Book - Chemical embryology 2-5 (1900)
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Needham J. Chemical Embryology Vol. 2. (1900)
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Chemical Embryology - Volume Two
Section 5. Biophysical Phenomena in Ontogenesis
5-1. The Osmotic Pressure of Amphibian Eggs
A considerable number of workers have turned their attention to measurements of osmotic pressure in eggs and embryos, in the attempt to throw some Hght either, in the case of aquatic embryos, on the relation between the embryo and its environment, or, in the case of birds, for instance, on the relations as regards water and salts, between the different components of the ovum.
The best known part of the work has been done on the eggs of amphibia. The fundamental observation was made by Backmann & Runnstrom in 1909 that the osmotic pressure of frog's egg-Breis was very different according to the stage of their development. They obtained the following figures :
A (depression of the freezing-point) (°C.) Ripe ovarial eggs ... ... -0-48
Fertilised eggs ... ... —0-045
Embryos of 5 days ... -0-23
Tadpoles of 20 days ... —0-405
Serum of adult ... ... -0-465
From these simple observations, which have often been confirmed, all the subsequent work took its origin. They were in many ways interesting, for the A of ordinary pool fresh water was found to be — o-o6, so that after the eggs were shed from the oviduct they evidently adjusted their osmotic pressure to equal that of their immediate environment. Then, later, they acquired some kind of independence, and by the time of hatching were well on their way to attaining the adult osmotic pressure. The question as to how the initial fall in osmotic pressure was achieved was not easy to answer. It might be accounted for, of course, on the supposition that the eggs took up water from their hypotonic medium, and diluted their contents, but if this had been so the increase in volume would have been far greater than was actually found. Backmann & Runnstrom preferred to assume that the egg-colloids were rapidly gelated after the eggs were shed, and inorganic ions adsorbed on to them, a process which would certainly lower the osmotic pressure, but would have to be reversible. Whatever the mechanism, the facts showed that in its earliest stages the frog's egg was poikilosmotic, becoming homoiosmotic as it developed; a conclusion of general interest in view of all the work reviewed in the last section, which demonstrates a passage from poikilothermicity to homoiothermicity in the case of embryos during their development.
In later papers Backmann & Runnstrom extended their researches at length. They found that amphibian eggs would not develop normally in solutions isotonic with adult serum or ovarial eggcontents, a strong indication that the behaviour of the osmotic pressure with time was a physiological phenomenon. This behaviour they studied at shorter intervals, and obtained the curves shown in Fig. 177. The isotonicity of the freshly fertilised egg was maintained, they found, during the formation of the blastula, and through gastrulation, but a rise occurred in the late period of the latter, continuing steadily for some time until the osmotic pressure reached about half the final value. Here there was a change in the rate of increase, which became much slower. Neither the closure of
a Bialascewic3 A (°)
© Backmann Si.as80ciabes A (°)
• Davenport water content
■ Schaper ?> >'
•^90
70
50
•2 2 Fertilisation
10 12 14 16 Days
Fig. 177.
22 24 26 28 30
the neural groove nor hatching affected it, and the rise continued without change until the osmotic pressure of adult serum was attained on or about the 25th day. These time relations held for Rana temporaria, but they were found to be applicable with variations to the eggs of many other amphibia. Backmann & Runnstrom found that parthenogenesis by hypertonic solutions induced at any rate the first of these changes, none of which, indeed, took place in unfertilised eggs. Micrometer measurements demonstrated that during the first few days exceedingly slight changes take place in egg-volume, quite insufficient in magnitude to account for the fall by simple dilution. E.g. :
% increase in diameter 64-cell stage->blastula ... 0-5
Blastula-^closure of blastopore 0-4
Closure^" 1 2 hours after closure 4-9
Diameter of unfertilised egg 3'537 mm.; fertilised 3*79 mm.
During the later ascent of the osmotic pressure cur\'e, there was a certain growth in volume, but Backmann & Runnstrom only gave a few fragmentary figures for this. The chorionic or perivitelline liquid seemed to be hypotonic to the 5-day old embryo, but hypertonic to the pool water, i.e. about — 0-15°. Backmann & Runnstrom regarded the change in the osmotic pressure of the egg as not of any recapitulatory significance, because some experiments which they made on land frogs gave the same results. Although the eggs normally developed on dry land, they showed the same osmotic pressure changes. They were more inclined to see in these effects an adaptation mechanism which had been retained by the land frogs although of no further use.
