CHANGES I N VOLUME AND SURFACE OF URECHIS EGGS UPON FERTILIZATION

ALBERT TYLER William G. Eerckhoff Laboratories of the Biological Sciences, California

Institute of Technology, Pasadena, California, and William G. Kerckhoff Marine Laboratory, Corona del Mar, California

TWO PLATES (THIRTY-SIX FIGURES)

The volume change of eggs upon fertilization has been measured by various investigators. Loeb ('08) noted no change in volume f o r the egg of Strongylocentrotus. McClendon ('10) found the fertilized egg of Arbacia to be the same size as the unfertilized egg. However, Glaser ( '13, '14) reported a decrease in volume for the Arbacia egg, and found the same to be true for Asterias. But Chambers ('21) attributed the apparent loss in volume to a flattening of the unfertilized egg as it lies on the bottom of a glass dish. He used a technique which presumably avoided flattening and reported for the eggs of Asterias and Arbacia no change as the fertilization membrane is formed and then a slight increase up to the time of segmentation. Glaser ('24) re- peated his measurements on Arbacia, using a method which likewise allowed no flattening and again obtained a decrease in volume. He accounted for Chambers's results by assum- ing a cylindroid distortion f o r unfertilized eggs suspended from one point, and presented measurements to demonstrate that view. Snyder ('25) measured the volume change in eggs of Echinarachnius and of Asterina, without taking into account any flattening of the eggs. The Asterina eggs showed a marked decrease in volume. The Echinarachnius eggs were not observed sufficiently before the first division, but gave an increase in diameter during the early cleavage stages.

155

156 ALBERT TYLER

The determination of volume change is important in that it gives a measure of the osmotic changes occurring within the egg upon fertilization. If the osmotic pressure within the egg increases upon fertilization, an increase in volume is to be expected; if the osmotic pressure drops, a decrease in volume should occur. However, if the shape of the egg and the tension of the membrane change at the same time, the situation becomes more complicated.

The results of the measurements on eggs of Urechis reported here show a slight increase in volume upon fertili- zation accompanied by a very much more marked decrease in surface area. At the same time the eggs undergo interesting changes in shape.

The behavior of Urechis eggs upon normal fertilization has been previously described (Tyler, '31). I n connection with the volume and surface measurements, it is necessary to amplify the description somewhat, especially in regard to the changes in shape of the egg.

The unfertilized egg (figs. 1, 11, and 21) has a shape simi- lar to that which would be produced in a spherical rubber ball by indenting the surface at one point. The depth of the indentation averages about 46 per cent of the long diameter of the egg, varying from about 41 to 55 per cent. Upon fer- tilization, the indentation rounds out, but the manner in which this occurs differs in different eggs. The eggs can be grouped in three classes according to the changes in shape that occur upon fertilization. The egg of type I (figs. 1 to 10) becomes spherical at about three minutes after insemination, and then dents in again at about six minutes after insemina- tion. The extent of the second indentation is less than that of the unfertilized egg. The indentation then disappears again about ten minutes after fertilization and the egg gradu- ally attains a spherical form. The egg of type I1 (figs. 21 to 36) behaves similarly, except that the initial rounding out is not complete. The indentation diminishes in size, but does not disappear. It enlarges again, and then finally disappears at about ten minutes after fertilization. The egg of type I11

VOLUME AND SURFACE CHANGES ON FERTILIZATION 157

(figs. 11 to 20) rounds out within about three minutes and does not become indented again. Normal embryos are pro- duced by all three types of eggs.

The last type of behavior is characteristic of eggs freshly removed from freshly collected animals. Eggs from animals that have been in the laboratory for a few days or eggs that have stood in sea water for over an hour usually exhibit the first or second type of behavior-if the indentation is rela- tively small it rounds out completely the first time, if it is large the initial rounding out is not complete. It is inter- esting to note that a similar situation may be produced in a hollow spherical rubber ball if it is indented by removing some of the air, and kept in that condition f o r a few weeks. If the indentation be forced out by introducing air, it reap- pears as soon as the pressure is removed.

