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Mechanical properties of the surface of the sea urchin egg at fertilization and during cleavage
Printed in Sweden Copyright Q 1974 by Academic Press, Inc. All rights of reproduction in any form reserued
Experimental Cell Research 89 (1974) 320-326
MECHANICAL
PROPERTIES
OF THE SURFACE
EGG AT FERTILIZATION
AND DURING
OF THE SEA URCHIN CLEAVAGE
Y. HIRAMOTO Misaki Marine Biological Station, Miura-shi, Kanagawa-ken and Biological Laboratory, Tokyo Institute of Technology, Meguro-ku, Tokyo, Japan
SUMMARY I. Changes in stiffness of the cell surface at fertilization and during cleavage in sea urchin eggs were determined by the magnetic particle method. 2. The stiffness of the cell surface increased at fertilization, reached a maximum after about 1.5 min, then decreased and reached a minimum about 4 min after insemination, followed by a gradual increase, in the eggs of Temnopleurus toreumaticus at 25.5 to 26.5”C. 3. The stiffness of the cell surface increased during the diaster stage, reached a maximum 2 to 3 min before the onset of cleavage, then decreased to a minimum about 1 min before the onset of cleavage, increased again, reached a maximum during cleavage and then diminished, in the eggs of Temnopleurus toreumaticus at 25.5 to 26.5”C. A similar stiffness change was observed in the eggs of Hemicentrotus pulcherrimus at 17 to 19°C occurring almost in parallel in both the equatorial and polar surfaces.
A dramatic
change in the structure of the protoplasm occurs at the surface of the sea urchin egg at fertilization, accompanied by changes in its various physical and chemical
properties. Mitchison & Swann [13] found that the stiffness of the cell surface, determined by sucking the surface into a fine
pipette (elastimeter), increased at fertilization followed by a decrease.A transient increase in the stiffness of the egg as a whole was observed by the author, using a compression method [6]. Brown [2] and Zimmerman et al. [21] found a change at fertilization in the cortical gel strength, which was determined from the time required to dislodge pigment granules from the cortex with centrifugal force under high hydrostatic pressure. There is a considerable number of reports concerning the changes in stiffness of the cell as a whole and of the cell surface during Exptl Cell Res 89 (1974)
cleavage in sea urchin eggs (cf [l 11).According to some authors [3-7, 13, 19, 201, the stiffness increasesbefore the onset of cleavage and decreasesduring cleavage. In other cases [7, 81,two stiffness maxima have been found, one occurring shortly before the onset of cleavage and the other during cleavage, with a minimum between the two maxima. It is interesting, in connection with the mechanism of cleavage, to know whether any variations in stiffness exist between different regions of the cell surface at cleavage.In seaurchin eggs, Wolpert [19] reported a significant difference in stiffness between the polar surface and the equatorial surface, whereas Mitchison & Swann [13] did not find any variations. In amphibian eggs, Sawai & Yoneda [15] found variations between the polar surface and the surface near the cleavage furrow, whereas Selman & Waddington [16] obtained similar
hivision of Gicrometer scale indicates 5 pm.
values of stiffness at these surfaces. Marsland [12] reported a variation in cortical gel strength between the equatorial and the polar region in sea urchin eggs. The magnetic particle method, which was used by the author [lo, 111 to determine the mechanical properties of sea urchin egg protoplasm, is useful for determining the stiffness of the cell surface by measuring the relation between the deformation of the surface and the force applied to an iron particle
embedded in the cortical region of the cell. It is possible, by this method, to compare the values for the stiffness of the cell surface in different regions of the cell. This paper deals with the changes in stiffness of the cell surface at fertilization and changes in both the equatorial (furrow) and the polar regions
during cleavage.
MATERIALS
AND
METHODS
The materials used were the eggs of the sea urchins, Temnopleurus toreumaticus and Hemicentrotus pulcherrimus. For measuring the eggs at fertilization, the jelly of the unfertilized eggs was removed by treating them with acidified sea water before experimentation. In measurements of eggs during cleavage, the fertilization membrane and the hyaline layer were removed by treating the eggs with 1 M urea solution for 1 to 4 min shortly after insemination.
