Presence of rRNA in the heavy bodies of sea urchin eggs: An in situ hybridization study with the electron microscope

Experimental conditions have been found, in which the presence of rRNA can be demonstrated by in situ hybridization at the electron microscope level in the heavy bodies of sea urchin eggs. The specificity of hybridization has been controlled by ribonuclease digestion and by competition experiments with unlabelled rRNA.     

Multiple molecular forms of acid nitrophenyl phosphatase in sea urchin eggs and embryos

Acid nitrophenyl phosphatases from sea urchin eggs and embryos were investigated by gel filtration. Four different forms were found in Hemicentrotus pulcherrimus, and three forms in Anthocidaris crassispina and Pseudocentrotus depressus. The first and second forms (designated AcP-1 and AcP-2) had the highest activity in the range of pH 5.6–6.0. The third (designated AcP-3) had an apparent optimum pH between pH 4.3 and 4.8, and the fourth (designated AcP-4) showed the maximum activity at pH 3.0. AcP-1 was much more thermolabile than AcP-2 and AcP-3 at 56°C. NaF inhibited AcP-2, AcP-3, and AcP-4 but not AcP-1. AcP-1, AcP-2, and AcP-3 were observed in the three species, whereas AcP-4 was not detected in A. crassispina and P. depressus. AcP-1, AcP-2, and AcP-3 were separted by gel filtration. AcP-1 and AcP-2 of A. crassispina and H. pulcherrimus were studied in developing embryos. The behavior of these forms in gel filtration changed during development, from unfertilized eggs to the pluteus stage.     

Two acid phosphatases in sea urchin eggs and embryos

• 1.1. Two types of acid phosphatases from sea urchin eggs and embryos were studied in three Japanese species, Hemicentrotus pulcherrimus, Anthocidaris crassispina and Pseudocentrotus depressus.
• 2.2. Acid phosphatase type 1, designated AcP-1, hydrolysed only flavin mononucleotide besides p-nitrophenylphosphate. The activity of AcP-1 was not inhibited by NaF and tartrate. This enzyme showed molecular weight between 14,000 and 16,000 by gel filtration through Sephadex G-75.
• 3.3. The higher molecular weight type of acid phosphatase, designated AcP-2, showed relatively high substrate specificity toward ADP and ATP. Molecular weight of AcP-2 ranged from 42,000 to 48,000 by gel filtration through Sephadex G-100.
• 4.4. Some properties of AcP-2 from Sphaerechinus granularis embryos are also described.

     

Hexokinase activity from eggs of the sea urchin Arbacia punctulata

1. The hexokinase activity of homogenates of eggs and embryos of the sea urchin Arbacia punctulata has been measured. Expressed as micrograms glucose consumed at 20°C., per hour per milligram of protein the following values were obtained: unfertilized eggs, 67; fertilized eggs, 72; 24 hour plutei, 94; 48 hour plutei, 226. The concentration of the enzyme in the eggs is small and may be calculated to be about 0.001 per cent of the dry weight of unfertilized eggs. 2. The hexokinase activity of the egg homogenate was virtually all recovered in the supernatant fraction when the homogenate was centrifuged at 20,000 x g for 30 minutes and was found to have the following properties: The concentrations for half maximal hexokinase activity with various substrates were, approximately: Glucose, 0,00003 M; fructose, 0.00075; mannose, 0.00007; 2-desoxyglucose, 0.00025. The relative rates of phosphorylation of various sugars by the supernate fraction when saturated with substrate were, approximately: Glucose, 1.0; mannose, 1.2; fructose, 1.8; 2-desoxyglucose, 2.0; glucosamine, 0.6. Adenosinediphosphate and glucose-6-phosphate inhibited the enzyme. No evidence for more than one hexokinase in the Arbacia extracts was found.     

Dynein isoforms in sea urchin eggs

Biochemical and immunological analysis of unfertilized sea urchin eggs has revealed the presence of at least two distinct isoforms of cytoplasmic dyneins, one soluble and the other microtubule-associated. The soluble enzyme is a 20 S particle with a MgATPase activity that can be activated 5-fold by nonionic detergents. It contains heavy chain polypeptides that 1) comigrate with the dynein heavy chains of embryonic cilia; 2) cross-react with antibodies against flagellar dynein; and 3) are cleaved by UV irradiation in the presence of MgATP and sodium vanadate into specific peptide fragments. The soluble egg dynein is, therefore, closely related to axonemal dynein and may be a ciliary precursor. Egg microtubule preparations contain a distinct dynein-like polypeptide, previously designated HMr-3 (Scholey, J.M., Neighbors, B., McIntosh, J.R., and Salmon, E.D. (1984) J. Biol Chem. 259, 6516-6525). HMr-3 binds microtubules in an ATP-sensitive fashion; it sediments at 20 S on sucrose density gradients, and it is susceptible to vanadate-sensitized UV cleavage. However, HMr-3 can be distinguished from the soluble cytoplasmic dynein on the basis of its weak cross-reactivity with antiflagellar dynein antibodies, its heavy chain composition on high resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis, its low specific ATPase activity, and the molecular weight of its vanadate-induced UV cleavage fragments. HMr-3 may represent a dynein-like polypeptide that is distinct from the pool of ciliary dynein precursors.     