METHOD

The first determinations of volume were made by measur- ing certain dimensions of the egg with a screw micrometer and assuming the shape of the egg to be that of a sphere with a se,gment removed by another intersecting sphere of either the same or slightly different radius of curvature. However, the actual shape of the egg departs somewhat from that type of object, especially in the region where the turning in occurs. Also models of rubber balls of similar shape gave volumes calculated on the above basis that were about 5 per cent less than their actual volumes as measured by displacement in a cylinder of water. Another disadvantage of the ocular micrometer method is that after fertilization the egg is changing in shape while the measurements are being made.

In order to obviate these difficulties, the measurements were made photographically. This gives instantaneous readings, and the volumes and surfaces can be obtained quite accu- rately from projections of the photographs. The photo- graphs were made on motion-picture camera film, and the set-up was arranged so as to give a constant magnification. The scale of a stage micrometer was photographed before

THE JOURNAL O F EXPERIMENTAL 206LOGY, VOL. 63, NO. 1

158 ALBERT TYLER

and after each series. The photographs were projected on cross-section paper (the necessary precautions being taken against distortion of the image) and enlarged to 2500 times the diameter of the egg.

It is obvious that the indented eggs must be photographed in such a position that the outline of the indentation is obtained. Normally, however, the eggs tend to come to rest in the dish with the indentation down. The eggs were there- fore watched through the ocular of a prism divider while they were being photographed and manipulated into the proper position whenever necessary by means of a fine pipette.

The volumes of the indented eggs were obtained from the projections of their outlines by an adaptation of Pappus’ theorem. If we assume the shape of the egg to be that of the solid generated by revolving the area represented by one of the symmetrical halves of the photograph (fig. 1) about the axis of the egg, then-

in which y is the distance of the center of gravity of the area, A, from the axis, and

(1) V = S r r y A

or approximately

(3) Y =

and substituting in (1)-

~ z (y’ AA) A

(4) V = 2rz(y.AA)

in which AA is the area of successive small rectangles and y is the corresponding distance from the axis. By taking a sufficiently large number of small rectangles, the measured volume can be made to approach the actual volume of the solid of revolution as closely as desired.

I n the measurements reported here the number of AA’s determined depended on the diameter of the egg, and was usually about fifty-five to sixty. An estimate of the accuracy

VOLUME AND SURFACE CHANGES ON FERTILIZATION 159

thus obtained was derived from a comparison of the volumes of spherical eggs determined in that fashion with the volumes calculated from their diameters. For five eggs the differ- ences were found to be less than 1 per cent, and the average of the volumes determined graphically was found to be 0.4 per cent less than that determined from the diameters.

When the indentation disappears, the fertilized egg gen- erally remains flattened in that region for some time before becoming spherical (e.g., figs. 9, 10, 33). Its shape is similar to that of a sphere with a segment removed by a plane. The expression for the volume of such an object is

V = 2 z R R ” + ? r R 2 h - - h 3 7r

3 3

in which h is the distance from the center to the plane. The volumes of six eggs of that shape, determined both graph- ically and by use of the above formula, were found to differ by less than 1 per cent, with an average difference of 0.6 per cent.

The volumes of the flattened eggs were therefore deter- mined from the dimensions, as were also those of the spherical eggs, whereas those of the indented eggs were determined by the graphical method (equation 4).

The surfaces of the indented eggs were at first determined by means of Pappus’ theorem. The values obtained were found to agree within 1.5 per cent with those calculated from the long diameter assuming the surface area to be equal to that of a sphere of the same diameter. Since relatively large changes in surface were found to occur upon fertilization, this method could be used with sufficient accuracy for most of the measurements.

SYMMETRY O F T H E EGG

I n all of the calculations the eggs are assumed to be radially symmetrical. I f the eggs become flattened under their own weight as they lie in the dish and the extent of flattening varies after fertilization the results would be materially affected. Vl&s (’26) reported flattening in the

160 ALBERT TYLER

118.0

case of the sea-urchin egg and made use of it in determining the surface energy of the egg. More recently, McCutcheon, Luck&, and Hartline ('30) made measurements of Arbacia eggs in order to determine whether they were distorted under their own weight. They measured the diameters of eggs lying on the bottom of a water-tight chamber, and then, turn- ing the chamber over, they measured the same eggs suspended from the top. No significant differences in diameter were observed.

