The Fertilization Reaction in the Sea-Urchin Egg

1949 ◽  
Vol 26 (2) ◽  
pp. 164-176
Author(s):  
LORD ROTHSCHILD ◽  
M. M. SWANN
Keyword(s):  
Light Scattering ◽  
Sea Urchin ◽  
Sea Water ◽  
Initial Increase ◽  
Point Of Entry ◽  
Sperm Suspension ◽  
Fertilization Membrane ◽  
A New Technique ◽  
Dark Ground ◽  
Cortical Change

1. After insemination of unfertilized eggs of Psammechinus miliaris and before the elevation of the fertilization membrane, a change in cortical structure is propagated over the egg surface or through the cytoplasm. When the fertilizing spermatozoon has become attached, the cortex progressively scatters more light when viewed under dark ground illumination. The cortex, which before fertilization is scarcely distinguishable from the cytoplasm, is white after the reaction is completed. 2. At 18° C. the change covers the egg surface in about 20 sec. 3. The conduction velocity is not uniform, there being a reduction in rate when the change is about halfway round the egg, after which there is a marked increase. 4. The cortex of the unfertilized egg shows a weak positive radial birefringence which disappears at the same time as the dark ground scattering appears. 5. Other changes in the egg structure occur at the same time, and are visible under dark ground illumination. These are: (a) a conical erection appears transiently at the site of sperm attachment; (b) the shape of the egg changes during the reaction; (c) there is a localized and transient decrease in light scattering at the point of entry of the spermatozoon, after the initial increase in cortical light scattering; (d) the fertilization membrane first appears at the point on the surface where the cortical change is initiated. 6. To investigate the possibility that the observed change might be the block to polyspermy, measurements were made of the speeds at which sea-urchin spermatozoa swim. A new technique has been evolved for this purpose. Spermatozoa were photographed under a special dark ground illumination system, with exposures of 0.25 sec. The heads of the spermatozoa trace out tracks on the film, the mean translatory speed being 190 µ/sec. The motion is helicoidal, the frequency of oscillation of the illuminated sperm heads being about 40/sec. 7. By treating the sperm suspension as an assembly of gas molecules, a rough estimate has been made of the frequency of collisions between the spermatozoa and an egg. For sperm densities of 105, 106 and 107/ml., and for the observed translatory sperm speeds, the number of collisions between spermatozoa and those parts of the egg surface unaffected by the propagated change, after it has been initiated, is 1.6, 16 and 160 respectively. 8. Some preliminary experiments on the insemination of oocytes have been carried out to assess the probability of a sperm-egg collision being successful. The results were: (a) oocytes react to insemination by the extrusion of blebs or papillae, each of which is associated with a spermatozoon; (b) this reaction is inhibited by insemination in Ca-free sea water and partially inhibited by acidified sea water. It is concluded that the extrusion of papillae represents an abortive fertilization reaction, the oocyte cortex being unable to propagate a block to polyspermy. The number of papillae extruded is less than the number of sperm-oocyte collisions. 9. The experiments on the insemination of oocytes favour the possibility that attachment of the spermatozoon to an egg is not followed by fertilization unless there exists a particular orientation, on a molecular scale, between the egg and sperm surfaces, and provided there has been no previous interaction between the spermatozoa and Gynogamone II. The low probability of fertilization that this implies is compatible with the hypothesis that the observed cortical change may be the block to polyspermy. Further experiments are needed to resolve this question.

