Characteristics of Sea-Urchin Mitotic Apparatus Isolated Using a Dimethyl Sulphoxide/Glycerol Medium

1974 ◽  
Vol 16 (3) ◽  
pp. 481-497
Author(s):  
A. FORER ◽  
A. M. ZIMMERMAN
Keyword(s):  
Sea Urchin ◽  
Phosphate Buffer ◽  
Dry Matter ◽  
Room Temperature ◽  
Dimethyl Sulphoxide ◽  
Mitotic Apparatus ◽  
Isolation Medium ◽  
Hexylene Glycol ◽  
Brain Microtubules ◽  
Glycerol Medium

A method for isolating sea-urchin zygote mitotic apparatus (MA) is described which is based on the Filner-Behnke method of isolating brain microtubules. MA were isolated in 50% (v/v) glycerol, 10%(v/v) dimethyl sulphoxide, 5 mM MgCl2, 0.1 mM EGTA, and 5 mM Sørensen's phosphate buffer at a final pH of 6.8. MA stored at room temperature in isolation medium had stable birefringence, stable microtubules and stable solubility properties (in 0.5 M KCl) over a period of 10 days to 2 weeks. These MA also seem to have more dry matter per volume than do MA isolated using hexylene glycol. The biggest disadvantages of the method are that zygotes often are difficult to lyse, and that cytoplasmic debris the same size as the MA sometimes contaminates the MA pellet.

Spindle birefringence of isolated mitotic apparatus analysed by pressure treatment

1976 ◽  
Vol 20 (2) ◽  
pp. 309-327
Author(s):  
A. Forer ◽  
A.M. Zimmerman
Keyword(s):  
Electron Microscopy ◽  
Sea Urchin ◽  
Dimethyl Sulphoxide ◽  
Pressure Treatment ◽  
Mitotic Apparatus ◽  
Isolation Medium ◽  
Hexylene Glycol ◽  
X 24 ◽  
Induced Changes

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.

Induction of cleavage in nucleated and enucleated frog eggs by injection of isolated sea-urchin mitotic apparatus

1978 ◽  
Vol 31 (1) ◽  
pp. 117-135
Author(s):  
Y. Masui ◽  
A. Forer ◽  
A.M. Zimmerman
Keyword(s):  
Sea Urchin ◽  
Room Temperature ◽  
Rana Pipiens ◽  
Sea Urchins ◽  
Cooperative Interaction ◽  
Nuclear Materials ◽  
Cytological Examination ◽  
Mitotic Apparatus ◽  
Frog Brain ◽  
Hexylene Glycol

Mitotic apparatus (MA) were isolated in glycerol-dimethylsulphoxide solution (MTME) from zygotes of sea urchins (Stronglyocentrotus purpuratus). Freshly isolated MA were stored in 1/10 strength MTME for varying periods of time and were then injected into unfertilized frog (Rana pipiens) eggs. These injections induced 40–60% of the recipient frog eggs to initiate cleavage, resulting in the formation of blastula cell clusters. The cleavage-inducing activity of MA stored in 1/10 MTME at room temperature decreased with time of storage in 1/10 strength MTME, and disappeared by about 6 h. There was no change in the ultrastructure of MA during storage. MA isolated and stored in MTME at room temperature had a constant level of cleavage-inducing activity during the first 48 h of storage, but this activity slowly declined upon further storage; almost no activity was left after 3 weeks. MA isolated in hexylene glycol (HG) and immediately transferred into MTME were compared with MA isolated in MTME; both MA had the same cleavage-inducing activity on the day of isolation, after which the MA isolated in HG quickly lost activity. On the other hand, MA isolated and stored in HG had little cleavage-inducing activity when tested 3 h following isolation. Cleavage-inducing agent (CIA) isolated from frog brains induced cleavage and blastula formation when injected into nucleated frog eggs, but had no such activity when injected into enucleated frog eggs. MA isolated in MTME induced cleavage and blastula formation in enucleated frog eggs as well as in nucleated frog eggs. Cytological examination revealed that blastula cells which developed from MA-injected enucleated eggs contained Feulgennegative nuclei, whereas cells which developed from CIA-injected nucleated eggs contained Feulgen-positive nuclei. These results suggest that sea-urchin nuclear materials participate in mitosis in frog eggs. Isolated MA which had been stored in MTME for 3 weeks and which exhibited little cleavage-inducing activity were injected together with frog brain CIA into either normal or enucleated eggs; normal recipient eggs cleaved with significantly higher frequencies (70%) than those injected with CIA alone (40%). Furthermore, enucleated eggs injected with CIA alone failed to cleave, while those injected with MA and CIA together cleaved with significant frequencies (overall 29%). This result suggests a cooperative interaction between CIA and the inactivated MA to restore the cleavage-inducing activity of MA.

