Acid release at meiotic maturation of oocytes in the polychaete annelid Sabellaria alveolata.

ABout 1 pmole of acid per egg is released when prophasic oocytes undergo maturation under the action of sperm, proteases or ionophore A 23187. No similar acid release occurs at fertilization of matured oocytes. These findings are compared with data on Urechis and sea urchin.     

Acid release at meiotic maturation of oocytes in the polychaete annelidSabellaria alveolata

Summary About 1 pmole of acid per egg is released when prophasic oocytes undergo maturation under the action of sperm, proteases or ionophore A 23187. No similar acid release occurs at fertilization of matured oocytes. These findings are compared with data onUrechis and sea urchin.     

Zinc increases the longevity of unfertilized sea urchin eggs

Summary We have found that Zn²⁺ prevented lysis of unfertilized sea urchin eggs, and the eggs retained the ability to form fertilization membranes and to divide. SDS polyacrylamide gel electrophoresis showed that proteolysis of several proteins accompanied egg lysis, but Zn²⁺ inhibited this proteolysis. Therefore, Zn²⁺ blocks protease activity directly or indirectly and thereby prolongs the longevity of unfertilized sea urchin eggs.     

Effect of valinomycin on mitotic activity and ciliary movement during embryonic development]

The K+ ionophore valinomycin very quickly arrests cleavage in sea urchin and mouse eggs at concentrations ranging between 10 and 3 micron. Development of Axolotl and Xenopus eggs is not arrested before the blastula or gastrula stage. The motility of sea urchin sperm, blastulae and gastrulae is suppressed, within a few minutes, by 1-9 micron valinomycin.     

Sperm chymotrypsin-like enzymes of different inhibitor-susceptibility as lysins in ascidians

Summary Inhibitory effects of three peptidyl phenylalaninals on fertilization and on chymotrypsin-like enzyme activity of sperm in three species of ascidians were examined. The results suggest that a sperm chymotrypsin-like enzyme is indispensable for the fertilization in each of the ascidians, and that these enzymes have different susceptibilities to inhibitors.12 November 1986Acknowledgments. We are indebted to Dr M. Hoshi of Tokyo Institute of Technology for his helpful discussion. This work was supportd by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan.     

The problem of sea urchin egg fertilization and its implications for biological studies.

The analysis of sea urchin egg fertilization shows that several phenomena common to other biological systems are involved: cell recognition, cell fusion, exocytosis and initiation of mitotic activity. Both the role of calcium ions in cell fusion and exocytosis and the function of the cell surface in the initiation of mitotic activity appear to have general applicability. The study of fertilization can contribute to the elucidation of these processes and, reciprocally, progress in this field can help to advance our understanding of the mechanisms of fertilization in sea urchins and other organisms.     

The problem of sea urchin egg fertilization and its implications for biological studies

Summary The analysis of sea urchin egg fertilization shows that several phenomena common to other biological systems are involved: cell recognition, cell fusion, exocytosis and initiation of mitotic activity. Both the role of calcium ions in cell fusion and exocytosis, and the function of the cell surface in the initiation of mitotic activity appear to have general applicability. The study of fertilization can contribute to the elucidation of these processes and, reciprocally, progress in this field can help to advance our understanding of the mechanisms of fertilization in sea urchins and other organisms.     

Timing of action of sperm proteases in ascidian fertilization

Summary The timing of action of three sperm proteases, acrosin, spermosin, and a chymotrypsin-like enzyme, in the fertilization of the ascidian,Halocynthia roretzi, was examined by adding specific protease inhibitors at various times after insemination. The results indicate that the last two enzymes both function at the early stage of the process of sperm penetration through the egg investment, while acrosin functions at the late stage.Acknowledgments. We are indebted to Dr M. Hoshi of Tokyo Institute of Technology for his helpful discussion, and to Dr T. Someno of Nippon Kayaku Kogyo Co. for his generous gifts of Z-Val-Pro-Arg-H and leupeptin. This work was supported by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan, and from Naito Research Foundation.     