The exnerimental arrangements were generally the same as those of the pr&ious experiments on the mechanical nrouerties of the endoplasm 191. For observation &d-photography, an egg was supported with a pair of small pieces of glass plate forming a sharp angle in a chamber filled with sea water on the stage of a microscope. An electromagnet was placed close to the chamber so that the extension of its axis would pass through the center of the egg. By the method described in a previous paper [9], an iron particle 5 to 7 ,um in diameter was introduced into the egg and brought to the region near the cell surface where the stiffness was to be determined. The particle was drawn in an outward direction normal to the cell surface by switching on the electromagnet. The magnitude of the force annlied to the particle was co&olled by the current-of the electromagnet and its distance from the particle. Because the direction of the force was fixed -in the experimental set-up, the direction of the force relative to the cell was adjusted by turning the egg with a glass hook manoeuvered by a micromanipulator. The deformation of the egg surface by applying a force to the particle was recorded with a 16 mm cin& camera (Bolex H16) or a 35 mm automatic camera (Robot 24a) at an appropriate magnification obtained with a 20 x objective and a 10 x or 15 x ocular (Nikon). The magnitude of the force applied to the particle was determined by the method described in the previous paper [9]. Experimental temperatures were 25.5 to 26.5”C for Temnopteurus eggs and 17 to 19°C for Hemicentrotus em.
RESULTS
Deformation of the egg by force applied to a part of the cell surface When
the iron
particle
in the protoplasm
near the surface of the sea urchin egg was Exptl Cell Res 89 (1974)
322 Y. Hiramoto drawn in an outward direction normal to the cell surface by switching on the electromagnet, the surface bulged out, leaving a narrow gap between the surface and the particle as shown in fig. 1b. The particle gradually approached the surface if application of the force was continued, until finally it came into contact with the surface. When the force was removed, the bulge gradually diminished at a decreasing rate and almost disappeared within 30 set if the duration of the application of force was 1 set or shorter (cf fig. 1c). The speed of the particle near the cell surface was much less than that in the central region of the cell, showing a high consistency of the protoplasm in the cortical region. The response of the cell surface to applied force was qualitatively similar in all the eggs, irrespective of the time before or after fertilization.
Changesin stiffness of the cell surface at fertilization The experimental procedure for measuring the change in stiffness of the egg surface at fertilization was as follows. A force of a definite magnitude and 1 set duration was applied once or twice, at an interval of 1 or 2 min, to an iron particle near the surface of an unfertilized sea urchin egg in the direction normal to the surface. Then the egg was inseminated by adding a small amount of sperm suspension to the medium, and forces of the same magnitude and the same duration were repeatedly applied at intervals of 0.5 1 or 2 min for 15 min. The magnitudes of the forces used in different eggs varied from 1.7 to 6.6 x 1O-4 dyne. The fertilization membranes elevated shortly after insemination and became stiff within several minutes. Since the distance between the cell surface and the elevated membrane was greater than the height of the bulge produced by the applied force, it seems likely that the Exptl Cell Res 89 (1974)
Fig. 2. Abscissa: time after insemination (min); ordinate: stiffnessof cell surface.
Changein stiffnessof the cell surfaceat fertilization in a T. toreumaticus eg,g. The stiffness is remesented by the reciprocal of & height of the bulge &m-l) formed by application for 1 set of a force of 2.7 x lo+ dynes, in the direction normal to the cell surface, to an iron particle embeddedin the cortex. Temperature 26.5”C.
presence of the membrane around the egg scarcely affected the formation of the bulge. Fig. 2 shows an example of the changes in stiffness represented by the reciprocal of the height of the bulge formed by applied force of a definite magnitude (2.7 x 1O-4 dynes in this case) and 1 set duration. As fig. 2 shows, the stiffness of the cell surface rises soon after insemination, reaches a maximum about 1.5 min after insemination, then falls, reaches a minimum about 4 min after insemination and gradually rises thereafter. Similar changes were observed in all the 7 eggs examined, and typical times for the maximum and the minimum stiffness were 1.5 min (range 1.0-2.0 min) and 4 min (range 3.0-5.0 min) after insemination, respectively. These times for the maximum and minimum coincide with those of the endoplasmic consistency determined by the magnetic particle method [lo]. The stiffness of the cell surface at the minimum was greater than that before insemination, whereas the minimum endoplasmic consistency was less than the consistency of the endoplasm of the unfertilized egg [lo]. The fertilization membrane gradually elevated over the entire surface before the stiffness reached its maximum.
