Hironobu Ozaki

A glycoprotein in the accessory cell of the echinoid ovary and its role in vitellogenesis

SummaryA high-molecular-weight glycoprotein with a sedimentation coefficient of 22.6 has been isolated and characterized from the accessory cells in the previtellogenic ovary of the echinoid Dendraster excentricus. This glycoprotein is similar to the major yolk glycoprotein of the mature egg in its electrophoretic mobility under non-denaturing conditions, high mannose-type glycan, amino acid composition, constitutive glycopeptides, and immunological determinants. Previous histological and electron microscopical analyses led to the hypothesis that vitellogenesis involves a translocation of material from the accessory cell in the ovary to the oocyte. Because of the close similarities of the accessory cell glycoprotein to the yolk glycoprotein of the mature egg, we conclude that the glycoprotein in the accessory cell is a precursor to the major glycoprotein of the egg yolk. This conclusion is further supported by our additional finding that the accessory cell of another echinoid, Strongylocentrotus purpuratus, also contains a high-molecular-weight (24 S) glycoprotein which shows similarities to the yolk glycoprotein of the mature egg in the carbohydrate moiety and the constitutive glycopeptides.

MOLECULAR PROPERTIES AND DIFFERENTIATION OF ACETYLCHOLINESTERASE IN SEA URCHIN EMBRYOS

The two molecular forms of acethylcholinesterase (EC 3.1.1.7) in sea urchin embryos were characterized by several physical methods. The sedimentation coefficients determined by sucrose gradient centrifugation are 7.6S and 10.6S. The Stokes radii determined by gel filtration are 65 Å and 91 Å. From these parameters, molecular weights were estimated as 190,000 and 380,000; the one is twice as large as the other. Both forms have similar electric property and buoyant density in a CsCl gradient. When the enzyme solution was concentrated, the 10.6S form became predominant. These results suggest that the two forms are monomer and dimer. The sea urchin enzymes resemble globular forms of acetylcholinesterase of the electric organ of fishes.

Changes in the histones of the sea urchin Strongylocentrotus purpuratus at fertilization

Abstract Both sperm and eggs of the sea urchin Strongylocentrotus purpuratus contain specific histones in place of some of the histones found during later development. Whether these specific histones are lost upon fertilization or are retained is not known. Therefore, we have examined the histones present in the zygote nucleus to determine the fate of the gamete histones. Nuclei of zygotes which have completed DNA replication in preparation for the first mitosis were isolated by sucrose density gradient centrifugation. Histones were extracted from the isolated nuclei, and were analyzed by acid-urea and SDS polyacrylamide gel electrophoreses, and by two-dimensional electrophoresis in which both gel electrophoresis systems were combined. Electrophoretic patterns of the zygote histones were compared with those of sperm, unfertilized eggs and embryos. The results show that the zygote histone pattern is identical with the unfertilized egg histone pattern. Neither the sperm histones H1, H2A, or H2B, nor the embryonic H1, H2A, or H2B, are present in the zygote pattern. The egg and the zygote do contain a unique H2A and H2B, but not an H1. After fertilization, sperm specific histones are not present on the DNA. Egg histones become associated with both the sperm DNA and the newly replicated DNA. The association of the embryonic histones with the DNA, therefore, occurs sometime later in development.

Developmental studies of sea urchin chromatin. Chromatin isolated from spermatozoa of the sea urchin Strongylocentrotus purpuratus.

Abstract Chromatin was isolated from spermatozoa of the sea urchin Strongylocentrotus purpuratus. The isolated chromatin shows less absorptivity ratio of 230 nm : 260 nm and possesses less protein than does embryonic chromatin. The ratio of histone : DNA is 1.02; nonhistone : DNA 0.13; RNA : DNA 0.04. Sperm chromatin melts in two steps with Tms 70°C and 84°C in 2.5 × 10−4, M EDTA in contrast to embryonic chromatin with a single Tm = 72°C. Disc electrophoresis of basic proteins of sperm revealed one minor component with extremely fast mobility and three major components. The one with the slowest mobility is characteristic of sperm. The embryo has in turn its characteristic histone which also migrated slowly in disc electrophoresis. Both of these unique histone fractions are selectively extracted from chromatin by 5% perchloric acid. Amino acid analyses of these chromatographically purified unique fractions show that both contain a large amount of lysine, while that from sperm, in addition, contains also a large amount of arginine. Minimal molecular weights of 33,000 for sperm and 16,200 for embryo unique histone were estimated from these analyses. Sperm chromatin supports a level of RNA synthesis in vitro with exogeneously supplied RNA polymerase about 2% that of the corresponding free DNA.