Backmann & Sundberg next confirmed de Varigny, who had stated in 1888 that solutions of different osmotic pressure were isotonic with frog's eggs at different stages of their development, without giving any experimental figures^. They also compared their gradually rising curve for osmotic pressure during larval life with the figures for water-content previously found by Davenport and Schaper. The result is shown in Fig. 178. There was undoubtedly a rise in watercontent, which came to a level plateau at about 94 per cent, by the 20th day at the latest, and this rise was accompanied by the rise in osmotic pressure. Backmann & Sundberg regarded this finding as very significant, reveahng as it did something of the mechanism by which the water-content of the embryonic cells was raised in the later stages. The entry of the water was, they thought, the factor which assured the maintenance of osmotic pressure within reasonable Hmits, as the safine ions, and perhaps other crystalloids, passed into solution
- 1 It must be remembered throughout this section that experiments of the type of de Varigny's, unlike freezing-point measurements, only give the osmotic pressure of the substances within the egg to which the membrane is impermeable.
Fig. 178.
from the adsorbed condition. Backmann & Sundberg found that if unfertilised eggs of Rana temporaria were placed in distilled water there was no sudden fall of osmotic pressure as in the fertilised eggs, but a gradual swelling due to water-absorption and a decrease of A from — 0-48° to — 0'35° in 3 hours, i.e. to the stage reached normally by embryos by about the 20th day of development. This showed that, whatever the explanation of the normal behaviour might be, it was not due to simple absorption of water. From these premises it was to be expected that organs or tissues of frog embryos would react differently to salt solutions, if taken from different developmental stages, and this was found to be the case by Harrison, who observed that explants of frog spinal cord from 4-5-day embryos would not grow in 0-7 per cent, salt solution (isotonic with adult serum), but would do so perfectly well in 0-4 per cent. Backmann & Sundberg pointed out that this corresponded with a A of — 0-245°, ^^^ ^^ could be explained perfectly on the basis of Fig. 178.
The work was continued by Backmann, who measured with a micrometer the swelling of frog's eggs in different solutions. Previously some observations on this point had been made by Wilson and by Tonkov, but they were more concerned with the morphological changes caused by salt solutions. This technique afforded a means of checking the measurements already made of the osmotic pressure of the egg-contents, only now Backmann worked with the eggs of Bufo vulgaris and Triton cristatus. In both cases the unfertilised eggs remained without change of diameter for many hours in solutions of A — 0-44°, but the fertilised eggs (morulae) remained without change of diameter in solutions of A — 0-02°. The former eggs placed in the latter solution swelled up by 2-5 per cent, in 48 hours, the latter eggs placed in the former solution shrank by i • 2 per cent, in 24 hours. There was thus every indication that the results previously obtained on the frog held also for the toad and the newt. In these cases also, unfertilised eggs would have a A of — 0-45° and the fertihsed ones — 0-02°. The only difference between the frog on the one hand and the toad and the newt on the other was that, in the former case, there was no perceptible increase of volume after fertihsation, and, in the two latter cases, there was a slight normal increase.
In 1900 Bataillon had studied the conditions under which the eggs of the lamprey, Petromyzon planeri, would develop. He found that the fertilised eggs of this cyclostome would not develop properly in salt solutions above 0-2 per cent. In 0-5 per cent, the process would not go further than the gastrula stage, in o-6 per cent, only as far as the morula, while in o-8 per cent, no further than the i6-cell stage. From these data Backmann calculated that the osmotic pressure of the egg-interior in the morula stage of Petromyzon must be about A — 0-125°. -^o figures are available for the A of the adult serum of Petromyzon planeri, but Dekhuysen got a value of — 0-49° for Petromyzon fluviatilis, from which it is probably legitimate to conclude that events proceed in the cyclostome tgg just as in that of amphibia. The only fact still wanting is the osmotic pressure of unfertilised ovarial Petromyzon eggs. Bataillon's data on Ascaris eggs might be treated in a similar way.