Stiffness of sea urchin egg surface
323
equatorial surface was determined during the first half of cleavage. Fig. 3a shows two representative examples of the changes during cleavage in stiffness of the polar surface, represented by the reciprocal of the height of the bulge caused by the application of forces of a definite magnitude for 1 sec. As shown in fig. 3a the stiffness 1100 rises during the diaster stage, reaches a maximum 2 to 3 min before the onset of cleavage, falls to a minimum about 1 min before the onset of cleavage, rises again, reaches a second maximum during cleavage and falls thereafter. Fig. 3b shows two representative examples of the changes in stiffness Fig. 3. Abscissa: time after onset of cleavage (min); ordinate: stiffness of cell surface (circles connected of the equatorial surface during the first half with lines, left scale) and diameter of cleavage furrow (continuous curve, right scale, per cent of diameter of of cleavage, and those of the polar surface egg before the onset of cleavage). Change in stiffness during the second half, in the same eggs.It is of the cell surface before, during and after cleavage in noted in this figure that the stiffness change four eggs of T. toreumaticus. The stiffness is represented by the reciprocal of the of the equatorial surface is similar to that of height of the bulge @m-l) formed by application of a the polar surface during the first half of force of a definite magnitude (8.5 x low4 dynes in A, 6.4 x lo+ dynes in B, 4.1 x lo-* dynes in C and 6.7 x cleavage. It is not certain whether the melOA dynes in D) and 1 set in duration. l , Furrow surface stiffness; 0, polar surface stiffness. 26°C in chanical properties of the cell surface are A and B and 25.5”C in C and D. exactly equal to each other at the equatorial and polar surfaces of the same egg at the same stage, because it was impossible by the Change in stiffness of the cell surface present method to determine the stiffness in during cleavage two different regions at the same time, and The stiffness of the cell surface was deter- becausethe stiffness was not strictly comparamined by repeatedly applying forces of a ble between the equatorial surface and the definite magnitude and 1 set duration at polar one since their shapes are different. intervals of 0.5 to 2 min, starting before the In Hemicentrotus eggs, the cell was deappearance of the cleavagefurrow, i.e. during formed by a constant force continuously diaster stage and continuing through and applied to the polar surface in the direction after cleavage. In each case the force was normal to the surface. The application of the applied to the cell surface at the equator and force was started before or shortly after the at the pole. The determination of the stiffness onset of cleavage and was continued during of the equatorial surface was stopped before and after cleavage.An example of this expericompletion of cleavage, because the particle ment is shown in fig. 4. It is noted in fig. 4 could not be kept in position during the that the deformation of the cell surface by the application of the force. In some experi- applied force decreasesduring the first half ments, the stiffness of the polar surface was of cleavage (fig. 4a-c) and increases during determined during the second half of cleavage the second half (fig. 4d-f). Using photousing an egg in which the stiffness of the graphs of the egg deformed by a force such as
*r
Exptl Cell Res 89 (1974
324 Y. Hiramoto
Fig. 4. Change in shape of a dividing H. pulcherrimus
egg deformed by a force applied to the polar surface in the direction of the spindle axis. Application of the force (1.3 y IOW dynes) was started before the onset of cleavage and continued during and after cleavage. (a-f) show the egg 2, 5, 9, 13, 18 and 24 min after the start of the application of the force, respectively. Temperature 18°C. Smallest division of micrometer scale indicates 5 pm.
those shown in fig. 4, it is possible to determine the surface forces of the cell in the polar region of a dividing egg, assuming that the surface forces are the same in both blastomeres, one deformed by the force applied to the particle and the other without applied force. In the present study, the angles formed by the spindle axis and the tangents at the cell surface 15 pm away from the spindle axis were determined in both blastomeres (0 and 8’ in the inset of fig. 5). From Exptl Cell Res 89 (1974)
the balance between the surface forces (T dyne/cm) and the force (F dyne) applied to the particle, 0.003 nT.cos 19=0.003 7cT.cos 8’ -F.