Experimental determinations of tubulin in the in vivo mitotic apparatus of sea urchin zygotes

1980 ◽  
Vol 58 (11) ◽  
pp. 1277-1285 ◽  
Author(s):  
Arthur Forer ◽  
D. E. Larson ◽  
A. M. Zimmerman
Keyword(s):  
Sea Urchin ◽  
Gel Electrophoresis ◽  
Dry Matter ◽  
Strongylocentrotus Purpuratus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mitotic Apparatus ◽  
Cell Lysate ◽  
Hexylene Glycol

Mitotic apparatus (MA) were isolated from zygotes of a sea urchin (Strongylocentrotus purpuratus), using hexylene glycol (pH 6.4) as lysing–stabilizing agent. Protein was measured in the MA pellet and in the remainder of the cell lysate (using the Lowry procedure). Tubulin was measured in the MA pellet and in the remainder of the cell lysate (using microdensitometry of stained gels after sodium dodecyl sulphate – polyacrylamide gel electrophoresis). From these data we calculated the maximum possible amounts of tubulin in the isolated MA and in the MA in vivo; in these calculations we assumed that all the tubulin in the cell is associated with the MA, and we assumed that, as reported in the literature, the MA lose 90% of their dry matter during the isolation. We conclude that tubulin probably comprises less than 7% of the protein in the in vivo MA, and, even if there are very large errors, tubulin is considerably less than haf the protein in the MA.

The Concentrations of Dry Matter in Mitotic Apparatuses in Vivo and After Isolation From Sea-Urchin Zygotes

1972 ◽  
Vol 10 (2) ◽  
pp. 387-418
Author(s):  
A. FORER ◽  
R. D. GOLDMAN
Keyword(s):  
Sea Urchin ◽  
Dry Matter ◽  
Mass Medium ◽  
Dry Mass ◽  
Interference Microscopy ◽  
High Mass ◽  
Isolation Medium ◽  
High Ph ◽  
Cytoplasmic Material

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 (at pH 5.3). 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.

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

1976 ◽  
Vol 20 (2) ◽  
pp. 329-339
Author(s):  
A. Forer ◽  
A.M. Zimmerman
Keyword(s):  
Electron Microscopy ◽  
Sea Urchin ◽  
Half Life ◽  
Cold Treatment ◽  
Pressure Treatment ◽  
Mitotic Apparatus ◽  
Isolation Medium ◽  
Spindle Structure ◽  
Cold Pressure

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.

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

1976 ◽  
Vol 22 (1) ◽  
pp. 115-131
Author(s):  
A. Forer ◽  
V.I. Kalnins ◽  
A.M. Zimmerman
Keyword(s):  
Room Temperature ◽  
Polyacrylamide Gel Electrophoresis ◽  
Temperature Treatment ◽  
Mitotic Apparatus ◽  
Intense Staining ◽  
Hexylene Glycol ◽  
Tubulin Binding ◽  
The Stability ◽  
Spindle Fibres ◽  
Binding Component

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.

scholarly journals THE MITOTIC APPARATUS

1962 ◽  
Vol 15 (2) ◽  
pp. 279-287 ◽  
Author(s):  
R. E. Kane
Keyword(s):  
Fine Structure ◽  
Granular Material ◽  
Sea Urchin ◽  
Osmium Tetroxide ◽  
Mitotic Apparatus ◽  
Isolation Medium ◽  
Sea Urchin Egg ◽  
Dividing Cells

The fine structure of the mitotic apparatus isolated from the sea urchin egg has been investigated. The isolation was accomplished by lysis of metaphase eggs in a 1 M solution of hexanediol, buffered at pH 6. The fine structure of the isolated apparatus was studied after fixation with osmium tetroxide directly in the isolation medium. The spindle is composed of fine fibrils, approximately 20 mµ in diameter, which appear tubular. Similar fibrils, radially oriented, are found in the aster. If the isolated mitotic apparatus is exposed to water at pH 6 before fixation, the structure is considerably modified. The most pronounced effects are an increase in the number of large membrane-bounded vesicles and in the amount of free granular material present. The conditions necessary for the fixation of the mitotic apparatus in dividing cells are discussed in the light of these observations on the isolated unit.