The eggs of Urechis were examined with the aid of a Zeiss prism rotator in order to determine this same point. By

T.IBLE 1

Diameters of unfert i l ized and fertiEized eggs observed f r o m above and from the side

18 109.2 109.8

D NFEXTILIZED

Top view

119.8 117.0 120.0 118.6 119.6 118.2 117.8 119.6

Side view

118.6 115.4 121.6 116.8 118.4 119.0 118.0

FERTILIZED

11 12 13 14 15 16 17

Top view

107.2 106.4 107.4 108.8 110.0 108.6 108.8

Side view ~~

107.4 105.8 108.2 108.0 109.6 108.6 107.8

I l l G L l l i U U I U J W G I I I U U G "I U V b I I b I I D I I " I l " " I I b U I Ul lU Y G I u'l l iaCI UILIIII-

eters of the same egg. Table I gives the results of such measurements made on unfertilized and fertilized eggs of Urechis. The precision of the measurements is relatively low, since a low magnification must be used with the prism rotator. At the magnification employed, sixty diameters, the microm- eter could not be read to better than about k0.5 p. However, this is sufficient to detect any serious flattening, if it were present. The results show that the vertical and horizontal diameters are equal within the limits of error of the apparatus.

VOLUME AND SURFACE CHANGES ON FERTILIZATION 161

RESULTS O F MEASUREMENTS

The changes in volume, surface area, and membrane area were followed photographically in seventeen eggs up to forty-five minutes after fertilization. Six of the eggs were of type I, five of type 11, and six of type 111. The results are given in tables 2, 3, and 4, and are expressed in per-

TABLE 2

Eggs of t y p e I . T h e indentation disappears at three minutes, reappears a t six minutes , and finally disappears a t ten minutes

TIME ABTER FERTILIZATION

IN M I N U T E S

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

10.00 12.00 15.00 25.00 35.00 45.00

VOLUME IN PER C E N T OF T H A T O F

UNFERTILIZED EGG P.E.,,

100.0 - 100.3 0.2 102.6 0.4 104.7 0.6 104.1 0.3 105.9 0.4 104.2 0.2 101.7 0.3 102.3 0.3 104.6 0.6 105.5 0.3 104.7 0.1 105.6 0.7 105.1 0.3 105.3 0.3 104.6 0.7

S U R F A C E A R E A I N P E R C E N T O F T H A T OF

UNFERTILIZED EQG P.E.,

100.0 - 97.1 0.5 91.9 0.2 84.2 1.0 84.1 0.6 85.8 0.2 85.4 1.1 89.8 0.7 94.5 0.4 96.7 0.4 93.2 0.5 90.7 0.5 91.4 0.9 86.8 0.3 84.7 0.2 84.4 0.4

MEMBRANE AREA IN PER C E N T O F THAT OF

UNFERTILIZED EGG P.E.,

100.0 - _ _ _ - - - 86.8 0.5 87.6 0.8 96.4 0.6

101.0 0.5 102.1 0.3 103.4 0.2 102.3 0.3 103.5 0.3 106.2 0.5 108.8 0.8 110.6 0.7 109.8 0.5

Average volume of six unfertilized eggs = 6.215 X 10' cubic micra. hverage surface area of six unfertilized eggs = 4.303 X 10' square micra. Average membrane area = 4.600 X l o 4 square micra. Temperature 21.0"C.

centages of the volumes and surface areas of the unfertilized eggs, and each value represents the mean of five o r six eggs. The probable error of this mean relative volume o r surface area is also given.

In table 2 the results of the measurements on the eggs of type I are presented. It is seen that the volume is increased by 4.7 per cent at three minutes after fertilization, which is the time of disappearance of the indentation. The volume

162 ALBERT TYLER

remains at about this value until six minutes after fertiliza- tion. At this time the indentation reappears and the volume drops to about 102 per cent of the volume of the unfertilized egg at seven to eight minutes after fertilization. The volume then increases to 5.5 per cent more than the unfertilized volume at ten minutes after fertilization, which is the time at which the second indentation finally disappears. It remains at about this value through the time at which the first polar body is given off (thirty minutes after fertilization) and takes a slight drop after the second polar body is given off (forty minutes after fertilization).