Some Experiments on Sea-urchin Eggs with Desoxynucleic Acids from Sea-urchin Sperms

Development ◽  
1953 ◽  
Vol 1 (3) ◽  
pp. 261-262
Author(s):  
Sven Hörstadius
Keyword(s):  
New York ◽  
Sea Urchin ◽  
Sea Water ◽  
Water Injection ◽  
Dark Field ◽  
The Other ◽  
Columbia University ◽  
Sea Urchin Eggs ◽  
King's College ◽  
Fertilization Membrane

Dr. I. Joan Lorch, of King's College, London, and I have made some experiments on sea-urchin eggs with desoxynucleic acids (DNA) prepared from sperms of several sea-urchin species by Professor Erwin Chargaff, of Columbia University, New York. Unfertilized eggs did not react when put into a solution of DNA in sea-water. Injection of a small amount of DNA dissolved in Callan's solution had the following consequences. If the DNA did not mix with the cytoplasm but remained as a distinct droplet, the egg could be fertilized. The droplet moved slowly towards the surface and ran out of the egg. This sometimes only occurred after several cleavages. Such eggs developed normally. If, on the other hand, the DNA mixed with the cytoplasm the egg became activated. A fertilization membrane was raised. The surface layer in dark field changed in colour from yellow to white as is the case upon fertilization.

Studies on the Respiration of Sea-Urchin Spermatozoa: III. Respiratory Quotient

1961 ◽  
Vol 38 (2) ◽  
pp. 249-257
Author(s):  
H. MOHRI ◽  
K. HORIUCHI
Keyword(s):  
Sea Urchin ◽  
Respiratory Quotient ◽  
Sea Water ◽  
Direct Method ◽  
O2 Uptake ◽  
Present Material ◽  
A Value ◽  
Sperm Suspension ◽  
Co2 Output ◽  
The Difference

1. The respiratory quotient of sea-urchin spermatozoa has been determined with Pseudocentrotus depressus, Anthocidaris crassispina and Hemicentrotus pulcherrimus. 2. The R.Q. of sea-urchin spermatozoa, measured by the Warburg direct method, has been reported to be near unity. This was also the case with the present material when the suspending medium was sea water, the R.Q. being 0.8-1.0. It was found, however, that the pH of sperm suspensions was markedly different in the presence and absence of alkali to absorb C02. 3. When the pH of the suspension was fixed by such buffers as 0.025 M-glycyl glycine, the R.Q. measured by the above method was about 0.7. This is in accord with the results of earlier metabolic studies, which indicated that endogenous phospholipids are the main substrates for the respiration of sea-urchin spermatozoa. 4. The O2 uptake of the present material, however, was found to be little affected by variation in pH. The difference in the R.Q. values obtained with ordinary sea water and buffered sea water, therefore, cannot be explained in terms of pH. 5. When the spermatozoa were suspended in ordinary sea water, the utilization of endogenous phospholipids was much reduced in the absence of alkali, while in buffered sea water the change in phospholipids was almost the same, with and without the absorption of CO2. 6. Determination of the R.Q. by the first method of Dickens & Šimer, in which the O2 uptake and the CO2 output were measured with one and the same sperm suspension, gave a value of about 0.7 with both ordinary and buffered sea water.

The Physiology of Sea-Urchin Spermatozoa

1950 ◽  
Vol 27 (1) ◽  
pp. 59-72
Author(s):  
LORD ROTHSCHILD ◽  
P. H. TUFT
Keyword(s):  
Sea Urchin ◽  
Sea Water ◽  
Dilution Effect ◽  
O2 Uptake ◽  
Time Curve ◽  
Sperm Density ◽  
Negative Results ◽  
Catalytic Function ◽  
Sperm Suspension ◽  
Dense Suspensions