(1)
Therefore, T = 106 F/(cos 19’- cos 0)
(2)
Fig. 5 shows an example of the results, which indicate decrease in the surface force before the onset of cleavage, increase during
Stiffness of sea urchin egg surface 325
0'
I 0
rIO
I 20
;-iO
Fig. 5. Abscissa: time after onset of cleavage(min); ordinate: surfaceforce in polar region (circlesconnec-
ted with lines, left scale, dyne/cm), and furrow diameter (continuous curve, right scale,per cent of egg diameter before the onset of cleavage). Changein surfaceforce at the polar region of a H. pulcherrimus egg before, during and after cleavage. The surfaceforces were calculated from the relation betweenthe force applied to the polar surfacein the direction of the spindle axis and the shapeof the egg, assumingthat the surfaceforces (T) at the surface 15 ,um away from the spindle axis are the samein both blastomeres(cf inset). Temperature18S”C. the first half of cleavage and decrease during the second half and after cleavage. The change is similar to the change in stiffness of the cell surface described above. DISCUSSION In the present study, a method has been devised for determining the stiffness of the cell surface by means of the magnetic particle method [9]. It may be considered that the stiffness values obtained by this method depend mainly on the mechanical properties of the cortex and the cell membrane and that they are scarcely affected by the values of endoplasmic consistency, because the displacement and the velocity of the iron particle in the region near the cell surface in response to applied force are far smaller than those of the same particle at the center of the cell in both unfertilized eggs and fertilized eggs. It is also considered that the values for the consistency of the endoplasm determined by the magnetic particle method [9, lo] are affected little if at all by the stiffness of the cell surface, because the cell surface is not detectably deformed by the movement of the particle in
the central part of the cell. Therefore, the similarity between the change in endoplasmic consistency at fertilization reported in the previous paper [lo] and the change in stiffness of the cell surface shown in the present paper may indicate that structural changes occur in parallel at the cell surface and in the endoplasm. Mitchison & Swann [13] found that the stiffness of the cell surface increased at fertilization, reached a peak within the first 3 min after insemination, followed by a gradual decrease in the next 5 min using sea urchin eggs treated with trypsin, which inhibited the elevation of the fertilization membrane. The stiffness of the sea urchin egg treated with trypsin determined by the compressionmethod increased at fertilization, reached a low peak 2 to 3 min after insemination and a high peak about 5 min after insemination followed by a decrease, and then increased again or retained an almost steady level [6]. It has been shown in the present study that change in stiffness of the cell surface, which is more or less similar to the stiffness changes described above, occurs at fertilization in normal eggs. The breakdown of cortical granules, which is the most dramatic visible change at fertilization in sea urchin eggs [5, 141, occurs within the first 20 to 40 set after the attachment of the fertilizing spermatozoon [ 11; the elevation of the fertilization membrane follows this cortical change. It seems likely that the stiffness of the cell surface reaches the peak after the completion of cortical granule breakdown, because about 1.5 min has elapsed after insemination and the fertilization membrane is elevating over the entire surface at the time of the stiffness peak. The gradual increase in stiffness after the minimum (about 4 min after insemination) may be related to the formation of the hyaline layer observed at this stage. It has been shown in the present study that Exptl Cell Res 89 (1974)
326 Y. Hiramoto both the stiffness of the cell surface determined by applying a force for 1 set and the surface force in the polar region determined from the deformation of the cell by a continuously applied force change in a fashion similar to the changes in the intracellular pressure, surface forces and the resistance to centrifugal force observed in the eggs of some specieslof sea urchin [7, 81. The increase in stiffness of the cell surface and the surface force during the first half of cleavage may indicate that cleavage results from neither active expansion of the polar membrane alone [17] nor active relaxation of its tension [18], as has been discussed elsewhere [8]. It is interesting, in connection with the mechanism of cleavage, to know whether variations in mechanical properties exist between different parts of the cell surface, especially between the equatorial and polar regions. It has been shown in the present study that the change in stiffness of the polar surface is closely similar to that of the equatorial surface during the first half of cleavage, but a question is left whether or not the stiffness is exactly the samein these two surfaces, because the present method does not permit a strict comparison of stiffness in surfaces of different shapes.
Exptl Cell Res 89 (1974)
The author wishes to thank Professor J. C. Dan of Ochanomizu University for her critical reading of the manuscript. This work was mainly carried out during the author’s stay at Misaki Marine Biological Station and supported in part by Research Expenditure of the Ministry of Education.