Later, Backmann, Sundberg & Jansson studied the effect of excess and lack of oxygen on the osmotic pressure curve during embryonic and larval life in the frog. Their results (which were never reported in full) are shown on Fig. 177. Development in almost pure oxygen led to a precocious augmentation of the osmotic pressure rise. They did not state, however, whether development in almost pure oxygen led to any shortening of the hatching time, or to more rapid differentiation or growth. Lack of oxygen was produced by causing the eggs to develop in water covered by a thick layer of paraffin oil. In such circumstances, the osmotic pressure of the eggs followed the usual course, until at about the 5th day the rise began to cease, and the gastrulae died shortly afterwards with an osmotic pressure of from A — 0-09° to A — 0-055°^. The membranes seemed to lose their elasticity from the 3rd day onwards, and there was great swelling. Unfertilised eggs placed under similar conditions cytolysed after only 24 hours, their osmotic pressure having fallen to isotonicity with the pond water. Backmann went on to study the effects of temperature. Kept at 30 to 40° the eggs swelled a good deal, but in spite of that they had an osmotic pressure by the 3rd day which was up to normal, or a little above it (A — 0-048 at 40° and A — 0-040 at 30°). Kept at 5 to 6° the development was much retarded, and the rise in osmotic pressure, though at first behaving quite normally, was in its second phase much drawn out. Thus on the 21st day the eggs at the low temperature would have a A of — 0-30°, while eggs at normal temperature (17°) would have — 0-39°, and eggs at normal temperature but of the same morphological stage as the cold ones would have — 0-25°. Backmann concluded that all these experiments showed a close association between morphological development and osmotic pressure, the latter entity being unable to vary more than a little independently of the former. As the temperature coefficient for osmotic pressure is exceedingly small, that entity must be dependent in its turn on some process with a marked temperature coefficient, i.e. whatever reaction or reactions were controlling the growth-process as a whole (see also on this p. 910).
1 Thus lack of oxygen abolishes the mechanism which maintains the osmotic pressure difference, but the parallel with the vitelline membrane of the hen's egg is not close for here the embryonic development is affected too (see p. 817).
Thunberg criticised the statement of Backmann & Runnstrom that frog's eggs would not develop in solutions isotonic with the adult blood serum, and cited some not very good work of Overton's in support of the opposite view. But the observations of Backmann and his associates were later confirmed by Bialascewicz, who found the fall of osmotic pressure on fertilisation, the subsequent rise in osmotic pressure and the corresponding rise in watercontent. Citing the experiments of Siedlecki, who had found the embryos of the Javanese frog {Polypedatus reinwardtii) incapable of developing in normal pond water if removed a day or two early -0-350from their envelopes, Bialascewicz concluded that the perivitelHne liquid was not by any means ordinary water, but contained osmotically active substances which could not diffuse out through the eggmembrane, and so maintained a definite pressure gradient
Fig- 179
involving constant tension on the envelopes. The values which Bialascewicz obtained for osmotic pressure are shown in Fig. 179. He also made osmotic pressure determinations on ovarial eggs of other anura {Rana esculenta — 0-446°, Bombinator igneus — 0-445°). For ovarial eggs his A was — 0-444°, ^^^ ^^^ adult serum -- 0-479°, a difference which he emphasised as resembling the corresponding difference in avian eggs. He explained the initial fall of osmotic pressure in the egg itself rather by an excretion of osmotically active substances into the newly formed perivitelline space than by an adsorption on to colloidal particles. Thus the perivitelHne fluid would be a salt solution of definite strength, and he had himself previously shown that the embryos of Salamandra and Molge would develop only in Ringer-Locke solution if removed from their envelopes before hatching, and not at all in pond water. With regard to the later rise in osmotic pressure, Bialascewicz pointed out that the adsorption explanation of Backmann & Runnstrom could not be right in view of the fact that the last traces of the yolk disappear some time before the osmotic pressure has reached its final level. On other grounds, moreover, Bialascewicz rejected any association between osmotic pressure and water-content, and certainly the curves in Fig. 178 do not go very well together. He preferred to postulate an increase in osmotically active substances derived from the food, and an important regulatory action on the part of the kidneys, organs now (in the later stages) quite functional in selectively retaining or excreting crystalloids.