The effect of estrogen on protein synthesis in echinoid coelomocytes

Abstract 1. 1. A similar high molecular weight glycoprotein is found in an asteroid egg, Pisaster giganteus , and in echinoid eggs, Dendraster excentricus and Strongylocentrotus purpuratus . 2. 2. Estrogen induced the synthesis of a novel protein in echinoid coelomocytes, 82 K daltons in D. excentricus and 78 K daltons in S. purpuratus ; but it did not increase yolk glycoprotein precursor synthesis nor protein synthesis as a whole in these cells during treatment up to 24 hr. However, the induced novel protein may ultimately lead to increased transcription of the yolk glycoprotein precursor gene. Estrogen therefore could be involved in the regulation of reproduction in echinoids, as it is in asteroids and vertebrates.

YOLK PROTEINS OF THE SAND DOLLAR DENDRASTER EXCENTRICUS

Yolk granules were isolated from eggs of the sand dollar Dendraster excentricus by density gradient centrifugation. Isolated granules were homogenous by the electron microscopic criterion. Proteins contained in the granules were analyzed by polyacrylamide gel electrophoresis under both denaturing and native conditions. SDS-polyacrylamide gel electrophoresis resolved the proteins into 3 size classes of polypeptides: molecular weights over 90,000, around 40,000, and around 30,000. PAS reagent stained only those polypeptides present in the size class with the highest molecular weights. A histochemical test for phosphoprotein was negative with any of the polypeptides. Triton X-100 gel electrophoresis revealed that proteins in the yolk granules fundamentally consisted one large molecular weight glycoprotein and a group of small proteins. Two-dimensional electrophoresis has shown that the glycoprotein is an oligomer consisting of glycoprotein subunits, and that all small proteins are monomers. The glycoprotein and small proteins may form lipoprotein complexes in the yolk granules.

Temporal pattern of RNA synthesis in animalized sea urchin embryos

Abstract The kinetics of 3H-uridine incorporation has been studied in sea urchin embryos developing normally and in the presence of concentrations of zinc which strongly animalize whole embryos. Cleavage stage embryos of both groups incorporate uridine at a low rate which increases after hatching in the controls and begins to level off at the prism stage. This acceleration is delayed by about 6 h in the animalized embryos but the same rate is achieved by these during later development. This difference is not due to any differential permeability to precursor, nor can it be correlated with a difference in cell number between control and animalized embryos. The results are discussed in terms of the knowledge concerning the control of sea urchin embryogenesis.

Nuclear histones of unfertilized sea urchin eggs

Abstract Histones have been isolated from the nuclei of unfertilized eggs of the sea urchin Strongylocentrotus purpuratus . The electrophoresis of these histones exhibits a pattern different from that of the sperm or embryo of the same species.

A comparison of protein synthetic patterns in normal and animalized sea urchin embryos

Abstract Newly synthesized proteins from normal and animalized sea urchin embryos were compared by the technique of double labeling. Total embryonic protein was solubilized in SDS, urea, and 2-mercaptoethanol. The proteins were examined by coelectrophoresis on an SDS-polyacrylamide gel. The gels were sliced and the radioactivity determined. A standardized ratio of the isotopes served as the basis of comparison. A comparison of newly synthesized proteins from normal embryos 24 and 48 h old showed a shift from larger to smaller molecular weight proteins. Animalized embryos showed a similar shift. When normal and animalized embryos of the same ages were compared, differences were found. The differences were distributed over the entire range of molecular weights. These results show that although differences in protein synthesis between animalized and normal embryos are evident by 24 h, most of the changes in protein synthesis that occur in normal embryos are unaffected by animalization.

RNA complementary to unique DNA sequences in normal and animalized sea urchin embryos

Abstract Early sea urchin development can be experimentally manipulated so that abnormal embryos, called animalized embryos, develop with exaggeration of their ectodermal characteristics and suppression of their mesentodermal characteristics. In order to test whether this alteration in development involves changes in the pattern of embryonic gene transcription, embryonic RNA was examined by RNA/DNA hybridization. Rapidly labelled RNA from both types of embryo hybridized to non-repetitive DNA readily, but much less readily to repetitive DNA. In order to determine the complexity of the RNA in normal and animalized embryos, purified radioactive non-repetitive DNA was incubated with a large excess of RNA. RNA was isolated from unfertilized eggs and from normal blastulae and prism larvae and from animalized embryos of comparable ages. The complexity of transcription increases during the development of both normal and animalized embryos. Experiments in which RNAs isolated from two stages were combined indicated that extensive homology exists between the populations, although some differences were detected between embryos of different ages as well as between normal and animalized embryos of the same age. This evidence indicates that animalization involves alterations in the pattern of embryonic gene expression and that this abnormal development provides a convenient experimental system for the study of gene regulation in embryonic development.