodermal plate occurs normally. When gastrulae in later stage having pseudopodia are chilled, the pseudopodia withdraw and the archenteron begins to retract, resulting in exogastrulation. The exogastrulae are also induced when the larvae are raised in the presence of colchicine, vinblastine, cytochalasin B or cytochalasin C. The formation of exogastrula induced by chilling is presumed to be caused by the depolymerization of microtubules in the secondary mesenchyme cells and their pseudopodia. The fully invaginated archenteron of the late gastrula, even when it is chilled, remains within the blastocoel and does not evaginate.
The effectiveness of low temperature treatment in inducing exogastrulation may be related to the environmental temperature at the breeding season of the animal.     

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⁺.     

Oocyte Mitochondria: Strategies to Improve Embrbryogenesis

Abstract  Mitochondria play a central role to provide ATP for fertilization and preimplantation embryo development in the ooplasm. The mitochondrial dysfunction of oocyte has been proposed as one of the causes of high levels of developmental retardation and arrest that occur in preimplantation embryos generated using Assisted Reproductive Technology. Cytoplasmic transfer (CT) from a donor to a recipient oocyte has been applied to infertility due to dysfunctional ooplasm, with resulting pregnancies and births. However, neither the efficacy nor safety of this procedure has been appropriately investigated. In order to improve embryogenesis, we observed the mitochondrial distribution in ooplasma under the several conditions using mitochondrial GFP-transgenic mice (mtGFP-tg mice) in which the mitochondria are visualized by GFP. In this report, we will present our research about the mitochondrial distribution in ooplasm during early embryogenesis and the fate of injected donor mitochondria after CT using mtGFP-tg mice. The mitochondria in ooplasm from the germinal vesicle stage to the morula stage were accumulated in the perinuclear region. The mitochondria of the mtGFP-tg mouse oocyte transferred into the wild type mouse embryo could be observed until the blastocysts stage, suggesting that the mtGFP-tg mice oocyte is very useful for visual observation of the mitochondrial distribution in the oocyte, and that the aberrant early developmental competences due to the oocyte mitochondrial dysfunction may be overcome by transferring the "normal" mitochondria.     

Mass Isolation of Germinal Vesicles from Starfish Oocytes*

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.     

Anion Channel Blockers Inhibit the Acrosome Reaction of Echinoderm Sperm

Two types of anion channel blockers, SITS (4-acetamide-4'-isothiocyanostilbene-2,2'-disulfonic acid) and DIDS (4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid), inhibited jelly-induced acrosorne reaction in starfish and sea urchin. In starfish sperm, both of the blockers reversibly inhibited the formation of acrosomal process but they had no effect on either the acrosomal exocytosis or acid release from the sperm. Complete acrosome reaction occurred even in Cl⁻- and SO₄²⁻-free artificial seawater whereas HCO₃⁻was required for the acrosomal exocytosis. Importance of anion transport in acrosome reaction is discussed.     

Induction of the Acrosome Reaction in Starfish

In the starfish, Asterias amurensis , at least two distinct components of the egg jelly are required for inducing the acrosome reaction: a sulfated glycoprotein named acrosome reaction-inducing substance (ARIS) and a diffusible organic substance(s) named Co-ARIS. The following evidence suggested that ARIS and Co-ARIS cooperatively activate CA-channels of the sperm plasma membrane and eventually induce dramatic changes in sperm morphology, the acrosome reaction. 1) Pronase digest of ARIS (P-ARIS) and Co-ARIS, either as a pure or a crude preparation (Fraction M₈), were fully effective in combination for induction of the acrosome reaction in normal sea water, although they were not effective individually. P- ARIS alone induced the acrosome reaction fully in high Ca²⁺ sea water and markedly at high pHs, whereas Fraction M₈ alone did not induce the reaction even in these conditions. The reaction was not induced by increase in either the Ca²⁺ concentration or the pH of sea water, but was markedly induced in the absence of jelly components by raising both the pH and Ca²⁺ concentration together. 2) The ionophore A23187 induced the acrosome reaction appreciably when present alone and fully in the presence of monensin or Fraction M₈. Monesin alone was ineffective. 3) The jelly or a combination of ARIS and Fraction M₈ caused abrupt Ca²⁺ -uptake by the sperm. The Ca-channel blockers verapamil and diltiazem inhibited the jelly-induced acrosome reaction.     