1. When a suspension of sea-urchin semen in sea water is diluted, the total O2 consumed and the rate of O2 uptake per spermatozoon are greater than before dilution. This is the Dilution Effect, first described by Gray (1928 a). A further investigation of this phenomenon has been made, using the semen of Echinus esculentus. 2. The form of the O2 uptake-time curve of such suspensions varies according to the ratio semen: sea water. 3. In very dense suspensions (d ≥ 5 x 109 sperm/ml.), the apparent low O2 uptake per unit quantity of spermatozoa is mainly due to inadequate O2 saturation of the lower layers of the suspension, in which the spermatozoa are virtually unable to respire. Such suspensions are not suitable for experiments in Warburg or Barcroft manometers. 4. In dense suspensions (4 x 108 < d ≤ 109 sperm/ml.) the Dilution Effect was observed when sea water was added to the suspension, in such proportions that the sperm density was only reduced by a factor of 1.14. No Dilution Effect occurred when isotonic ‘Analar’ NaCl was added in the same proportions. On the basis of the small dilution involved, and the negative results with ‘Analar’ NaCl, it is concluded that the Dilution Effect is not exclusively due to the sperm having more space to move after dilution and therefore being able to expend more energy. 5. The Dilution Effect occurs when isotonic ‘Analar’ NaCl containing 1 p.p.m. CuCl2 is added to a dense sperm suspension, in the above proportions. Stimulation of O2 uptake is proportional, over certain limits, to the amount of CuCl2 added. Both CuCl, ZnCl2, and to a lesser extent CuSO2, have similar effects. 6. In a particular experiment, egg-water and Cu salts were about equally efficacious in increasing the O2 uptake of a sperm suspension. 7. The characteristic decline in the O2 uptake of dense suspensions can be partly reversed by the addition of Cu salts. 8. In dilute suspensions (d < 4 x 108 sperm/ml.), there is no Dilution Effect when sea water or CuCl2 are added in the above proportions. On the other hand, the decline in O2 uptake can be partly arrested by the addition of Cu salts. 9. Diethyldithiocarbamate markedly inhibits the O2 uptake of unwashed sea-urchin spermatozoa diluted with sea water. The inhibition does not take place in the presence of added CuCl2. Cu may therefore have a catalytic function in the metabolism of sea-urchin semen. 10. The possibility that Cu lack is responsible for the reduced O2 uptake of spermatozoa in dense suspensions is considered.

scholarly journals The Physiology of Sea-Urchin Spermatozoa

1956 ◽  
Vol 33 (1) ◽  
pp. 155-173
Author(s):  
LORD ROTHSCHILD
Keyword(s):  
Sea Urchin ◽  
Sea Water ◽  
Usnic Acid ◽  
Dilution Effect ◽  
O2 Uptake ◽  
Sperm Suspension ◽  
Dense Suspensions ◽  
Cast Doubt ◽  
Dilute Suspensions ◽  
Glycyl Glycine

1. Sea-urchin spermatozoa (Echinus esculentus) are extremely sensitive to changes in the pH of the suspending medium, their respiration being proportional to pH between 7.6 and 8.4. 2. In a manometric experiment in which semen was diluted 1/25 with sea water (CO2 absorbed), the pH of the suspension was 7.5 at the beginning of the experiment and 8.2 at the end, after 180 min. incubation at 15° C. 3. Sea water, buffered with glycyl glycine, 0.025 M and brought to pH 8.3, was added to a sperm suspension whose pH was 7.5 at the beginning of the experiment, after a period of incubation. There was a pronounced respiratory Dilution Effect. When the sea water was buffered with glycyl glycine and brought to pH 7.8, and this diluent was added to the same sperm suspension, there was a negligible respiratory Dilution Effect. 4. The O2 uptake of suspensions prepared in buffered sea water at pH 8.3 was markedly higher than that in the same buffered sea water at pH 8.0. 5. These observations cast doubt on the reality of the respiratory Dilution Effect when observed in experiments in which sea water is the suspending medium and respiration is measured by a method involving the absorption of CO2. An exception to this generalization was observed in buffered sea water at a low pH, 7.6. In this case, the O2 uptake of dilute suspensions was greater, per unit number of spermatozoa, than that of dense suspensions. 6. 2,4-dinitrophenol, 6 x 10-5M, stimulated the O2 uptake and depressed the motility of spermatozoa more in dense than in dilute suspensions. Versene, 10-3M, reversed the action of 2,4-dinitrophenol. 7. The results of other workers on adding suspensions of usnic acid to sea-urchin spermatozoa were not confirmed. This substance is more toxic to spermatozoa in dilute than in dense suspensions.