The changes in surface area of the same eggs are just the reverse of the volume changes and are of much greater mag- nitude. As the indentation rounds out, the surface area drops to about 84 per cent of the surface area of the unfer- tilized egg. Then as the indentation reappears the surface area increases to about 97 per cent, and finally, after the second rounding out, it drops to about 85 per cent. It appears then that as a result of fertilization the volume of the egg shows a slight increase, whereas the surface area shows a marked decrease.

The unfertilized egg of Urechis is surrounded by a rela- tively tough membrane of about 0.3 1-1 in thickness. It is separated from the surface of the egg by a perivitelline space of about 1.0 p in thickness. The question may now be raised as to whether membrane elevation in this egg is due to the surface area of the membrane remaining constant while the surface of the egg decreases. An investigation of the changes in membrane area shows that this is not the case. The measurements were made to the inner surface of the mem- brane, and included the perivitelline space. Thus the diam- eter used to calculate the surface area of the membrane is 2 p greater than that used to calculate the surface area of the unfertilized egg. The results are presented in the last sec- tion of table 2. Membrane elevation does not occur until four to five minutes after fertilization, so the changes in mem- brane area up to that time are practically identical with the

VOLUME AND SURFACE CHANGES ON FERTILIZATION 163

changes in surface area of the egg. Thus the membrane of the egg apparently shrinks immediately after fertilization and does not regain its original size until seven minutes after fertilization. The membrane area then increases to about 110 per cent of that of the unfertilized egg.

The changes in volume and surface of the eggs of type I1 are quite similar. In this type of egg, as noted above, the indentation does not disappear completely the first time. It diminishes markedly in size up to three and one-half minutes after fertilization, then enlarges up to about seven minutes, and disappears at ten minutes. Different eggs of this type differ in the extent to which the indentation rounds out initially. For the egg shown in figures 21 to 36 the initial rounding out is almost complete (figs. 25 and 26). But in other cases the initial rounding out may not proceed past the stage shown in figure 23.

The results of measurements on five eggs of this type are given in table 3. The volumes show a slight increase up to three minutes after fertilization, then a decrease to seven minutes after fertilization and finally an increase as the indentation disappears. The volume after the extrusion of the polar bodies is slightly less than before that time. The surface area shows a decrease to 91.6 per cent at three min- utes and then as the indentation rounds out and the polar bodies are given off it drops to 82.4 per cent. Thus the decrease in surface area is even greater in this ease than in the previous one.

The changes in surface area of the membrane are in the same direction as in the previous type of egg. Membrane elevation occurs at five to six minutes after fertilization, and the changes in membrane area up to this time are practically identical with the changes in surface area of the egg. The membrane then enlarges to its original size at seven to eight minutes after fertilization and at the time of polar body extrusion reaches a value 10 per cent greater than that of the unfertilized egg. Thus it is again seen that the membrane shrinks after fertilization and then later swells.

1% ALBERT TYLER

In the eggs of type 111, as pointed out above, the indenta- tion rounds out at three minutes and does not reappear later. The results of measurements on six eggs of this type are given in table 4.

The volume again shows a slight increase at three to four minutes after fertilization when the egg rounds out, followed

TSBLE 3

Eggs of t y p e I I . T h e indentat ion diminishes (but doesn't disappear) u p t o three and one-half minutes, enlarges u p t o seven minutes , and finally disappears a t t e n minutes

TIME AFTER FERTILIZATION

IN MINUTES

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

10.00 12.00 15.00 25.00 35.00 45.00

VOLUME I N PER CENT O F T H A T O F

UNFERTILIZED EQQ P.E.,

100.0 - 99.8 0.2

101.6 0.4 103.4 0.4 102.5 0.2 101.9 0.6 101.2 0.5 101.9 0.1 103.8 0.6 103.0 0.4 105.2 0.1 104.8 0.4 106.7 0.4 105.6 0.2 104.8 0.5 104.7 0.6