The work of Backmann and his collaborators, and of Bialascewicz, was again confirmed by Przylecki, who paid special attention to the role of the perivitelline fluid. In contradiction with Backmann, however, he found that fertilisation was not the governing factor in the lowering of internal osmotic pressure in the eggs of Rana temporaria and of Triton cristatus, but that this occurred whether fertihsation took place or not. Absorption of water is not responsible, but rather the excretion of salts and water to form the perivitelline fluid. Przylecki (working on Triton taeniatus) agreed with Bialascewicz in not getting such a high freezing-point as Backmann for the just-fertilised eggs, i.e. — 0-20 instead of — 0-045°. I^i a second paper, Przylecki studied the conditions necessary for the formation of the perivitelHne space in the unfertilised frog's tgg. The chief of these was the hypotonicity of the surrounding medium, a difference in freezing-point of only a few hundredths of a degree, however, being sufficient to set the process in motion. The trigger mechanism is probably the entry of a small amount of external water into the egg. The egg has to remain at least 30 minutes in the hypotonic medium, but after from 5 to 7 hours in a wet chamber the eggs, placed in water, can still develop perivitelHne spaces. Electrolyte solutions of all kinds hinder the formation of the perivitelHne space, from A — o- 1° or o-o8° onwards. Oxygen seemed to be necessary, for eggs in hydrogen and water would not produce the space, although all other conditions were favourable. Augmentation of 10° doubles the speed with which the perivitelline space is produced.
Work on the frog's egg was continued by Voss in 1926. By micrometric measurements he found that the size of the perivitelline space was directly dependent on the osmotic pressure gradient between the inside and the outside of the tgg. In distilled water it was greatest, in 0-004 P^^ cent, sodium chloride less, and so on. Natural isotonicity was with 0-05 per cent, sodium chloride; the space was then normal in size. Over a considerable range it acted as an accurate biological osmometer, but ceased to do so when the concentration of salts outside became very high. During development the permeabiHty of the egg-membranes varied ; shortly after fertilisation, salts were let out, but not in the later stages. The more eggs developed in a given volume of water, the more ions diffused out in the early stages, and the smaller the perivitelline spaces were. As for the egg-jelly, it consists of two concentric portions, both of which are very sensitive to the concentration of ions in the external water, as may be judged by their rapidity of sweHing after the eggs are laid (see p. 323). Hykes has since found that in ordinary water frog's eggs develop quicker when freed from their jellies than they do inside them. In distilled water, however, they will not develop except within the jellies.
Voss's work agreed very well in general with that of McClendon. McClendon parthenogenetically fertihsed the eggs of the leopard frog, Ranapipiens, by electrical stimulation in distilled water, and then estimated the salt content of the water after a definite interval. The results were as follows:
Chlorine (as c.c. Total ash in
i/ioo JV) in water mgm. after
after 7 hours 7 hours
Unfertilised ... i-2 6-6
Fertilised 2-05 1 9-6 1
Stimulated ... i-q8j ^ °' ii-o
10-3
The ash apart from the chloride contained sodium, lithium, potassium, calcium, magnesium, sulphate and carbonate, but no phosphates. There was no doubt that the diffusion of salts from the fertilised eggs (whether naturally or artificially) was nearly double that from the unfertilised eggs. McClendon had previously shown that in the case of sea-urchin's eggs a great many parthenogenetic agents caused an increase in permeabiHty of the egg-membranes, and he regarded this as a general phenomenon. He thought that fertilisation, by allowing increased permeability, permitted the eggs to live, while if they were not fertilised they would swell up and die. Experiment showed that the mean diameter of unfertilised eggs placed in water for 30 minutes was 1-52 mm., while the mean diameter of fertilised ones was only 1-47 mm. These relations confirmed Backmann's results; Backmann, indeed, had found that the unfertilised salamander egg burst in 2 to 7 hours, if placed in water, but not if placed in salt solution. McClendon observed that, if the fertilised eggs were placed in salt solutions, development often ceased at the gastrula stage, and he concluded that the exit of salts from the egg was a physiological necessity.
