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.

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.

Studies of the cleavage in the frog egg

Development ◽  
1969 ◽  
Vol 21 (1) ◽  
pp. 119-129
Author(s):  
T. Kubota
Keyword(s):  
Sea Urchin ◽  
Sea Urchins ◽  
Cleavage Furrow ◽  
Mitotic Apparatus ◽  
Animal Pole ◽  
Sea Urchin Eggs ◽  
Rana Nigromaculata ◽  
Vegetal Pole ◽  
Egg Cortex

In sea-urchin eggs, once karyokinesis reaches metaphase or anaphase, the cleavage furrow can be formed even if the mitotic apparatus is destroyed (Swann & Mitchison, 1953) or removed (Hiramoto, 1956). A similar result was obtained in frog eggs (Kubota, 1966). In amphibian eggs a much longer time is available for performing experiments than in sea urchins as the furrow first appears at the animal pole and slowly travels toward the vegetal pole. Taking advantage of this situation, Waddington (1952) and Dan & Kuno-Kojima (1963) performed various kinds of operations to elucidate the roles of the egg cortex and the inner cytoplasm in furrow formation, and Selman & Waddington (1955) also made cytological observations of the process. In the present paper a shift of the inner cytoplasm relative to the cortex and its influence on the course of the furrow was analysed for eggs of the frog Rana nigromaculata.

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.

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.

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.

Evidence for Association of Vibrio Echinoideorum with Tissue Necrosis on Body Shell of the Green Sea Urchin Strongylocentrotus Droebachiensis

2021 ◽  
Author(s):  
Jonathan Hira ◽  
Klara Stensvåg
Keyword(s):  
Sea Urchin ◽  
Virulence Genes ◽  
Vibrio Vulnificus ◽  
Sea Urchins ◽  
Opportunistic Pathogen ◽  
Significant Degree ◽  
Strongylocentrotus Droebachiensis ◽  
Host Immune System ◽  
Progressive Development ◽  
Green Sea Urchin

Abstract “Sea urchin lesion syndrome” is known as sea urchins disease with the progressive development of necrotic epidermal tissue and loss of external organs, including appendages on the outer body surface. Recently, a novel strain, Vibrio echinoideorum has been isolated from the lesions of green sea urchin (Strongylocentrotus droebachiensis), an economically important mariculture species in Norway. V. echinoideorum has not been reported elsewhere in association of with green sea urchin lesion syndrome. Therefore, in this study, an immersion based bacterial challenge experiment was performed to expose sea urchins (wounded and non-wounded) to V. echinoideorum, thereby mimicking a nearly natural host-pathogen interaction under controlled conditions. This infection experiment demonstrated that only the injured sea urchins developed the lesion to a significant degree when exposed to V. echinoideorum. Pure cultures of the employed bacterial strain was recovered from the infected animals and its identity was confirmed by the MALDI-TOF MS spectra profiling. Additionally, the hemolytic phenotype of V. echinoideorum substantiated its virulence potential towards the host, and this was also supported by the cytolytic effect on red spherule cells of sea urchins. Furthermore, the genome sequence of V. echinoideorum was assumed to encode potential virulence genes and were subjected for in silico comparison with the established virulence factors of Vibrio vulnificus and Vibrio tasmaniensis. This comparative virulence profile provided novel insights about virulence genes and their putative functions related to chemotaxis, adherence, invasion, evasion of the host immune system, and damage of host tissue and cells. Thus, it supports the pathogenicity of V. echinoideorum. In conclusion, the interaction of V. echinoideorum with injured sea urchins appears to be essential for the development of lesion syndrome and therefore, revealing its potentiality as an opportunistic pathogen.

Host use pattern and life history of Liopetrolisthes mitra, a crab associate of the black sea urchin Tetrapygus niger

2000 ◽  
Vol 80 (4) ◽  
pp. 639-645 ◽  
Author(s):  
J.A. Baeza ◽  
M. Thiel
Keyword(s):  
Sea Urchin ◽  
Black Sea ◽  
Sea Urchins ◽  
Austral Summer ◽  
Average Density ◽  
The Black Sea ◽  
Reproductive Pattern ◽  
Host Use ◽  
Austral Spring ◽  
Use Pattern

