Katsutoshi Ishihara

Toad egg-jelly as a source of divalent cations essential for fertilization.

Dejellied uterine eggs of the toad Bufo bufo japonicus are not fertilizable in 1/20 De Boers solution (1/20 DB), but are fertilized when inseminated in a uv-solubilized jelly (UVJ) or the dialyzate of UVJ (UVJD). The present study was carried out to define this fertilization-supporting activity of egg-jelly. Dejellied eggs were fertilized in a high frequency when inseminated in a medium containing the ashes obtained by heating UVJD at 600 degrees C for 16 hr. Similarly, a reconstituted salt solution (RSS), which mimics the ionic composition of UVJD, supported a high rate of fertilization. To be effective in fertilization, however, RSS had to be present at the time of insemination. Analyses of individual salts revealed that dejellied eggs are successfully fertilized in CaCl2 and/or MgCl2 at 1-5 mM, only slightly in KCl at 10 mM, but not at all in NaCl at any of the concentrations tested. The activity of UVJD was lost reversibly when divalent cations were chelated by EDTA. The fertilization of dejellied eggs is therefore possible in a medium without any organic components of egg-jelly, provided that 2-5 mM Ca2+ or Mg2+ is present. Sperm were motile in media containing cations below 20-25 mM, regardless of the ionic composition. The egg-jelly possessed cations in a concentration of about 130 mM, but most ions were lost from intact jelly on immersion of eggs in water for 2-3 min, accompanied by the acquisition of fertilizability by sperm. Examination of the behavior of salts on dialysis or gel-filtration of jelly molecules revealed that the jelly retains Ca2+ and Mg2+, and possibly K+ as well, but not Na+ and Cl-. We propose that toad egg-jelly plays a function in fertilization by retaining Ca2+ and/or Mg2+ around each egg at the level necessary for successful sperm entrance into the egg.

An analysis of acid polysaccharides produced at fertilization of sea urchin

Abstract A substance rich in acid polysaccharides is produced at fertilization of sea urchin, Hemicentrotus pulcherrimus. The substance (designated as fertilization product, FP) was found to have a similar composition as in other species of sea urchins, containing fucose, mannose, glucose, galactose, hexosamine, sulfate, cations and 16 amino acids. The fertilization product was located at the egg surface and extruded into the perivitelline space and surrounding medium at fertilization. The acidic character of the fertilization product would be due to sulfate groups rather than acidic amino acids. Sixty-two per cent or more of “fertilization acid” were accounted for by the release of the acid polysaccharides.

RELEASE OF ACID POLYSACCHARIDES FOLLOWING FERTILIZATION OF SEA URCHIN EGGS. A CHEMICAL STUDY OF CHANGES IN THE CELL SURFACE.

Abstract 1. 1. At the time of fertilization Japanese sea urchin eggs released organic substances into the surrounding sea water, which reduced KMnO 4 and were non-dialyzable, thermostable, and alcohol-insoluble. 2. 2. The fertilization product of the Pseudocentrotus depressus egg contained acid polysaccharides composed of glucose, galactose, mannose, hexosamine, sulfate and probably also amino acid. The fertilization product of the Anthocidaris crassispina egg contained acid polysaccharides composed of the same constituents except for glucose and mannose. 3. 3. No release of free sulfates from the eggs of the above two species could be demonstrated. 4. 4. Fifty to 100 per cent of the amount of fertilization product appeared in the suspension medium during the first 5 min after insemination, the remainder appearing within 5 to 10 min after insemination. The normal eggs retained the same substance in perivitelline space formed at fertilization. 5. 5. The pH of the surrounding sea water markedly decreased shortly after fertilization in unbuffered artificial sea water; the drop in pH of the suspension medium of trypsin-treated eggs and urea-treated eggs was not as great as that of the suspension medium with intact egg, suggesting that other compounds with buffering action may also have been released by the treated eggs. 6. 6. It is highly probable that the release of the fertilization product corresponds, in part at least, to the acid formation occurring very soon after fertilization.