Bialascewicz's views on the importance of the perivitelline fluid were accepted by McClendon, who analysed it in the cases o^ Amblystoma and Cryptorhyncus , and found salts and organic substances, but only traces of protein (o-i6 per cent, dissolved solids). Bialascewicz's finding, that immediately after fertilisation there was a measurable decrease in the diameter of the egg-cell, evidently reflected the formation of the perivitelline fluid. Backmann & Runnstrom had suggested that the fluid might be secreted by the suckers of the embryo, but the case of the salamander, which has no suckers, is contrary to this view. The normal course of events in the amphibian egg, therefore, after laying, is as follows : if fertilisation occurs, the osmotic pressure of the egg drops sharply to a low value, perhaps because of the intracellular fixation of osmotically active substances, but more probably because some of these are excreted into the perivitelline fluid, and to a certain extent into the surrounding medium. Then there occurs a gradual rise, leading back to the initial value, probably brought about by the increase of osmotically active substances produced in metabolism, and not by any absorption of salts from without. The skin of the adult frog absorbs water at a rapid rate, and if it were not for the action of the kidney the animal would die of oedema. The pronephros develops and is believed to be functional well before hatching, so the frog embryo and larva is well protected against this possibility.
5-2. The Genesis of Volume-regulation
More recently the problem of the origin of water regulation in amphibia has been investigated by Adolph, who studied the change in volume or weight when different developmental stages of Rana pipiens were placed in various solutions. The unfertilised eggs sometimes swelled, and sometimes shrank, but in none of the experiments did the change in volume follow exactly the concentration of the environmental salt solution. This was in contradiction with Backmann's results, but, although his were regular, they never exceeded plus or minus 5 per cent., while Adolph's were of the order of 30 per cent. As volume decreased in some dilute salt solutions, Adolph concluded that the egg-membranes were not completely permeable to sodium chloride. The next stage investigated was the yolk-plug stage. "The results were unexpectedly irregular and allowed of no precise definition of the relation between volume and concentration. Possibly the egg is comparatively semipermeable in the dilute solutions, but in stronger ones is made completely permeable to solutes." This would explain the relationships seen in Figs. 180, i8i and 182, taken from Adolph's paper. After hatching, the response became more regular, and predictions of the results could be made. Adolph concluded that from the unfertilised tgg up to metamorphosis no marked changes in regulatory activity occurred in Rana pipiens. The only definite diflference was that the eggs and embryos lost relatively less in volume when immersed in strong salt solutions (such as o-20 M), and the hatched larvae lost relatively more.
Fig. 180. Unfertilised eggs. A, Loss in volume; B, gain in volume.
Adolph also made a very interesting observation in the later stages. Many observers had found that adult frogs gained weight in dilute salt solutions, but that tadpoles lost weight in solutions of the same concentration. Adolph found that there was a change over as regards this property about i| days after the appearance of the fore limbs, and, in his opinion, it was quite sharp. At exactly the same time well-marked morphological changes occurred in the skin.
Fig. 181. Embryos at the yolk-plug stage.
"Extrusion of eggs from the body", says Adolph, "is followed by a gradual increase of volume by entrance of water from the freshwater medium. Fertilisation changes all this and enables electrolytes to get out of the egg so that now concentration is sacrificed while volume is kept constant. Before hatching, the ability of the embryo to hold its electrolytes is regained and the ability to absorb solutes from a medium very dilute in them is acquired (Krizenecki). During this period the ability to regulate volume is sacrificed slightly, but with gain in mass it becomes possible for the larva to hold the volume as well as the concentration, and the losses are exactly compensated for on return to water. Soon after hatching, the tadpole is very independent of its medium with respect to both concentration and volume. At metamorphosis a change occurs in the mode of regulating in various solutions. This is the ontogenetic history of the water relations of tadpoles." Adolph pointed out, with reference to the increasing water regulation apparent from the results of Backmann and his collaborators, that the most important factor was probably the embryonic ectoderm, for there is no evidence that the egg-membranes play any part in regulating the distribution of water between the body of the embryo and the environment of the egg.