S U R F A C E A R E A I N P E R C E N T OF THAT OF

UNFERTILIZED EQG

P.E.,

100.0 - 97.8 0.3 94.8 0.6 91.6 0.5 92.9 0.5 93.9 0.1 94.8 0.3 97.6 0.4 94.1 0.8 89.8 0.8 85.0 0.3 86.6 0.1 83.7 0.4 83.0 0.3 82.4 0.4 82.4 0.4

MEMBRANE AREA fN PER C E N T O F THAT OF

UNFERTILIZED EQG P.E.,

100.0 - _ - - - - - - _ 94.5 0.1 96.0 0.4

100.4 0.3 99.1 0.6

103.4 0.7 104.7 0.5 105.2 0.2 106.9 0.2 109.8 0.6 108.7 0.5 110.3 0.7

Average volume of five unfertilized eggs = 6.329 X 106 cubic micra. Average surface area of five unfertilized eggs = 4.448 X 10' square micra. Average membrane area = 4.753 X l o 4 square micra. Temperature 21.0"C.

by a slight drop at five minutes. Later, the volume increases to a value 5 per cent greater than that of the unfertilized egg and then drops to 3 per cent when the polar bodies are ex- truded. The drop at five minutes is only slightly greater than the probable error, but it may be of some significance, inaa- much as it coincides with a similar drop in the types of eggs previously considered. It is also of interest to note that mem- brane elevation begins at this time.

VOLUME AND SURFACE CHANGES ON FERTILIZATION 165

The surface area of this type of egg also shows a decrease up to four minutes after fertilization while the volume is increasing. But after that time its changes follow those of the volume in direction. The magnitude of the change in surface area is not as great as in the previous cases, its value after the extrusion of the polar bodies being 13 per cent less than that of the unfertilized egg.

TABLE 4

Eggs of t y p e III. Tho intlrntntion disappears at three' minutes

TIME AFTER FERTILIZATION

I N MINUTES

VOLUME I N PER CENT OF THAT OF

UNFERTILIZED EGG P . E . ,

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

10.00 12.00 15.00 25.00 35.00 45.00

100.0 - 98.9 0.6

101.4 0.4 103.3 0.6 103.0 0.7 101.6 0.6 102.6 0.1 102.4 0.2 102.3 0.6 103.1 0.4 103.7 0.2 105.3 0.3 105.1 0.6 104.0 0.2 103.0 0.2 103.1 0.3

SURFACE AREA I N PER CENT OF THAT OF

UNFERTILIZED EGG P.E.,

100.0 - 94.7 0.5 92.6 0.6 88.8 0.6 87.7 0.1 86.0 0.3 86.5 0.4 86.4 0.7 86.4 0.2 86.9 0.2 87.2 0.3 88.0 0.1 88.8 0.6 88.1 0.7 87.0 0.2 87.0 0.2

MEMBRANE AREA I N PER CENT OF THAT O F

UNFERTILIZED EGG P.E.,

100.0 - - - - - - - - - 91.4 0.4 98.2 0.8

101.9 0.7 103.7 0.5 104.6 0.5 104.0 0.8 107.6 0.3 109.0 0.7 111.8 0.6 110.3 0.3 111.5 0.3

Average volunie of six unfertilized eggs = 6.229 X 10' cubic micra. Average surface area of six unfertilized eggs = 4.133 X lo4 square micra. Average membrane area = 4.398 X 10' square micra. Temperature 21.O"C.

I n regard to the changes in membrane area, this type of egg behaves similarly to the other two types. The membrane first shrinks after fertilization and then swells until about twenty-five minutes after fertilization when it attains a roughly constant value 11 per cent greater than that of the unfertilized egg.

THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 63, NO. 1

166 ALBERT TYLEI:

DISCUSSION

The three types of eggs described thus agree in showing a slight increase in volume accompanied by a marked decrease in surface as the indentation rounds out initially. The first two types of eggs then tend to go back to their original size and shape, and again show a slight increase in volume and a marked decrease in surface as the indentation rounds out the second time.