The exogastrula-inducing peptides in embryos of the sea urchin, Anthocidaris crassispina — isolation and determination of the primary structure

Four exogastrula-inducing peptides, A, B, C, and D have been isolated from the homogenates of embryos of the sea urchin, Anthocidaris crassispina, with successive chromatographic fractionations. The complete amino acid sequences of the peptides A and D were determined by analysis of the peptides generated by their digestion with lysyl endopeptidase. They were composed of 52 and 53 amino acid residues, and their molecular weights were calculated to be 5754 and 5737, respectively. The sequences of peptides A and D were DSVYQCNRDTNSCDGFGKCEKSTFGRTTGQYICNCDDGYRNNAYGGCSPRTE, and DTVARCERDTKNCDGHGTCQLSTFGRRTGQYICFCDAGYRKPNSYGGCSPSSA, respectively. The biological significance of the exogastrula-inducing peptides was discussed.

EFFECTS OF CHEMICAL DISRUPTION ON THE BIOLOGICAL ACTIVITIES OF SEA URCHIN EGG JELLY1

In an attempt to relate the biological activities of sea urchin egg jelly to the structural characteristics of the acid glycoprotein molecule, the jelly was oxidized with H2O2 and sodium periodate, and digested with trypsin and pronase. The non‐dialyzable products of H2O2 and periodate oxidation, and a fucose‐rich fraction isolated from enzyme‐digested jelly by column chromatography, were tested for their capacity to induce sperm agglutination and acrosome reaction in Hemicentrotus pulcherrimus.

Isolation and characterization of fucose sulfate from jelly coat glycoprotein of sea urchin egg

Fucose sulfate was isolated from the egg jelly glycoproteins of two kinds of sea urchins, Hemicentrotus pulcherrimus and Pseudocentrotus depressus, by mild acid hydrolysis and paper chromatography followed by charcoal and Sephadex G-25 column chromatography. The yields of fucose sulfate were 24 and 20% of the total fucan sulfate from H. pulcherrimus and P. depressus, respectively. On the basis of chemical analysis, periodate oxidation and infrared spectroscopy, the structure of the fucose sulfate of the jelly coat glycoproteins derived from two kinds of sea urchins was proposed to be L-fucose-4-sulfate.

Induction of the acrosome reaction on the surface of de-jellied sea urchin eggs

Abstract The vitelline coat of sea urchin eggs was disrupted by DTT and trypsin after removal of the jelly layer. Thereafter the percentage of acrosome reaction was determined and the fertilization rate was estimated, employing the treated eggs. Electron microscopical investigation of these eggs showed that the vitelline coat was disrupted but no morphological difference was observed between eggs treated with DTT and those treated with trypsin. However, the fertilizability of the eggs was markedly decreased by the treatment with trypsin. In contrast, DTT treatment did not affect the fertilizability of the eggs, indicating that some surface substance(s) necessary for fertilization which were not eliminated by DTT were digested by trypsin. At the same time, the percentage of acrosome reaction of supernumerary spermatozoa in the presence of variously treated eggs was estimated as an index of the acrosome reaction-inducing activity of the egg surface. The acrosome reaction of spermatozoa actually occurred at the surface of de-jellied and DTT-treated eggs. However, the eggs treated with trypsin lost the capacity to induce the acrosome reaction. The surface substance which induces the acrosome reaction and renders the eggs fertile was removed by trypsin and found in the supernatant fraction. The necessity of an acrosome reaction for fertilization was demonstrated by the fact that the low fertilizability of trypsin-treated eggs was brought back to the control level by insemination with spermatozoa previously treated with egg water to evoke the reaction of the acrosomes.

