Benzohydroxamic acid induces polyspermic fertilization in the sea urchin Arbacia punctulata

Benzohydroxamic acid (BHA) is a competitive inhibitor of the sea urchin sperm peroxidase. We now report that addition of BHA to fertilization cultures of Arbacia punctulata promotes polyspermy. This effect is dose and sperm density dependent. The cortical reaction (elevation of the fertilization envelope) is not retarded by BHA. BHA must be added to the cultures before the eggs complete the cortical reaction at 60 sec post insemination in order to induce polyspermy. Since sea urchin eggs release H2O2 during the cortical reaction at fertilization, these findings support our hypothesis that the sperm peroxidase has a functional role in helping to prevent polyspermy.     

Insemination of immature sea urchin (Arbacia punctulata) eggs

Nuclei from osmotically opened erythrocytes and erythroblasts were injected into nucleated or enucleated Xenopus laevis eggs. Although the cleavage pattern of the recipient eggs which started to divide was normal in about half of the cases, nuclei from erythrocytes injected into nucleated or enucleated eggs never promoted development beyond the early gastrula stage. In contrast, nuclei from osmotically opened erythroblasts injected into enucleated eggs promoted development to early tadpole stages (stages 29–36). Frequently, injection of osmotically broken erythroblasts injected into nonenucleated eggs gave rise to triploid larvae which all died at roughly the same early tadpole stages (29–36). Surprisingly, development did not proceed to the stage of advanced organogenesis (stages 44–47), which is easily reached by gynogenetic haploids: The presence of the haploid genome derived from the egg pronucleus did not significantly improve the developmental capacity. Embryos obtained by single injection of erythrocyte nuclei into nucleated eggs were unable to pass the gastrula stage. To invalidate the interpretation that the observed arrest in development was related to nuclear damage during injection of the recipient eggs, single unbroken erythrocytes and unbroken erythroblasts were transferred into nucleated and enucleated eggs. No cleavage was observed in both classes of eggs injected with unbroken erythrocytes. In contrast, erythroblasts were found to induce cleavage in the recipient eggs at a frequency of about 11%. To ascertain that the nucleus of unbroken erythroblasts participated in development, the 1-nucleolar marker was used. Diploid embryos with only one nucleolus present were found following injection of unbroken erythroblasts into enucleated eggs from 2nu females. Triploid 2nu embryos were detected following injection of (diploid) 1nu erythroblasts into nonenucleated eggs from 2nu females. The most advanced development stages reached by these embryos did not, however, differ from the best results found in the first class of experiments: Nuclei from erythroblasts injected undamaged into nucleated or enucleated eggs never developed into a normal tadpole. Serial transfer experiments were performed using normally gastrulating embryos which had developed, following the injection of 1nu unbroken erythroblasts into recipient eggs. These donors for serial transfer experiments were checked for the presence of the 1nu marker. In addition they had passed through a normally cleaving eight-cell stage. No improvement in developmental capacity as compared to first transfer experiments could be found.     

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.     

Characterization of yolk platelets isolated from developing embryos of Arbacia punctulata

Yolk platelets, a major organelle of sea urchin eggs and embryos, were isolated from Arbacia punctulata and biochemically characterized over the course of development to the pluteus stage. Fractionation by sucrose gradient centrifugation revealed yolk platelets in two major density classes. The low-density yolk platelet fraction could be obtained as a very homogeneous preparation and was highly enriched in acid phosphatase activity, while depleted of mitochondrial (cytochrome c oxidase) and plasma membrane (phosphodiesterase) marker enzymes. The chemical composition of low-density yolk platelets prepared from eggs and embryos at various stages of development remained unchanged in terms of phospholipid, triglyceride, hexose, sialic acid, RNA, and protein. However, analysis of the major yolk platelet glycoproteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a number of stage-specific changes. These glycoproteins were found to be major glycoproteins of crude embryo lysates and were predominantly of the polymannose, N-linked type. The predominance of polymannose-type glycoproteins in yolk platelets was further demonstrated by their staining with concanavalin A-colloidal gold in Lowicryl-embedded sections of embryos. These studies represent the first systematic biochemical characterization of intact yolk platelets and the changes in them during early embryonic development.