Maitotoxin, A Presumed Calcium Channel Activator, Induces the Acrosome Reaction in Mussel Spermatozoa

Maitotoxin, a presumed activator of the voltage-sensitive calcium channel, induced the acrosome reaction in the mussel, Mytilus edulis at physiological pH and in the starfish, Asterias amurensis at pH 9.5. The induction of acrosome reaction by maitotoxin depended upon external Ca²⁺ and was inhibited by two types of calcium channel blockers; verapamil and diltiazem. These results suggest that the activation of the voltage-sensitive calcium channel takes an important part in the initiation of acrosome reaction in Mytilus and other animals.     

Intracellular pH Changes of Starfish Sperm Upon the Acrosome Reaction

The acrosome reaction is accompanied by ionic changes such as increases in intracellular Ca²⁺ and intracellular pH (pH_i). Since the two jelly components essential for inducing the acrosome reaction, ARIS and Co-ARIS, were shown to activate Ca-channels (accompanying paper), we examined the jelly components to determine which was responsible for the pH_i-increase using 9-aminoacridine as a probe of pH_i. This paper presents evidence that an oligopeptide(s) is responsible for the pH_i-increase. The pH_i of swimming sperm is 7.4-7.5. Within 20 sec after the addition of jelly, their pH_i increased rapidly by 0.06 pH unit, then decreased by 0.2–0.3 pH unit, and reached a plateau level within 3 min. Similar changes in pH_i were observed on addition of a Pronase digest of ARIS (P-ARIS) and a diffusible fraction of jelly (Fraction M₈) together. Fraction M₈, but not ARIS or Co-ARIS increased the pH_i, and activated sperm respiration in sea water at pH 6.5. The two activities of Fraction M₈ depended upon Na⁺ but not Ca²⁺, and were susceptible to Pronase digestion. Fraction M₈ is also known to enhance induction of the acrosome reaction by the Ca-ionophore A23187. These results suggest that the egg jelly contains a peptide(s) that is not obligatory for the acrosome reaction but facilitates the reaction by increasing the pH_i of the sperm. The significance of the pH_i-increase upon the acrosome reaction is discussed.     

Presence of two glycolytic gene clusters in a severe pathogenic line of Candidatus Phytoplasma asteris

Phytoplasmas are plant-pathogenic bacteria that are associated with numerous plant diseases. We have previously reported the complete genomic sequence of Candidatus Phytoplasma asteris, OY strain, OY-M line, which causes mild symptoms. The phytoplasma genome lacks several important metabolic genes, implying that the consumption of metabolites by phytoplasmas in plants may cause disease symptoms. Here we show that the approximately 30-kb region including the glycolytic genes was tandemly duplicated in the genome of OY-W phytoplasma, which causes severe symptoms. Almost duplicated genes became pseudogenes by frameshift and stop-codon mutations, probably because of their functional redundancy. However, five kinds of genes, including two glycolytic genes, remained full-length ORFs, suggesting that it is advantageous for the phytoplasma to retain these genes in its lifestyle. In particular, 6-phosphofructokinase is known as a rate-limiting enzyme of glycolysis, implying that the different number of glycolytic genes between OY-W and OY-M may influence their respective glycolysis activities. We previously reported that the phytoplasma population of OY-W was higher than that of OY-M in their infected plants. Taking this result into account, the higher consumption of the carbon source may affect the growth rate of phytoplasmas and also may directly or indirectly cause more severe symptoms.