All three types of eggs also agree in showing a decrease in the size of the membrane as the indentation rounds out fol- lowed by an increase as the membrane separates off from the surface of the egg. This initial shrinkage of the membrane may be due to one or both of two causes. The elastic tension of the membrane may increase upon fertilization or the mem- brane may be under tension initially and the shrinkage due to a change in the contents of the egg.

I n order to obtain evidence bearing on this last point some rough measurements were made of the changes in viscosity of the egg on fertilization. A hand centrifuge of 16 em. radius was used f o r the determination. It was operated at 2000 r.p.m. The eggs were centrifuged for various lengths of time, ranging from thirty seconds to two minutes before fer- tilization and at various times after fertilization. The unfer- tilized and the fertilized eggs were usually centrifuged at the same time on the machine. The results show that the fer- tilized eggs stratify much more readily than the unfertilized eggs. Thus it required two minutes’ centrifuging to give an indication of stratification in the unfertilized egg, whereas at two minutes o r more after fertilization the same result was obtained after one to one and one-half minutes’ centrifuging. Two minutes’ centrifuging at two or more minutes after fertilization gave a quite clear-cut oil cap of about 20 I.I in diameter and a definite pigment zone. It appears, then, that a decrease in viscosity does occur upon fertilization.

Attempts were also made to determine whether the mem- brane of the egg is originally under tension, by immersing the egg in hypertonic solutions of cane sugar in sea water.

VOLUME AND SURFACE CHANGES ON FERTILIZATION 167

The egg decreases in volume in the hypertonic solution, and the indentation becomes larger. However, the long diameter of the egg remains constant and the surface area does not change. If the membrane were initially under tension one might expect a decrease in its area in the hypertonic sea water. But the failure to decrease in area may be due to some factors in the structure of the egg that determine that the long diameter remains constant as the egg shrinks, instead of the shrinkage taking place uniformly throughout the egg. It cannot then be concluded from this result that the membrane is not initially under tension. It may be noted in this connection that when the egg is allowed to swell in hypotonic sea water the swelling takes place first in the region of' the indentation, the long diameter and the surface area remaining constant up to the time at which the indenta- tion disappears. It may appear that the lower hemisphere of the unfertilized egg possesses a fairly rigid structure, the rigidity diminishing toward the indented region, so that changes in shape in hypo- or hypertonic solution take place mainly in the latter region.

The unfertilized eggs of Urechis are not entirely unique in possessing a large indentation. The eggs of Nereis limbata show a small indentation before fertilization and the extent of the indentation may be greatly increased by treating the eggs for about ten minutes with 40 per cent sea water and then returning them to normal sea water. The eggs of Cumingia tellinoides, after twelve minutes' treatment with 20 per cent sea water, also show an indentation even while in the dilute sea water.

If the tension at the surface were known, it should be pos- sible to calculate, from the changes in surface area, the work done in effecting the changes in shape recorded for the Urechis egg. This information would be important in deter- minations of the energy utilized upon fertilization and early development.

The decrease in surface area upon fertilization is also probably not unique for Urechis eggs. A good many marine

168 ALBERT TYLER

eggs are more spherical after fertilization than before. Such is the case, for example, with eggs of Dendraster excentricus, Patiria miniata, and Nereis limbata. If the volume remains constant or only increases slightly upon fertilization in these cases, then a decrease in surface area must occur.

I n another type of cell, the mammalian red blood cells, an interesting change from the typically biconcave discoidal shape to that of a sphere has been observed by Hamburger (,95) upon transfer from plasma to isotonic salt solution. Ponder ('29) studied the phenomenon more recently and found that it was necessary for the cells to be placed between two wetted flat surfaces, a certain minimum distance from each other, in order that the change may occur. The volume of the cell remains constaiit, so the change in shape involves a large decrease in the surface area. But the comparison of the change in shape of the Urechis egg with that of the mam- malian erythrocyte does not help very much since the ex- planation in the latter case is as obscure as in the former.