Tadpoles of 2-7gm. Forelegs appeared 1 day after 2daj/3 after 3da^s after 4 days after Tail disappeared Adults of 16gm.
Age Change of volume in o-o8 M NaCl related to age
Fig. 182. The development of the power of regulating body-volume in Rana pipiens.
Belehradek & Huxley subsequently found that the water-regulating power of Amblystoma ; imperfect during the larval state, assumes its adult efficiency during metamorphosis. Konopacki's conclusions are also in general agreement with those of Adolph. He studied the effects of distilled water on amphibian eggs and embryos, and showed the coming-into-being of a regulatory mechanism, probably resident in the embryonic ectoderm. Konopacki also confirmed many of Bialascewicz's results with reference to the function of the perivitelline liquid in the frog's egg.
5-3. The Osmotic Pressure of Aquatic Arthropod Eggs
The osmotic behaviour of the amphibian egg is to some extent paralleled by that of the eggs of cladocerans, on which Przylecki has made notable studies. He found that the youngest embryos in his series had the lowest osmotic pressures; thus at 6 hours it was A — 0-245° for Simocephalus vetulus, and — 0-186° for Daphnia magna. As development pro
ceeded, the pressure steadily rose, reaching at 54 hours — 0-752° in the former case, and at 84 hours — 0-739° in the latter. The curve is shown in Fig. 183 taken from Przylecki's paper. The eggs were parthenogenetically " fertilised," and the osmotic pressure was not determined by direct freezing-point measurements, but by observing how strong a v glucose solution was required
Fig.
to make no change in ^gg- or embryo-volume^. Przylecki regarded the rise in osmotic pressure as largely due to the formation of osmotically active substances in metaboUsm. At the 6th hour the egg-membrane is in a state of tension, which augments until the 24th hour ( 1 20 per cent.), but then ceases altogether up to the 60th hour, after which it rises again (to 167 per cent.), as is shown in Fig. 184. In spite of the mounting osmotic pressure, then, there is a period during which no increase in membrane tension takes place'^. At this time the membrane does not return to its original state if the pressure is relieved by pricking the Qgg, but has evidently expanded in a more plastic manner. At the 60th hour the outer membrane (egg-membrane) bursts, and the larval membrane begins to expand. The growth of the embryo in volume follows these limiting factors.
In a second paper, Przylecki confirmed the earlier results, working with fertilised eggs oi Daphnia pulex and Daphnia magna. All his results are shown in Fig. 183, from which it will be seen that the osmotic pressure of the perivitelline liquid rises pari passu with that of the embryo, but at a rather lower level.
^ And assuming that the permeability of the egg-membranes for glucose was the same as that for salts.
^ Strictly speaking, this is not tension, but rather the amount by which the elastic limit is exceeded.
The cladoceran embryo, hke that of the silkworm, passes through a hibernation period when part of its development has been completed. Przylecki found that during this time the osmotic pressure remained at the final level reached after the main rise, i.e. from -- 0-734° to — 0-74°, but after two months there was a lowering of 30 per cent, or so, followed by a rise until hatching. Przylecki estimated that of this lowering about 24 per cent, was caused by absorption of water, and 34 per cent, by excretion of osmotically active substances into the perivitelline fluid, as in the early stages of the frog's egg, while the remainder he could not account for. Przylecki gave no figures for the osmotic pressure of the unfertilised Daphjiia egg, but presumably it was somewhere about — 0-7°, in which case the events in amphibians are closely paralleled by those in cladocerans (see Fig. 185).