Action of colcemid in sea urchin eggs

The effects of Colcemid, the deacetyl-N-methyl derivative of colchicine, on the eggs of Arbacia punctulata were investigated. Colcemid in concentrations of 2.7 x 10^-5 M or greater blocks syngamy (the fusion of the pronuclei) in these eggs. Although a tenfold decrease in concentration of Colcemid usually permits the pronuclei to fuse, the subsequent division is blocked. In the sea urchin egg, the duration of presyngamy is about 15 min during which time there is no DNA synthesis. However, DNA synthesis is recorded in Colcemid-blocked cells prior to syngamy. Radioautographs of Colcemid-blocked cells which were immersed into thymidine-³H exhibited silver grains above each of the pronuclei. The action of Colcemid on Arbacia eggs is reversible. Nevertheless, exposures to 2.7 x 10^-5 M Colcemid for only 3 min, initiated 5 min after insemination, caused delays of 70 min in subsequent division. In general, cells are more sensitive to Colcemid prior to the time when the mitotic spindle is being assembled than at presyngamy stages. The results are discussed in terms of Colcemid action on pronuclear fusion and cell division.     

Ribosomal proteins of sea urchin eggs

Summary Ribosomal proteins from unfertilized eggs of three sea urchin species, Pseudocentrotus depressus, Hemicentrotus pulcherrimus, and Anthocidaris crassispina, were analyzed. Species-specific differences were observed in the profiles of large subunit proteins on two-dimensional slab gels, though the number of ribosomal proteins and the molecular weights of their counterparts were the same. The small subunit proteins revealed similarities in the electrophoretic profiles and in the phosphorylation patterns among these three species.     

Fertilization kinetics of sea urchin eggs

Eggs and sperm of the sea urchin Paracentrotus lividus of the Mediterranean are used for an in vitro study of fertilization kinetics. The results are analyzed in terms of two models. One of these models assumes that all sperm-egg encounters lead to permanent attachment; the other (less realistically) assumes that sperm continue their random search after an unsuccessful encounter. More than 100 spermatozoa per egg are needed to achieve a fertilization ratio of more than 95%. There are two explanations for this: only 1% of the egg surface is subject to fertilization, or only 1% of spermatozoa are intrinsically able to fertilize. In the same context, chemotactic attraction and the role of the jelly are discussed. Comparison with earlier work of Rothschild and Swann and of Hultin and Hagström clarifies some discrepancies between and within these papers.     

Localization of tropomyosin in sea urchin eggs

Localization of tropomyosin in sea urchin eggs was investigated immunohistochemically. A rabbit antiserum against tropomyosin prepared from lantern muscle of the sea urchin was used for the indirect immunofluorescence staining of unfertilized and fertilized eggs. The tropomyosin-specific fluorescence was observed at the peripheral region beneath the plasma membrane, mitotic apparatus and contractile ring. The mitotic apparatus isolated from sea urchin eggs was also stained with the anti-tropomyosin serum.     

Peroxidatic activity of catalase in the cortical granules of sea urchin eggs

The presence of peroxidatic activity of catalase in eggs of the sea urchins Hemicentrotus pulcherrimus and Temnopleurus toreumaticus was investigated by the ultrastructural cytochemical techniue and by biochemical assay on homogenates of eggs from before fertilization to the 2-cell stage. Biochemical assays showed that the unfertilized eggs had strong catalase activity whereas fertilized eggs had weak activity owing to the rapid decrease of activity after fertilization. The activity did not change from immediately after fertilization to the 2-cell stage. Cytochemical examination showed that the peroxidatic activity of catalase was mainly localized in the lamellae in the cortical granules. Disintegrated cortical granules with no lamellae and substances in the perivitelline space derived from breakdown of the cortical granules had no peroxidatic activity of catalase.     

RNA synthesis in unfertilized sea urchin eggs

We have examined the synthesis of messenger-like RNA in unfertilized sea urchin eggs. Most of the RNA synthesized is restricted to the nucleus and sediments from 16 to 30S. A small fraction can be isolated from the postmitochondrial supernatant and displays a sedimentation profile typical of embryonic mRNA with peaks at 9 and 18S. This cytoplasmic RNA is largely present as free RNPs and we estimate that less than 20% of the RNA is in polysomes. The RNA made in the egg is unstable and reaches a steady state with a half-time of about 30 min. We have examined the accumulation of RNA in the egg and have calculated a rate of synthesis of 1.4 × 10^?14 g of RNA/min/egg which is similar, on a per-nucleus basis, to that found in the just-fertilized egg and very early embryo. It is approximately 10 times greater than the rate of RNA synthesis in the blastula nucleus. We estimate that the RNA synthesized by the unfertilized egg amounts to a maximum of 3 × 10^?13 g of potential mRNA at the time of fertilization, or 10–15% of its immediate needs. This RNA cannot account for the increase in protein synthesis that occurs after fertilization, which must be the result of the translation of another population of more stable egg or oogenic mRNA that is kinetically distinct from the RNA we have measured. The steady-state level of labeled RNA present in the egg does not change upon fertilization until after the first cleavage, at about 2.5 hr after fertilization. Thus the RNA synthesis that occurs in the just-fertilized zygote appears to be merely a continuation (at least quantitatively) of the RNA synthesis taking place in the egg.