REFERENCES 1. Allen, R D & Griffin, J L, Exptl cell res 15 (1958) 163. 2. Brown, D E S, J cell comu nhysiol 5 (1934) 335. 3. Cole, R S & Michaelis, E-M, J cell cdmp physiol 2 (1932) 121. 4. Danielli, J F, Nature 170 (1952) 496. Endo, Y, Exptl cell res 3 (1952) 406. 2: Hiramoto. Y. Exntl cell res 32 (1963) 76. - J cell physiol 69 (1967) 216. ~ ’ ii. - Symp sot exptl bio122 (1968) 311. 9: - Exptl cell res 56 (1969) 201. - Ibid 56 (1969) 209. :7 - Biorheology 6 (1970) 201. 12: Marsland, D A, J cell camp physiol 13 (1939) 15. 13. Mitchison, J M & Swann, M M, J exptl biol 32 (1955) 734. 14. Moser, F, J exptl zoo1 80 (1939) 423. 15. Sawai, T & Yoneda, M, J cell biol 60 (1974) 1. 16. Selman, G G & Waddington, C H, J exptl biol 32 (1955) 700. 17. Swann, M M & Mitchison, J M, Biol rev 33 (1958) 103. 18. Wolpert, L, Int rev cytol 10 (1960) 163. 19. - Exptl cell res 41 (1966) 385. 20. Yoneda, M & Dan, K, J exptl bio157 (1972) 575. 21. Zimmerman, A M, Landau, J V & Marsland, D, J cell camp physiol 49 (1957) 395.
Received June 5, 1974
Experimental Cell Research 89 (1974) 320-326
MECHANICAL
PROPERTIES
OF THE SURFACE
EGG AT FERTILIZATION
AND DURING
OF THE SEA URCHIN CLEAVAGE
Y. HIRAMOTO Misaki Marine Biological Station, Miura-shi, Kanagawa-ken and Biological Laboratory, Tokyo Institute of Technology, Meguro-ku, Tokyo, Japan
SUMMARY I. Changes in stiffness of the cell surface at fertilization and during cleavage in sea urchin eggs were determined by the magnetic particle method. 2. The stiffness of the cell surface increased at fertilization, reached a maximum after about 1.5 min, then decreased and reached a minimum about 4 min after insemination, followed by a gradual increase, in the eggs of Temnopleurus toreumaticus at 25.5 to 26.5”C. 3. The stiffness of the cell surface increased during the diaster stage, reached a maximum 2 to 3 min before the onset of cleavage, then decreased to a minimum about 1 min before the onset of cleavage, increased again, reached a maximum during cleavage and then diminished, in the eggs of Temnopleurus toreumaticus at 25.5 to 26.5”C. A similar stiffness change was observed in the eggs of Hemicentrotus pulcherrimus at 17 to 19°C occurring almost in parallel in both the equatorial and polar surfaces.
A dramatic
change in the structure of the protoplasm occurs at the surface of the sea urchin egg at fertilization, accompanied by changes in its various physical and chemical
properties. Mitchison & Swann [13] found that the stiffness of the cell surface, determined by sucking the surface into a fine
pipette (elastimeter), increased at fertilization followed by a decrease.A transient increase in the stiffness of the egg as a whole was observed by the author, using a compression method [6]. Brown [2] and Zimmerman et al. [21] found a change at fertilization in the cortical gel strength, which was determined from the time required to dislodge pigment granules from the cortex with centrifugal force under high hydrostatic pressure. There is a considerable number of reports concerning the changes in stiffness of the cell as a whole and of the cell surface during Exptl Cell Res 89 (1974)
cleavage in sea urchin eggs (cf [l 11).According to some authors [3-7, 13, 19, 201, the stiffness increasesbefore the onset of cleavage and decreasesduring cleavage. In other cases [7, 81,two stiffness maxima have been found, one occurring shortly before the onset of cleavage and the other during cleavage, with a minimum between the two maxima. It is interesting, in connection with the mechanism of cleavage, to know whether any variations in stiffness exist between different regions of the cell surface at cleavage.In seaurchin eggs, Wolpert [19] reported a significant difference in stiffness between the polar surface and the equatorial surface, whereas Mitchison & Swann [13] did not find any variations. In amphibian eggs, Sawai & Yoneda [15] found variations between the polar surface and the surface near the cleavage furrow, whereas Selman & Waddington [16] obtained similar
hivision of Gicrometer scale indicates 5 pm.
values of stiffness at these surfaces. Marsland [12] reported a variation in cortical gel strength between the equatorial and the polar region in sea urchin eggs. The magnetic particle method, which was used by the author [lo, 111 to determine the mechanical properties of sea urchin egg protoplasm, is useful for determining the stiffness of the cell surface by measuring the relation between the deformation of the surface and the force applied to an iron particle
embedded in the cortical region of the cell. It is possible, by this method, to compare the values for the stiffness of the cell surface in different regions of the cell. This paper deals with the changes in stiffness of the cell surface at fertilization and changes in both the equatorial (furrow) and the polar regions
during cleavage.