Osmotic pressure seems to play a very important part in the normal embryonic life of cladocerans. Ramult, working on Daphnia pulex, found that if these animals, soon after the eggs had been laid in the brood-pouch, were transferred from pond water to sodium chloride solutions the embryos did not hatch at the proper time. During normal embryonic life, two membranesjbuj^t^ first the egg-membrane, and then the larval membrane. Although differentiation goes on continuously, growth does not, but has periods of slowness while waiting for the membranes to burst, the larval one being more extensible than that of the egg. In "closed development", artificially induced by a rise in osmotic pressure of the environment, differentiation proceeds normally in spite of the suppressed growth, so that well-formed dwarf embryos are produced. These will never hatch if the osmotic pressure of the exterior remains high, and will die within the membranes. The necessary minimum osmotic pressure for "closed development" is that of JV/30 NaCl. Embryos artificially Hberated from their membranes will continue to develop normally in either pond water or salt solution of the same strength as that which prevents hatching, or even in stronger salt solutions. The volume of the finished embryo in the cladocerans is at least three times as great as that of the egg before the bursting of the egg-membrane, and as this increase in volume can be completely inhibited by raising the osmotic pressure of the ex- ^ ^o^ ternal hquid, there can be no doubt that the cladoceran egg is arranged to absorb a great deal of water from its environment. "^1^ This property is of much interest and will be referred to again in the succeeding section (see p. 896). In distilled water the preponderance of the inner over the outer pressure at hatching would be about A — 0-75° but in pond water a little less, as the latter has itself an osmotic pressure of A — 0-02°. At external osmotic pressures of about A — 0-176°, 50 per cent, of the eggs go into "closed development", which shows that half of them can, if necessary, raise their internal osmotic pressure to higher than this. But they cannot go further, and though they may overcome JV/20 NaCl they will not manage JV/15 NaCl. Ramult's discovery of " osmotic hatching" in cladoceran eggs is paralleled by the fact that phyllopod eggs (e.g. Artemia salina) will not hatch in the strong salt solutions in which the adults normally live (Becking) . This is a remarkable instance of an experiment done for us by Nature.
Fig. 184.
The eggs of other arthropods have been little investigated from this point of view. Walther in 191 3 placed the eggs of the crab Telphusa fluviatilis in water to which magnesium salts were added, and found by microchemical methods that not until many days had elapsed was any magnesium to be found inside the egg-membranes. In other eggs, of course, the salt penetrates much more quickly, e.g. Stockard's magnesium (cyclopean) embryos. Roffo & Correa studied the osmotic properties of the egg-membrane of the mollusc Valuta brasiliense, and found that they showed no changes during the development of the embryo.
5-4. The Osmotic Pressure of Fish Eggs
A different state of affairs came to light, however, when fish eggs were investigated. Runnstrom, for instance, examined the eggs of Salmo by counting the number which developed normally in different solutions. Nordgaard had previously found that these eggs would not develop in sea water, or in 20 per cent, salt solution, though, if fertilisation had been done in fresh water, they could stand 9 per cent. Runnstrom and Svetlov measured the depression of the freezing-point of the egg-contents (excluding the perivitelline liquid) oi^ Salmo savelinus a.nd Salmo fario respectively, obtaining the following results :
AC)
Investigator
Oviduct eggs
-0-645
Runnstrom
Eggs 4 hours after fertilisation .
Hatched larvae ...
-0-580
Blood of full-grown fish .. .
-0-636
,,
Egg throughout development .
-0-500
Svetlov
Perivitelline liquid
-o-oio
,,
Evidently the changes were very small. The initial slight decrease can be accounted for, according to these workers, by an absorption of water from the hypotonic (fresh water) medium and an increase in the amount of perivitelline liquid, for after 4 hours in water there is a 6 per cent, increase in weight, just as, in the case of Salmo trutta, Miescher found an increase of weight of 10-83 per cent, in the same circumstances, and Bogucki an increase of 20 per cent, volume. In Osmerus eperlams, again, there was no fall or rise of osmotic pressure. Thus the eggs of fresh- water teleosteans are practically independent of the hypotonic medium in which they live, and the same independence is shown by marine teleosteans with regard to their strongly hypertonic medium, as will be shown below. On the other hand, as Nordgaard's investigations showed, the fresh-water teleost egg cannot be said to be indifferent to hypertonic solutions. Development in 8-5 per cent, salt solution would be development in a practically isotonic solution, and, as we have seen, this is as much as the Salmon egg will stand. Ramult has found, however, that Salmo trutta eggs are more resistant than other varieties, a fact probably connected with the adult habits of the fish which inhabits fresh and salt water indiscriminately.