Denaturation map of the ribosomal DNA of Lytechinus variegatus sperm.

Electron microscopy of the partially heat denatured ribosomal DNA (rDNA) from sea urchin (Lytechinus variegatus) sperm has demonstrated that it consists of repeating units of 3.6 +/- 0.2 micron, corresponding to a mol.wt of 7.2 +/- 0.4 x 10(6). Based on differential denaturability, each repeat unit is divided into 2 regions. The larger region of 2.47 +/- 0.11 micron (mol.wt 4.9 +/- 0.22 x 10(6)) corresponds in length to the ribosomal precursor RNA of sea urchins and the smaller, GC-rich, subunit of 1.16 +/- 0.09 micrometer (mol.wt 2.3 +/- 0.18 x 10(6)) is presumed to contain non-transcribed spacer sequences.     

Teratogenic effect of tolbutamide on the development of the sea urchin embryo (Paracentrotus lividus lamarck)

Summary The hypoglycemic agent tolbutamide was tested for its action on the cleavage and differentiation of the sea urchin embryo. Tolbutamide effects a strong selective action on the endoderm which becomes suppressed.     

Control of sperm motility and fertility: Diverse factors and common mechanisms

Spermatozoa generated in the testis are immature and incompetent for fertilization. During their journey toward the egg, the sperm acquire fertility and achieving fertilization. These sperm modifications to ensure fertilization are induced by many female or male extra-sperm factors: for example, sperm motility-activating factors from the egg jelly, sperm attractants from the eggs, and decapacitation factors from the seminal plasma. The factors controlling sperm fertility are myriad and species specific; they may be peptides, sugar chains, or small organic compounds. Nevertheless, the fundamental mechanisms underlying fertilization must be common among all animals; increase in [Ca²⁺]_i triggers all the steps in the process of fertilization, and cAMP plays important roles in many steps. Elucidating the dynamic functional and morphological changes in sperm cells is important for understanding the regulation of fertilization. Here, we introduce the diversity and generality of the control of sperm fertility. Received 28 April 2008; received after revision 13 June 2008; accepted 17 June 2008     

A preliminary report on the fine structure of Tripneustes esculentus eggs.

The fine structure of Tripneustes esculentus eggs was studied with the aid of an electron microscope. Cells obtained from this West Indies sea urchin showed cortical granules, mitochondria forming a rosette around lipid granules, endoplasmic reticula, Golgi apparatus and annulated lamellae. These structures appear identical to those seen in eggs of the Atlantic sea urchin: Arbacia punctulata.     

Satellite DNA (II) from sea urchin (Lytechinus variegatus) sperm

When DNA isolated from freshly collected sperm of sea urchin (Lytechinus variegatus) is centrifuged to equilibrium in CsCl, 2 heavy satellite bands appear beside the main band DNA. Satellite DNA (II) appears in between the main band DNA (rho = 1.695 g/cm3) and the rDNA satellite (rho = 1.722 g/cm3). Satellite DNA (II) has a buoyant density 1.710 g/cm3, corresponding to 50% GC content. It is speculated that the satellite DNA (II), which appears to be of high mol. wt, might contain the sequences complementary to histone mRNA.     

Fibrin membrane endowed with biological function. III. Fixing living cells in fibrin gel without impairing their functions

Summary Chlorella cells, sea urchin eggs and Paramecium were embedded in fibrin gel which was formed by fibrinogen-fibrin conversion in the presence of thrombin. The embedded Chlorella cells retain the ability of photosynthesis by illumination. The embedded sea urchin eggs develop to normal blastulae and gastrulae. Samples of Paramecium survive for more than several h beating their cilia. It is suggested that this technique of fixing living cells is useful for handling free cells as a mass like a tissue, and for holding free cells in micrurgical experiments.     