Fine structural morphology of immunocytes of some molluscs and insects

1990 ◽  
Vol 48 (3) ◽  
pp. 386-387
Author(s):  
S.G. Pal ◽  
G. Baur ◽  
B. Ghosh ◽  
S. Palit ◽  
S. Modak ◽  
...  
Keyword(s):  
Blood Cells ◽  
Phosphate Buffer ◽  
Room Temperature ◽  
Structural Information ◽  
Uranyl Acetate ◽  
Meretrix Meretrix ◽  
Lead Citrate ◽  
Bivalve Molluscs ◽  
Structural Morphology ◽  
Ultrathin Sections

In recent years some of the blood cells of several molluscs and insects are characterised as immunocytes. Similar cells from a few invertebrates from India have been looked into under conventional TEM to register the ultrastructural features. This type of study is first of its kind in the subcontinent. Immunocytes from bivalve molluscs Meretrix meretrix, Laroellidens marqinalis and two insect species, apterygote Ctenolepism a longicaudata and pterygote Gesonula punctifrons provide a new set of fine structural information which forms a basis of comparison with those studied earlier.Immunocytes have been collected from the fresh live species of bivalve molluscs and insects obtained locally at Calcutta. These were fixed in icecold 2% glutaraldehyde in 0.1M phosphate buffer (pH 7.2-7.4) for 1-2 hours at 4-5°C. Subseguently pellets were post-osmicated in 1% OsO4 at room temperature for 1-2 hours. Following dehydration these were embedded in Araldite mixture in plastic capsules and polymerization was effected for 2 days at 60°C. Ultrathin sections were cut in a ultrotome and sections were double stained with Uranyl acetate and lead citrate. These were viewed in a TEM.

The Physiology of Sea-Urchin Spermatozoa

1950 ◽  
Vol 26 (4) ◽  
pp. 396-409
Author(s):  
LORD ROTHSCHILD
Keyword(s):  
Sea Urchin ◽  
Seminal Plasma ◽  
Human Blood ◽  
Phosphate Buffer ◽  
Dry Weight ◽  
O2 Uptake ◽  
Low Concentrations ◽  
Initial Substrate ◽  
Rough Calculation ◽  
Initial Substrate Concentration

1. Spermatozoa and seminal plasma of Echinus esculentus contain catalase. 2. At 15° C., 4 ml. of a suspension of semen diluted with neutral phosphate buffer in the ratio 1:13 produced in 1 min. 90µl. O2 from an H2O2 solution containing 150 µl. O2. The dry weight of semen in the suspension was 45 mg. and the number of spermatozoa 8.55x109. Under the same conditions, seminal plasma obtained by centrifuging semen produced 50 µl. O2 in 1 min. The dry weight of seminal plasma in the suspension was 12 mg. Human blood, dry weight 229.3 mg./ml., must be diluted with phosphate buffer in the ratio 1:1700 to produce the same amount of O2 in 1 min. as the above suspension of semen. If catalatic activity is defined by the equation Ac = (gt)-1 In {a/(a-x)}, where g = weight in g./ml. of the catalase-containing material, t = 1 min., a = initial substrate concentration (H2O2), and x = amount of H2O2 decomposed in 1 min. at 15° C., Ac = 80-100, 150-200 and 6800 respectively for sea-urchin semen, sea-urchin seminal plasma and human blood. 3. The catalatic activity of semen and seminal plasma is strongly inhibited by hydroxylamine. 4. The O2 uptake and motility of sea-urchin spermatozoa is unaffected by M/5000 H2O2. Higher concentrations of H2O2, M/3000-5000, produce a pronounced ‘shock’ effect, from which the spermatozoa often completely recover. 5. Low concentrations of hydroxylamine, M/3000, reduce O2 uptake and motility. 6. Sea-urchin spermatozoa are almost instantly killed by combinations of hydroxylamine and H2O2, at concentrations which are relatively innocuous when the substances are added separately. 7. A rough calculation indicates that a single spermatozoon contains less than 500 molecules of catalase. 8. A new method of adding H2O2 to catalase-containing material in a manometer is described.

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