Sea Urchin Zygotes Research Articles

A possible bio-assay for chromosome movement in isolated mitotic apparatus

Abstract We isolated mitotic apparatus (MA) from sea urchin zygotes using several methods. The isolated MA were injected into nucleated frog eggs or into enucleated frog eggs, and 24 h later we determined whether or not the eggs had undergone normal cleavage. Normal cleavage occurred in about 50% of the cases when MA isolated in glycerol-dimethyl sulphoxide were injected into either nucleated or enucleated frog eggs. Likewise for MA isolated initially in hexylene glycol and transferred immediately into glycerol-dimethyl sulphoxide. No cleavage occurred when MA isolated in hexylene glycol (and stored in hexylene glycol) were injected into frog eggs. We discuss two possible interpretations of the results. In one interpretation cleavage of the frog eggs is a bioassay, measuring the ability of the isolated MA to support chromosome movement. In the other interpretation the isolated MA contribute only nuclear material and cleavage initiation factors, and there is no chromosome movement in the isolated MA per se.

Spindle birefringence of isolated mitotic apparatus: further evidence for two birefringent spindle components.

We studied sea-urchin zygote mitotic apparatus (MA) isolated in hexylene glycol, transferred immediately to a glycerol-dimethylsulphoxide medium, and stored for 2 weeks at room temperature. Treatment with 0-5 M KC1 caused loss of 45% of the birefringence, but microtubules remained intact (as seen electron microscopically in glutaraldehyde-fixed MA), and tubulin was not extracted (as determined by polyacrylamide gel electrophoresis). These results suggest that a non-tubulin component which is extracted by the KC1 contributes 45% of the MA birefringence. Further evidence for this conclusion came from indirect immunofluorescence experiments. Non-extracted (control) MA were fixed with formaldehyde and reacted with antibody against tubulin; there was intense staining of the spindle fibres and astral rays. Electron microscopically, however, microtubules were not present in formaldehyde-fixed MA. Since formaldehyde fixation caused breakdown of microtubules but the tubulin remained in the MA (as judged by reaction with antibodies) we suggest that after microtubule breakdown the tubulin remains in the MA because it is bound to a peri-microtubule spindle component (which we call 'substance gamma'). When KCl-extracted MA were fixed with formaldehyde and reacted with antibody against tubulin there was very little staining of spindle fibres and astral rays. Electron microscopically, formaldehyde caused microtubule breakdown, and since the tubulin is lost from formaldehydefixed, KC1-extracted MA (as judged by reaction with antibodies), we suggest that the tubulin-binding component, substance gamma, is extracted by the 0-5 M KC1. Pressure treatment caused the asters not to stain with antibody against tubulin, suggesting that the stability of substance gamma is different in different regions of the mitotic apparatus.

Spindle birefringence of isolated mitotic apparatus analysed by treatments with cold, pressure, and diluted isolation medium.

Mitotic apparatus (MA) were isolated from sea-urchin zygotes using glycerol-dimethyl-sulphoxide. Cold treatment had no effect on MA birefringence when MA were in isolation medium, but caused a 10-15% reduction of MA birefringence when MA were in quarter-strength isolation medium. Pressure treatment also caused a reduction in MA birefringence, but the cold and pressure treatments were not additive, suggesting that both treatments affected the same MA component. MA were not stable in quarter-strength isolation medium, and birefringence gradually decayed, with a half-life of about 40 h. Electron microscopy after cold treatment, or after decay of 55% of the MA birefringence showed abundant, normal-looking microtubules, suggesting that alterations in non-microtubule components cause the reductions in birefringence. Addition of EGTA eliminates the effect of cold treatment, suggesting that Ca2+ has a role in maintenance of spindle structure. We discuss possible reasons why isolated MA do not respond to cold treatment like MA in vivo.

Spindle birefringence of isolated mitotic apparatus analysed by pressure treatment.

Sea-urchin zygote mitotic apparatus (MA) isolated in a glycerol/dimethylsulphoxide medium were treated with pressure. Pressure treatment had no effect on spindle birefringence when MA were in full-strength isolation medium. After placing MA in quarter-strength isolation medium, pressures of 4-0 X 10(3)-1-8 X 10(4) lbf in.-2 (2 X 76 X 10(4)-I X 24 X 10(5) k N m-2) for 15 min caused reduction of birefringence which occurred in 2 steps: firstly 20-30% of the birefringence was lost, and then, at higher pressures, the rest of the birefringence was lost. Electron microscopy suggested that pressure-induced changes were in non-microtubule material. Pressure treatment had no effect on MA isolated with hexylene glycol when the MA were pressurized in hexylene glycol; but pressure treatment did cause loss of birefringence when MA isolated in hexylene glycol were transferred immediately into glycerol/dimethylsulphoxide medium and were subsequently treated with pressure (after dilution into quarter-strength glycerol/dimethyl-sulphoxide). We discuss the differences in response between isolated MA and in vivo MA, and we discuss the possibility that 2 components contribute to MA birefringence.

The concentrations of dry matter in mitotic apparatuses in vivo and after isolation from sea-urchin zygotes.

ABSTRACT We have measured the concentrations of dry matter in mitotic apparatuses (MA) in vivo and after isolation from the same cell type. The isolation medium was hexylene glycol plus buffer. The MA were from sea-urchin zygotes (Echinus esculentus Linn, and Psammechinus miliaris Gmelin), and measurements were made using interference microscopy. MA as isolated have much lower concentrations of dry matter than do MA in vivo. The dry mass concentrations of isolated MA vary with the pH of the isolation medium, ranging from about 20 % of the in vivo concentration (at pH 7·3) to about 60 % of the in vivo concentration (atpH5·3). The isolated MA were further characterized. Evidence is presented which suggests that non-specific cytoplasmic material adsorbs to MA, and thus that at least some of the material in isolated MA is not derived from in vivo MA. Some MA components are apparently changed during the isolation procedure: MA lysed in low pH (high mass) medium and quickly transferred to high pH (low mass) medium have higher concentrations of dry matter than do MA lysed in high pH medium. The isolation media as generally used do not have enough buffering capacity: the pH changes after the isolation. These data suggest that the isolation procedures need be improved before studies of isolated MA can give data relevant to the chemistry of in vivo MA. We discuss the problem of obtaining functional isolated MA, and also the relevance of our data to previous work on MA from other species.

Tubulin synthesis in fertilized sea urchin eggs

Sea urchin zygotes incorporate 14C-amino acids into tubulin (microtubule protein). The rate of tubulin synthesis increases after fertilization, but shows a pronounced depression during cell division. The amount of 3H-colchicine binding material in 15 000 g supernatants fluctuates in the same way.

Active polysomes in sea urchin eggs and zygotes: evidence for an increase in translatable messenger RNA after fertilization.

AbstractThe increase in polysomal material in sea urchin zygotes after fertilization occurs primarily as an increase in the number rather than in the sizes of the polysomes. This suggests that the protein synthesis system in eggs is messenger RNA limited and that polysomes form at fertilization as a result of the messenger RNA becoming available. While these findings do not rule out the possibility that the peptide initiation step is less efficient in eggs than in embryos, they do suggest that this step is not the principal cause of the inactivity of the protein synthesis system in eggs.