MATERIALS
AND
METHODS
The materials used were the eggs of the sea urchins, Temnopleurus toreumaticus and Hemicentrotus pulcherrimus. For measuring the eggs at fertilization, the jelly of the unfertilized eggs was removed by treating them with acidified sea water before experimentation. In measurements of eggs during cleavage, the fertilization membrane and the hyaline layer were removed by treating the eggs with 1 M urea solution for 1 to 4 min shortly after insemination.
The exnerimental arrangements were generally the same as those of the pr&ious experiments on the mechanical nrouerties of the endoplasm 191. For observation &d-photography, an egg was supported with a pair of small pieces of glass plate forming a sharp angle in a chamber filled with sea water on the stage of a microscope. An electromagnet was placed close to the chamber so that the extension of its axis would pass through the center of the egg. By the method described in a previous paper [9], an iron particle 5 to 7 ,um in diameter was introduced into the egg and brought to the region near the cell surface where the stiffness was to be determined. The particle was drawn in an outward direction normal to the cell surface by switching on the electromagnet. The magnitude of the force annlied to the particle was co&olled by the current-of the electromagnet and its distance from the particle. Because the direction of the force was fixed -in the experimental set-up, the direction of the force relative to the cell was adjusted by turning the egg with a glass hook manoeuvered by a micromanipulator. The deformation of the egg surface by applying a force to the particle was recorded with a 16 mm cin& camera (Bolex H16) or a 35 mm automatic camera (Robot 24a) at an appropriate magnification obtained with a 20 x objective and a 10 x or 15 x ocular (Nikon). The magnitude of the force applied to the particle was determined by the method described in the previous paper [9]. Experimental temperatures were 25.5 to 26.5”C for Temnopteurus eggs and 17 to 19°C for Hemicentrotus em.
RESULTS
Deformation of the egg by force applied to a part of the cell surface When
the iron
particle
in the protoplasm
near the surface of the sea urchin egg was Exptl Cell Res 89 (1974)
322 Y. Hiramoto drawn in an outward direction normal to the cell surface by switching on the electromagnet, the surface bulged out, leaving a narrow gap between the surface and the particle as shown in fig. 1b. The particle gradually approached the surface if application of the force was continued, until finally it came into contact with the surface. When the force was removed, the bulge gradually diminished at a decreasing rate and almost disappeared within 30 set if the duration of the application of force was 1 set or shorter (cf fig. 1c). The speed of the particle near the cell surface was much less than that in the central region of the cell, showing a high consistency of the protoplasm in the cortical region. The response of the cell surface to applied force was qualitatively similar in all the eggs, irrespective of the time before or after fertilization.
Changesin stiffness of the cell surface at fertilization The experimental procedure for measuring the change in stiffness of the egg surface at fertilization was as follows. A force of a definite magnitude and 1 set duration was applied once or twice, at an interval of 1 or 2 min, to an iron particle near the surface of an unfertilized sea urchin egg in the direction normal to the surface. Then the egg was inseminated by adding a small amount of sperm suspension to the medium, and forces of the same magnitude and the same duration were repeatedly applied at intervals of 0.5 1 or 2 min for 15 min. The magnitudes of the forces used in different eggs varied from 1.7 to 6.6 x 1O-4 dyne. The fertilization membranes elevated shortly after insemination and became stiff within several minutes. Since the distance between the cell surface and the elevated membrane was greater than the height of the bulge produced by the applied force, it seems likely that the Exptl Cell Res 89 (1974)
Fig. 2. Abscissa: time after insemination (min); ordinate: stiffnessof cell surface.
Changein stiffnessof the cell surfaceat fertilization in a T. toreumaticus eg,g. The stiffness is remesented by the reciprocal of & height of the bulge &m-l) formed by application for 1 set of a force of 2.7 x lo+ dynes, in the direction normal to the cell surface, to an iron particle embeddedin the cortex. Temperature 26.5”C.
presence of the membrane around the egg scarcely affected the formation of the bulge. Fig. 2 shows an example of the changes in stiffness represented by the reciprocal of the height of the bulge formed by applied force of a definite magnitude (2.7 x 1O-4 dynes in this case) and 1 set duration. As fig. 2 shows, the stiffness of the cell surface rises soon after insemination, reaches a maximum about 1.5 min after insemination, then falls, reaches a minimum about 4 min after insemination and gradually rises thereafter. Similar changes were observed in all the 7 eggs examined, and typical times for the maximum and the minimum stiffness were 1.5 min (range 1.0-2.0 min) and 4 min (range 3.0-5.0 min) after insemination, respectively. These times for the maximum and minimum coincide with those of the endoplasmic consistency determined by the magnetic particle method [lo]. The stiffness of the cell surface at the minimum was greater than that before insemination, whereas the minimum endoplasmic consistency was less than the consistency of the endoplasm of the unfertilized egg [lo]. The fertilization membrane gradually elevated over the entire surface before the stiffness reached its maximum.