The porcellanid crab Liopetrolisthes mitra is a common associate of the black sea urchin, Tetrapygus niger in north central Chile. The host-use pattern, population dynamics and reproductive pattern of L. mitra on sea urchins were examined between January 1996 and February 1997. Each month, between 60 and 95 per cent of all collected urchins hosted crabs, with the highest frequency of cohabitation occurring during the austral summer (January to March). Group sizes of crabs on individual urchins ranged from 1 to 25 crabs per host. The average density of crabs on the urchins ranged from 2 to 5.5 crabs per host. Large urchins were inhabited by crabs more frequently than small urchins but urchin size had no effect on the number or size of crabs. The sex ratio of adult crabs was ˜1:1 during most months. Reproduction occurred throughout the year but was most intense during the austral spring and summer (October to March), when the highest percentage of ovigerous females were found. Similarly, recruitment of L. mitra occurred throughout the year but reached a peak during austral summer and early autumn (January to May). All life stages of L. mitra including recently settled megalopae and reproductive adults were found on urchins. Size–frequency analysis indicated that many crabs live >1.5 years. The results of this study confirm that the association between L. mitra and T. niger is strong and persists throughout the benthic life of the commensal crab.

scholarly journals Effects of five southern California macroalgal diets on consumption, growth, and gonad weight, in the purple sea urchin Strongylocentrotus purpuratus

2014 ◽  
Author(s):  
Matthew C Foster ◽  
Jarrett E Byrnes ◽  
Daniel C Reed
Keyword(s):  
Sea Urchin ◽  
Sea Urchins ◽  
Strongylocentrotus Purpuratus ◽  
Macrocystis Pyrifera ◽  
Reproductive Capacity ◽  
Giant Kelp ◽  
Mixed Diet ◽  
Gonad Weight ◽  
Consumption Growth ◽  
Purple Sea Urchin

Consumer growth and reproductive capacity are direct functions of diet. Strongylocentrotid sea urchins, the dominant herbivores in California kelp forests, strongly prefer giant kelp (Macrocystis pyrifera), but are highly catholic in their ability to consume other species. The biomass of Macrocystis fluctuates greatly in space and time and the extent to which urchins can use alternate species of algae or a mixed diet of multiple algal species to maintain fitness when giant kelp is unavailable is unknown. We experimentally examined the effects of single and mixed species diets on consumption, growth and gonad weight in the purple sea urchin Strongylocentrotus purpuratus. Urchins were fed single species diets consisting of one of four common species of macroalgae (the kelps Macrocystis pyrifera and Pterygophora californica, and the red algae Chondracanthus corymbiferus and Rhodymenia californica (hereafter referred to by genus) or a mixed diet containing all four species ad libitum over a 13-week period in a controlled laboratory setting. Urchins fed Chondracanthus, Macrocystis and a mixed diet showed the highest growth (in terms of test diameter, wet weight and jaw length) and gonad weight while urchins fed Pterygophora and Rhodymenia showed the lowest. Urchins consumed their preferred food, Macrocystis at the highest rate when offered a mixture, but consumed Chondracanthus or Macrocystis at similar rates when the two algae were offered alone. The differences in urchin feeding behavior and growth observed between these diet types suggest the relative availability of the algae tested here could affect urchin populations and their interactions with the algal assemblage. The fact that the performance of urchins fed Chondracanthus was similar or higher than those fed the preferred Macrocystis suggests purple sea urchins could sustain growth and reproduction during times of low Macrocystis abundance as is common following large wave events.

scholarly journals Contrasting recruitment seasonality of sea urchin species in Gran Canaria, Canary Islands (eastern Atlantic)

2014 ◽  
Vol 15 (3) ◽  
pp. 475 ◽  
Author(s):  
S. GARCIA-SANZ ◽  
P. G. NAVARRO ◽  
F. TUYA
Keyword(s):  
Community Structure ◽  
Seasonal Variation ◽  
Sea Urchin ◽  
Sea Urchins ◽  
Late Summer ◽  
Paracentrotus Lividus ◽  
Abundant Species ◽  
Late Winter ◽  
Gran Canaria ◽  
Arbacia Lixula

Despite sea-urchins can play an important role affecting the community structure of subtidal bottoms, factors controlling the dynamics of sea-urchin populations are still poorly understood. We assessed the seasonal variation in recruitment of three sea-urchin species (Diadema africanum, Paracentrotus lividus and Arbacia lixula) at Gran Canaria Island (eastern Atlantic) via monthly deployment of artificial collectors throughout an entire annual cycle on each of four adjacent habitat patches (seagrasses, sandy patches, ‘urchin-grazed’ barrens and macroalgal-dominated beds) within a shallow coastal landscape. Paracentrotus lividus and A. lixula had exclusively one main recruitment peak in late winter-spring. Diadema africanum recruitment was also seasonal, but recruits appeared in late summer-autumn, particularly on ‘urchin-grazed’ barrens with large abundances of adult conspecifics. In conclusion, this study has demonstrated non-overlapping seasonal recruitment patterns of the less abundant species (P. lividus and A. lixula) with the most conspicuous species (D. africanum) in the study area.

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