Fractionation of jelly substance of the sea urchin egg and biological activities to induce acrosome reaction and agglutination of spermatozoa

Jelly coat substance surrounding the egg of the sea urchin, Hemicentrotus pulcherrimus, was fractionated by gel filtration and three fractions designated A, B, and C were obtained which mainly consisted of fucose sulfate, sialic acid, and protein, respectively. The biological activities of the fractions were examined for induction of acrosome reaction (AR) and agglutination of spermatozoa. Only fraction A, a fucose-rich glycoprotein fraction, had activities for both AR and agglutination. Fraction A was found to lose activity for AR but to retain activity for agglutination after pronase digestion. Pronase-digested fraction A was further fractionated by the same gel filtration and three fractions designated P1, P2, and P3 were obtained, which contained mainly fucose sulfate, sialic acid, and proteinous material, respectively. These fractions had no activity for AR but activity for agglutination resided in fraction P1, a fucose sulfate fraction. Furthermore, beta-elimination of the jelly substance was carried out to separate protein and fucose sulfate polysaccharide and three fractions designated E1, E2, and E3 were obtained by gel filtration, of which the fucose-rich fraction (E1) exhibited activities for both AR and agglutination, and the sialoprotein fraction (E2) retained activity only for AR. However, the activity for AR of both fractions was destroyed by pronase digestion. These results suggest that activity to induce AR resides in the protein moiety of fucose-rich glycoprotein and activity for agglutination resides in the fucose sulfate polysaccharide moiety of the same glycoprotein of the jelly substance.

Maternal Exogastrula‐Inducing Peptides (EGIPs) and Their Changes during Development in the Sea Urchin Anthocidaris crassispina

Exogastrula‐inducing activity was examined in eggs and embryos of the sea urchin Anthocidaris crassispina at various stages. During fractionation on a column of DEAE‐cellulose, the exogastrula‐inducing activity was found in the flow‐through fraction at all developmental stages. In particular, the activity present in the flow‐through fraction of unfertilized eggs represents the presence of maternal exogastrula‐inducing peptides (EGIPs). The flow‐through fractions from the column of DEAE‐cellulose were applied to a column of Sephadex G‐100 and the activities in the eluate were assayed. The active low‐molecular‐weight fraction was obtained in all cases with the exception of pluteus larvae, extracts of which contained another active fraction. Immunoblots of protein samples from eggs and embryos probed with antiserum against EGIP‐D indicated that there is a major immunoreactive protein that migrates with an apparent molecular weight of about 6 kDa in all cases with the exception of pluteus larvae, and that there are two major immunoreactive proteins that migrate with apparent molecular weights of 6 kDa and 35 kDa, respectively, in pluteus larvae.

Localization of an Exogastrula‐Inducing Peptide (EGIP) in Embryos of the Sea Urchin Anthocidaris crassispina

Exogastrula‐inducing peptides (EGIPs) are present in the unfertilized eggs and embryos of the sea urchin Anthocidaris crassispina. They induce exogastrulation when added exogenously to the embryos. The localization of EGIP‐D during embryogenesis has been explored using polyclonal antibodies against EGIP‐D. Immunofluorescent staining revealed that EGIP‐D is stored in the cytoplasm of immature oocytes and is concentrated into vesicles in unfertilized eggs. At fertilization, the vesicles containing EGIP‐D (EGIP‐vesicles) migrate to the cortical surface of the zygotes and are distributed in a ring‐like pattern at the apical surface of blastomeres, disappearing from basal surfaces and those adjacent to neighboring cells, during development from cleavage stages to larval stages. Mesenchyme cells also contain the vesicles but no such polarized distribution of vesicles is apparent. Acidic vesicles with a similar polarized distribution were examined by staining with acridine orange, which revealed that acidic vesicles were in close proximity to the surface of eggs at fertilization and were then distributed in a ring‐like pattern at the apical surface of blastomeres as are the EGIP‐vesicles. Furthermore, immunoelectron microscopy revealed that EGIP‐D is present in vesicles that are located at the apical surface of blastomeres. The significance of the localized distribution of EGIP‐D is discussed in relation to its function.