The Physiology of Sea-Urchin Spermatozoa

1948 ◽  
Vol 25 (4) ◽  
pp. 353-368
Author(s):  
LORD ROTHSCHILD
Keyword(s):  
Sea Urchin ◽  
Sea Water ◽  
Protective Action ◽  
Wave Length ◽  
Dilution Effect ◽  
O2 Consumption ◽  
O2 Uptake ◽  
Sperm Suspension ◽  
Dense Suspensions ◽  
Dilute Suspensions

1. An investigation has been made into the causes of the interrelated phenomena of Senescence and the Dilution Effect in the spermatozoa of Echinus esculentus. The Dilution Effect is the increase in O2 uptake and in movement observed when a concentrated sperm suspension, obtained by partial dilution of semen, is diluted with sea water. 2. The supernatant sea water obtained by centrifuging partially metabolized sperm suspensions contains no substances which depress the respiration or motility of the spermatozoa of the same species. This supernatant sea water has a small but definite protective action on spermatozoa in maintaining but not increasing their O2 consumption. The effect is enhanced by agitating the suspensions before centrifugation. 3. E. esculentus spermatozoa do not glycolyse aerobically, anaerobically, or in the presence of 5% O2 in CO. The absence of aerobic acid production proves that the reduced O2 uptake per sperm of dense suspensions is not due to a lowering of the pH of the medium. 4. As the Dilution Effect is observed in manometric experiments in which CO2 is continuously and adequately removed, neither accumulation of CO2 nor a reduction in pH resulting from the accumulation of CO2 are responsible for the reduced O2 uptake of dense suspensions. 5. The O2 uptake of dense sperm suspensions (> 109 sperm/ml.) is altered by changes in O2 tension. O2 consumption in 100% oxygen is nearly double that in 10% O2 in N2. An increase in O2 tension has no stimulating action on dilute suspensions. 6. An atmosphere of 100% O2 has a gradual toxic effect on dense and dilute sperm suspensions. 7. Carbon monoxide has a greater inhibitory action on dilute than on concentrated sperm suspensions. 8. The difference between the effects of varying O2 tensions or CO on dense and dilute suspensions is partly, or perhaps wholly, due to gaseous diffusion being a limiting factor. This may partly explain the Dilution Effect. 9. Apart from photo-reversibly inhibiting respiration, CO exerts an irreversible toxic influence on sea-urchin spermatozoa. 10. Illumination of spermatozoa with a wave-length of light outside the region of photo-chemical absorption by CO cytochrome oxidase enables visual examination to be made in the presence of CO. The O2 uptake of a suitable concentration of spermatozoa is 70% inhibited by 5% O2 in CO. The same inhibition occurs when the suspension is illuminated by light of wave-length 548 mµ, but there is no equivalent decrease in motility. 11. After motility and respiration have ceased sea-urchin spermatozoa still contain oxidizable substrates and the appropriate dehydrogenases as evidenced by their ability to reduce methylene blue in an evacuated Thunberg tube.

Counting Spermatozoa

1950 ◽  
Vol 26 (4) ◽  
pp. 388-395
Author(s):  
LORD ROTHSCHILD
Keyword(s):  
Light Scattering ◽  
Sea Urchin ◽  
Sea Water ◽  
Original Sample ◽  
Bull Semen ◽  
A Cell

1. The number of spermatozoa in a sample of sea-urchin semen (Echinus esculentus) can be accurately estimated by measuring the amount of light scattered and absorbed by a subsample composed of semen diluted with sea water. A ‘Spekker" photoelectric absorptiometer was used for the measurements, with a cell 1 cm. long. 2. The number N of spermatozoa in a sample of sea-urchin semen was found to be given by the equation N=8.0918x1010Y'±(1.590x109√Y')where Y' =0.3010-log10y' and y' = absorptiometer reading, when the original sample was diluted 200 times and the absorptiometer set to 2.000 for the blank. 3. The light-scattering and light-absorbing properties of washed and unwashed suspensions were found to be virtually identical. 4. The number of spermatozoa in a sample of bull semen was found to be given by the equation N=3.3515x109Y'±(5.283x107√Y'),when the original sample was diluted 30 times and the absorptiometer set to 2.000 for the blank.