Localization of vitelline-coat lysin purified from testis of a top shell,Turbo cornutus

Summary The vitelline-coat lysin purified from the testis ofTurbo cornutus was found, by an immunofluorescence technique, to be located in the acrosome of the sperm, which suggested that the lysin reacts with the vitelline-coat in an early phase of fertilization to allow the sperm to penetrate through the coat.We are grateful to Professor M. Akino (Tokyo Metropolitan University) for his helpful advice.     

Satellite DNA (II) from sea urchin (Lytechinus variegatus) sperm

Summary When DNA isolated from freshly collected sperm of sea urchin (Lytechinus variegatus) is centrifuged to equilibrium in CsCl, 2 heavy satellite bands appear beside the main band DNA. Satellite DNA (II) appears in between the main band DNA (=1.695 g/cm³) and the rDNA satellite (=1.722 g/cm³). Satellite DNA (II) has a buoyant density 1.710 g/cm³, corresponding to 50% GC content. It is speculated that the satellite DNA (II), which appears to be of high mol.wt, might contain the sequences complementary to histone mRNA.Acknowledgment. This work was carried out in the laboratory of Dr D. W. Stafford, Department of Zoology, University of North Carolina, Chapel Hill, N.C., USA. I thank him for his help and cooperation.     

Binding of Lens culinaris lectin to sea urchin embryo chromatin.

Lentin binds specifically to sea urchin embryo chromatin. This binding is saturable and inhibited by alpha-methyl-mannose. Scatchard plot analysis of the binding reaction suggests a single binding site.     

Effects of zinc and lithium ions on the strengthening cell adhesion in sea urchin blastulae

Summary Cellular adhesion in sea urchin blastulae, normal and vegetalized by treatment with lithium ions, strengthened as development proceeded. This tendency was arrested in the embryos animalized by treatment with zinc ions.The authors thank Mr T. Shinkai and S. Sone for their technical assistance.     

Sea urchin embryo as a model for analysis of the signaling pathways linking DNA damage checkpoint,DNA repair and apoptosis

DNA integrity checkpoint control was studied in the sea urchin early embryo. Treatment of the embryos with genotoxic agents such as methyl methanesulfonate (MMS) or bleomycin induced the activation of a cell cycle checkpoint as evidenced by the occurrence of a delay or an arrest in the division of the embryos and an inhibition of CDK1/cyclin B activating dephosphorylation. The genotoxic treatment was shown to induce DNA damage that depended on the genotoxic concentration and was correlated with the observed cell cycle delay. At low genotoxic concentrations, embryos were able to repair the DNA damage and recover from checkpoint arrest, whereas at high doses they underwent morphological and biochemical changes characteristic of apoptosis. Finally, extracts prepared from embryos were found to be capable of supporting DNA repair in vitro upon incubation with oligonucleotides mimicking damage. Taken together, our results demonstrate that sea urchin early embryos contain fully functional and activatable DNA damage checkpoints. Sea urchin embryos are discussed as a promising model to study the signaling pathways of cell cycle checkpoint, DNA repair and apoptosis, which upon deregulation play a significant role in the origin of cancer. Received 10 April 2007; accepted 23 April 2007     

Lithium and phorbol ester modify the activating capacity of ascidian spermatozoa

In this paper we have shown, using the whole-cell voltage clamp technique, that two parameters of the fertilization current in ascidian eggs may be modified by exposing spermatozoa to lithium or to phorbol ester. When spermatozoa were pre-treated in 250 mM lithium sea water for up to 30 min there was a significant increase in the mean initial slope of the fertilization current, from 116±90 to 169±84 pA/s (p<0.05). The peak current increased from 1371±1079 to 1719±1052 pA (p>0.05). Pre-treatment in 200–600 nM phorbol 12-myristate 13-acetate also increased the activating capacity of ascidian sperm, as monitored by a significant increase in the initial slope current in control eggs; however, there was no increase in peak current. Furthermore, we have shown, using NH₄Cl, that an increase in intracellular pH alone is insufficient to change the activating capacity of spermatozoa. This is the first report showing that the kinetics of an egg activation event depend upon the physiological status of the spermatozoon.