SUMMBRY

1. The eggs of Urechis exhibit three types of behavior in regard to the changes in shape upon fertilization. I n one type the indentation disappears at three minutes, reappears again at six minutes, and disappears finally at ten minutes. In the second type the initial rounding out of the indentation is not complete, but the rest of its behavior is the same as type I. In the third type the indentation rounds out at three minutes after fertilization and does not reappear.

2. The volume increases slightly upon fertilization in all three types. Fo r the first two types the reappearance (or enlargement) of the indentation is accompanied by a slight decrease in volume.

3. The surface area decreases markedly upon fertilization in all three types. For the first two types the reappearance (or enlargement) of the indentation is accompanied by a definite increase in surface area.

VOLUME A N D SURFACE CHANGES ON FERTILIZATION 169

4. The surface area of the membrane decreases initially upon fertilization in all three types and later increases to a value greater than that of the membrane of the tmfertilized egg. The initial decrease in the size of the membrane appears to be correlated with a decrease in the viscosity of the egg upon fertilization.

BIBLIOGRAPHY

CHAMBERS, R. 1921 Microdissection studies, 111. Some problems in the maturation and fertilization of the echinoderm egg. Biol. Bull., vol. 41, p. 318.

GLASER, 0. 1913 On inducing devclopmcnt in the sea-urchin (Arbacia punctulata), together with considcrations on the initiatory cffect of fertilization.

1914 The change in volume of Arbacia and Asterias eggs a t fer- tilization.

1924 Fertilization, cortex, and volume. Biol. Bull., vol. 47, p. 274. 1908 Uber die osniotischen Eigeiischaften und die Entstchung der Befruchtungsiiiembraii beim Seeigelei. Arch. Entwmech., Bd. 26, S. 82.

1910 Further proofs of the increase in permeability of the sea urchin’s egg to clcctrolytes at the beginning of development. Science, vol. 32, p. 318.

The osmotic properties of living cells (egg of Arbacia punctulata). Jour. Gen. Phys., vol. 14, p. 393.

OKKELBURG, P. 1914 Volumetric changes in the egg of the brook lamprey, Entosphenus (Lampctra) Wildcri (Gagc), after fertilization. Biol. Bull., vol. 26, p. 92.

PONDER, E. 1929 On the spherical form of the mammalian erythrocyte. Brit. J. Exp. Biol., vol. 6, p. 387.

SNYDER, C. n. 1925 Egg-volume and fertilization membrane. Biol. Bull., vol.

TYLER, A. 1931 The production of normal embryos by artifieial partheno- genesis in the echiuroid, Urechis.

The relation between cleavage and total aetiration in art i- ficially activated eggs of Urechis. 1926 Les tensions de surface et les dBformatioiis de l’oeuf d’Oursin. Arch. Phgs. Biol., T. 4, p. 263.

Science, vol. 38, p. 446.

Biol. Bull., vol. 26, p. 84.

LOEB, J.

MCCLENDON, J. F.

MCCUTCHEON, M., B. LUCKB, AND H . K. HARTLINE 1930

49, p. 54.

Biol. Bull., vol. 60, p. 187.

Biol. Bull., vol. 61, p. 45. 1931

V L ~ , F.

PLATE I

EXPLANATION OF FIGURES

Changes in shape upon fertilization

1 t o 10

11 t o 20

Photoniicrographs of a single egg of type I at 0, 1, 2, 3, 5 , 7, 8, 9,

Photomicrographs of a single egg of type 111 a t 0, 1, 2, 3, 5, 7; 12, 10, and 15 minutes after fertilization.

20, 25, and 35 minutes after fertilization.

170

VOLUXE AND SURFACE CHBNGES ON FERTILIZATION ALBERT TYLER

PLATE 1

1 7 1

PLATE 2

EXPLANATION OF FIGURES

Changes in shape upon fertilization

21 t o 36 Photomicrographs of a single egg of type I1 at 0, 1, 2, 3, 34, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, and 35 minutes after fertilization.

VOLUME AND SURFACE CHANGES ON FERTILIZATION AliRlRT TYLER

PLATE 2

173