Cytasters induced within unfertilized sea-urchin eggs

1983 ◽  
Vol 61 (1) ◽  
pp. 175-189
Author(s):  
R. Kuriyama ◽  
G.G. Borisy
Keyword(s):  
Electron Microscopy ◽  
Sea Urchin ◽  
Sea Water ◽  
Light And Electron Microscopy ◽  
Sea Urchin Eggs ◽  
Microtubule Organizing Centres ◽  
Treatment Step

Conditions that induce the formation of asters in unfertilized sea-urchin eggs have been investigated. Monasters were formed by treatment of eggs with acidic or basic sea-water, or procaine- or thymol-containing sea-water. A second treatment step, incubation with D2O-containing, ethanol-containing or hypertonic sea-water induced multiple cytasters. The number and size of cytasters varied according to the concentration of agents and duration of the first and second treatments, and also upon the species of eggs and the season in which the eggs were obtained. Generally, a longer second treatment or a higher concentration of the second medium resulted in a higher number of cytasters per egg. Asters were isolated and then examined by light and electron microscopy. Isolated monasters apparently lacked centrioles, whereas cytasters obtained from eggs undergoing the two-step treatment contained one or more centrioles. Up to eight centrioles were seen in a single aster; the centrioles appeared to have been produced during the second incubation. Centrospheres prepared from isolated asters retained the capacity to nucleate the formation of microtubules in vitro as assayed by light and electron microscopy. Many microtubules radiated from the centre of isolated asters, whether they contained centrioles or not. This observation is consistent with many other reports that microtubule-organizing centres need not contain centrioles.

Metabolic importance of Na+/K+-ATPase activity during sea urchin development.

1997 ◽  
Vol 200 (22) ◽  
pp. 2881-2892 ◽  
Author(s):  
P Leong ◽  
D Manahan
Keyword(s):  
Enzyme Activity ◽  
Atpase Activity ◽  
Sea Urchin ◽  
Sodium Pump ◽  
Sea Water ◽  
Strongylocentrotus Purpuratus ◽  
Lytechinus Pictus ◽  
Stimulation Of

Early stages of animal development have high mass-specific rates of metabolism. The biochemical processes that establish metabolic rate and how these processes change during development are not understood. In this study, changes in Na+/K+-ATPase activity (the sodium pump) and rate of oxygen consumption were measured during embryonic and early larval development for two species of sea urchin, Strongylocentrotus purpuratus and Lytechinus pictus. Total (in vitro) Na+/K+-ATPase activity increased during development and could potentially account for up to 77 % of larval oxygen consumption in Strongylocentrotus purpuratus (pluteus stage) and 80 % in Lytechinus pictus (prism stage). The critical issue was addressed of what percentage of total enzyme activity is physiologically active in living embryos and larvae and thus what percentage of metabolism is established by the activity of the sodium pump during development. Early developmental stages of sea urchins are ideal for understanding the in vivo metabolic importance of Na+/K+-ATPase because of their small size and high permeability to radioactive tracers (86Rb+) added to sea water. A comparison of total and in vivo Na+/K+-ATPase activities revealed that approximately half of the total activity was utilized in vivo. The remainder represented a functionally active reserve that was subject to regulation, as verified by stimulation of in vivo Na+/K+-ATPase activity in the presence of the ionophore monensin. In the presence of monensin, in vivo Na+/K+-ATPase activities in embryos of S. purpuratus increased to 94 % of the maximum enzyme activity measured in vitro. Stimulation of in vivo Na+/K+-ATPase activity was also observed in the presence of dissolved alanine, presumably due to the requirement to remove the additional intracellular Na+ that was cotransported with alanine from sea water. The metabolic cost of maintaining the ionic balance was found to be high, with this process alone accounting for 40 % of the metabolic rate of sea urchin larvae (based on the measured fraction of total Na+/K+-ATPase that is physiologically active in larvae of S. purpuratus). Ontogenetic changes in pump activity and environmentally induced regulation of reserve Na+/K+-ATPase activity are important factors that determine a major proportion of the metabolic costs of sea urchin development.