Stiffness of sea urchin egg surface
323
equatorial surface was determined during the first half of cleavage. Fig. 3a shows two representative examples of the changes during cleavage in stiffness of the polar surface, represented by the reciprocal of the height of the bulge caused by the application of forces of a definite magnitude for 1 sec. As shown in fig. 3a the stiffness 1100 rises during the diaster stage, reaches a maximum 2 to 3 min before the onset of cleavage, falls to a minimum about 1 min before the onset of cleavage, rises again, reaches a second maximum during cleavage and falls thereafter. Fig. 3b shows two representative examples of the changes in stiffness Fig. 3. Abscissa: time after onset of cleavage (min); ordinate: stiffness of cell surface (circles connected of the equatorial surface during the first half with lines, left scale) and diameter of cleavage furrow (continuous curve, right scale, per cent of diameter of of cleavage, and those of the polar surface egg before the onset of cleavage). Change in stiffness during the second half, in the same eggs.It is of the cell surface before, during and after cleavage in noted in this figure that the stiffness change four eggs of T. toreumaticus. The stiffness is represented by the reciprocal of the of the equatorial surface is similar to that of height of the bulge @m-l) formed by application of a the polar surface during the first half of force of a definite magnitude (8.5 x low4 dynes in A, 6.4 x lo+ dynes in B, 4.1 x lo-* dynes in C and 6.7 x cleavage. It is not certain whether the melOA dynes in D) and 1 set in duration. l , Furrow surface stiffness; 0, polar surface stiffness. 26°C in chanical properties of the cell surface are A and B and 25.5”C in C and D. exactly equal to each other at the equatorial and polar surfaces of the same egg at the same stage, because it was impossible by the Change in stiffness of the cell surface present method to determine the stiffness in during cleavage two different regions at the same time, and The stiffness of the cell surface was deter- becausethe stiffness was not strictly comparamined by repeatedly applying forces of a ble between the equatorial surface and the definite magnitude and 1 set duration at polar one since their shapes are different. intervals of 0.5 to 2 min, starting before the In Hemicentrotus eggs, the cell was deappearance of the cleavagefurrow, i.e. during formed by a constant force continuously diaster stage and continuing through and applied to the polar surface in the direction after cleavage. In each case the force was normal to the surface. The application of the applied to the cell surface at the equator and force was started before or shortly after the at the pole. The determination of the stiffness onset of cleavage and was continued during of the equatorial surface was stopped before and after cleavage.An example of this expericompletion of cleavage, because the particle ment is shown in fig. 4. It is noted in fig. 4 could not be kept in position during the that the deformation of the cell surface by the application of the force. In some experi- applied force decreasesduring the first half ments, the stiffness of the polar surface was of cleavage (fig. 4a-c) and increases during determined during the second half of cleavage the second half (fig. 4d-f). Using photousing an egg in which the stiffness of the graphs of the egg deformed by a force such as
*r
Exptl Cell Res 89 (1974
324 Y. Hiramoto
Fig. 4. Change in shape of a dividing H. pulcherrimus
egg deformed by a force applied to the polar surface in the direction of the spindle axis. Application of the force (1.3 y IOW dynes) was started before the onset of cleavage and continued during and after cleavage. (a-f) show the egg 2, 5, 9, 13, 18 and 24 min after the start of the application of the force, respectively. Temperature 18°C. Smallest division of micrometer scale indicates 5 pm.
those shown in fig. 4, it is possible to determine the surface forces of the cell in the polar region of a dividing egg, assuming that the surface forces are the same in both blastomeres, one deformed by the force applied to the particle and the other without applied force. In the present study, the angles formed by the spindle axis and the tangents at the cell surface 15 pm away from the spindle axis were determined in both blastomeres (0 and 8’ in the inset of fig. 5). From Exptl Cell Res 89 (1974)
the balance between the surface forces (T dyne/cm) and the force (F dyne) applied to the particle, 0.003 nT.cos 19=0.003 7cT.cos 8’ -F.