Localization and expression of msp130, a primary mesenchyme lineage-specific cell surface protein in the sea urchin embryo

Development ◽  
1987 ◽  
Vol 101 (2) ◽  
pp. 255-265 ◽  
Author(s):  
J.A. Anstrom ◽  
J.E. Chin ◽  
D.S. Leaf ◽  
A.L. Parks ◽  
R.A. Raff
Keyword(s):  
Cell Surface ◽  
Sea Urchin ◽  
Sea Water ◽  
Surface Protein ◽  
Specific Cell ◽  
Cell Surface Protein ◽  
Sea Urchin Embryo ◽  
A Cell ◽  
Mesenchyme Cells ◽  
Primary Mesenchyme Cells

In this report, we use a monoclonal antibody (B2C2) and antibodies against a fusion protein (Leaf et al. 1987) to characterize msp130, a cell surface protein specific to the primary mesenchyme cells of the sea urchin embryo. This protein first appears on the surface of these cells upon ingression into the blastocoel. Immunoelectronmicroscopy shows that msp130 is present in the trans side of the Golgi apparatus and on the extracellular surface of primary mesenchyme cells. Four precursor proteins to msp130 are identified and we show that B2C2 recognizes only the mature form of msp130. We demonstrate that msp130 contains N-linked carbohydrate groups and that the B2C2 epitope is sensitive to endoglycosidase F digestion. Evidence that msp130 is apparently a sulphated glycoprotein is presented. The recognition of the B2C2 epitope of msp130 is disrupted when embryos are cultured in sulphate-free sea water. In addition, two-dimensional immunoblots show that msp130 is an acidic protein that becomes substantially less acidic in the absence of sulphate. We also show that two other independently derived monoclonal antibodies, IG8 (McClay et al. 1983; McClay, Matranga & Wessel, 1985) and 1223 (Carson et al. 1985), recognize msp130, and suggest this protein to be a major cell surface antigen of primary mesenchyme cells.

scholarly journals THE EXTRACELLULAR RELEASE OF ECHINOCHROME

1946 ◽  
Vol 29 (5) ◽  
pp. 267-275 ◽  
Author(s):  
Herbert Shapiro
Keyword(s):  
Sea Urchin ◽  
Sea Water ◽  
Potassium Content ◽  
Red Pigment ◽  
Anaerobic Conditions ◽  
Outward Diffusion ◽  
Arbacia Punctulata ◽  
Extracellular Release

A study was made of the diffusion of the red pigment echinochrome from the eggs of the sea urchin, Arbacia punctulata, into sea water. Unfertilized eggs retained their pigment, over periods of hours. Outward diffusion of pigment from unfertilized eggs normally is entirely negligible, or does not occur at all. Enchancing the calcium or potassium content of the artificial sea water (while retaining isosmotic conditions) did not induce pigment release. Under anaerobic conditions, unfertilized eggs release pigment in small quantities. Fertilization alone brings about echinochrome release. Fertilized eggs invariably released pigment, whether in normal sea water, or sea water with increased calcium or potassium. This diffusion of the pigment began during the first cleavage, possibly soon after fertilization. The pigment release is not a consequence solely of the cell's permeability to echinochrome (or chromoprotein, or other pigment combination) but is preceded by events leading to a release of echinochrome from the granules in which it is concentrated within the cell. These events may be initiated by activation or by anaerobiosis. The phenomenon was not due to cytolysis.

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