(1)
Therefore, T = 106 F/(cos 19’- cos 0)
(2)
Fig. 5 shows an example of the results, which indicate decrease in the surface force before the onset of cleavage, increase during
Stiffness of sea urchin egg surface 325
0'
I 0
rIO
I 20
;-iO
Fig. 5. Abscissa: time after onset of cleavage(min); ordinate: surfaceforce in polar region (circlesconnec-
ted with lines, left scale, dyne/cm), and furrow diameter (continuous curve, right scale,per cent of egg diameter before the onset of cleavage). Changein surfaceforce at the polar region of a H. pulcherrimus egg before, during and after cleavage. The surfaceforces were calculated from the relation betweenthe force applied to the polar surfacein the direction of the spindle axis and the shapeof the egg, assumingthat the surfaceforces (T) at the surface 15 ,um away from the spindle axis are the samein both blastomeres(cf inset). Temperature18S”C. the first half of cleavage and decrease during the second half and after cleavage. The change is similar to the change in stiffness of the cell surface described above. DISCUSSION In the present study, a method has been devised for determining the stiffness of the cell surface by means of the magnetic particle method [9]. It may be considered that the stiffness values obtained by this method depend mainly on the mechanical properties of the cortex and the cell membrane and that they are scarcely affected by the values of endoplasmic consistency, because the displacement and the velocity of the iron particle in the region near the cell surface in response to applied force are far smaller than those of the same particle at the center of the cell in both unfertilized eggs and fertilized eggs. It is also considered that the values for the consistency of the endoplasm determined by the magnetic particle method [9, lo] are affected little if at all by the stiffness of the cell surface, because the cell surface is not detectably deformed by the movement of the particle in
the central part of the cell. Therefore, the similarity between the change in endoplasmic consistency at fertilization reported in the previous paper [lo] and the change in stiffness of the cell surface shown in the present paper may indicate that structural changes occur in parallel at the cell surface and in the endoplasm. Mitchison & Swann [13] found that the stiffness of the cell surface increased at fertilization, reached a peak within the first 3 min after insemination, followed by a gradual decrease in the next 5 min using sea urchin eggs treated with trypsin, which inhibited the elevation of the fertilization membrane. The stiffness of the sea urchin egg treated with trypsin determined by the compressionmethod increased at fertilization, reached a low peak 2 to 3 min after insemination and a high peak about 5 min after insemination followed by a decrease, and then increased again or retained an almost steady level [6]. It has been shown in the present study that change in stiffness of the cell surface, which is more or less similar to the stiffness changes described above, occurs at fertilization in normal eggs. The breakdown of cortical granules, which is the most dramatic visible change at fertilization in sea urchin eggs [5, 141, occurs within the first 20 to 40 set after the attachment of the fertilizing spermatozoon [ 11; the elevation of the fertilization membrane follows this cortical change. It seems likely that the stiffness of the cell surface reaches the peak after the completion of cortical granule breakdown, because about 1.5 min has elapsed after insemination and the fertilization membrane is elevating over the entire surface at the time of the stiffness peak. The gradual increase in stiffness after the minimum (about 4 min after insemination) may be related to the formation of the hyaline layer observed at this stage. It has been shown in the present study that Exptl Cell Res 89 (1974)
326 Y. Hiramoto both the stiffness of the cell surface determined by applying a force for 1 set and the surface force in the polar region determined from the deformation of the cell by a continuously applied force change in a fashion similar to the changes in the intracellular pressure, surface forces and the resistance to centrifugal force observed in the eggs of some specieslof sea urchin [7, 81. The increase in stiffness of the cell surface and the surface force during the first half of cleavage may indicate that cleavage results from neither active expansion of the polar membrane alone [17] nor active relaxation of its tension [18], as has been discussed elsewhere [8]. It is interesting, in connection with the mechanism of cleavage, to know whether variations in mechanical properties exist between different parts of the cell surface, especially between the equatorial and polar regions. It has been shown in the present study that the change in stiffness of the polar surface is closely similar to that of the equatorial surface during the first half of cleavage, but a question is left whether or not the stiffness is exactly the samein these two surfaces, because the present method does not permit a strict comparison of stiffness in surfaces of different shapes.
Exptl Cell Res 89 (1974)
The author wishes to thank Professor J. C. Dan of Ochanomizu University for her critical reading of the manuscript. This work was mainly carried out during the author’s stay at Misaki Marine Biological Station and supported in part by Research Expenditure of the Ministry of Education.
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Received June 5, 1974