The osmotic pressure of the contents of the teleost egg, therefore, is constant throughout development, and so differs profoundly from that of the amphibian zgg. Runnstrom noticed that the membranes of oviduct eggs were very fragile and easily broken, whereas immediately after laying they were hard and tough. Comparative centrifugation and experiments on solubiHty in 3 per cent, potassium hydroxide easily demonstrate this. In 10 per cent, salt solution the hardening does not take place. Obviously these changes in the egg-membranes are of great importance with respect to the osmotic behaviour of the teleost egg, and perhaps may be regarded as the causes of its special properties. Runnstrom concluded that another function of the membrane was protection against polyspermy. He also made experiments with potassium chloride solutions, finding the time taken for the salt to diffuse through the membranes and stop the heart-beat. In this way he observed that the egg-membranes were much less permeable than the skin of the hatched larvae, just as Loeb had previously done for Fundulus embryos. For further details see the section on Susceptibility and Resistance.
In considering the difference between the behaviour of teleosts and amphibia as regards osmotic pressure, it must be remembered that during development up to hatching, and for a short time afterwards, the water-content of both amphibian and teleost embryos (or rather embryo plus yolk) is steadily increasing. Yet amphibian eggs will not develop properly in solutions isotonic with the adult tissues, while those of teleosts will.
Further details are afforded us through the researches of Bogucki, who set up the egg-membranes in microdialysers and studied the passage of substances through them. In this way he found that they were penetrable by chlorides, monosaccharides and amino-acids, but not by proteins, polysaccharides or colloids such as Congo red. During development the permeability was not modified: the same amount of potassium chloride dialysing through in a given time at 10 or 30 days from fertilisation. The initial intake of water after fertilisation was correlated by Bogucki with the formation of the perivitelHne liquid, which just accounts for the change. Bogucki found that this absorption of water was stopped by hypertonic electrolyte solutions, but not by hypertonic solutions of non-electrolytes. The ordinary laws of diffusion and osmosis, therefore, will not explain the formation of the perivitelline liquid. This conclusion is in accordance with the views of Svetlov. Hayes found in the case of the Atlantic salmon, Salmo salar, that the egg membranes are impermeable to electrolytes or colloidal substances.
Gray has also worked on the trout tgg. He began by enquiring what the forces were which maintain the teleostean egg's high concentration of electrolytes against the low osmotic pressure of the fresh- water medium, and where they were located. The membranes could not, he thought, be impermeable to electrolytes, for the embryo could not then take in any inorganic substances from outside. Moore; Osborne; and Donnan had all urged that the presence of colloidal substances inside a cell would cause a differential distribution of inorganic ions within and without, but Gray was not convinced that this would explain the state of affairs in the trout egg.
Gray found experimentally that death of the eggs was accompanied by a marked increase of electrolytes in the surrounding water. Thus the resistance of the water in which eggs were standing was as follows :
Ohms Before shaking ... ... 3550
I hour after shaking ... 305
This was confirmed afterwards by Kronfeld & Scheminzki, who got less striking figures :
Ohms Before addition of alcohol 3200
3 hrs after addition of alcohol 2700
But during normal development no appreciable amount of electrolytes was lost from the eggs ; thus, in an experiment where the original resistance of the water was 6520 ohms, after 3 hours in contact with fertilised eggs it was 5000 ohms, a difference amply accounted for by carbon dioxide production. Direct measurement on egg-Breis before and after fertilisation showed that the amount of diffusible electrolyte within the egg is hardly affected at all by this event.
Ohms Unfertilised ... 1040
Fertilised ... 1070
Similar determinations done on eggs and embryos of different ages showed no change over 38 days, as seen in Fig. 186. Again, the osmotic pressure, cryoscopically determined, was the same before fertilisation as between the 3rd and the loth days, i.e. — 0-48°, though this figure was rather lower than that found by Bottazzi for the adult blood of the same species {Salmo fario), namely, — 0-567°. In the case of Fundulus, also, Loeb & Wasteneys found no difference in the depression of the freezing-point of the egg-contents before and after fertilisation (in both cases — 0-76°). On the other hand Bogucki found the A of trout eggs which had never been wetted, to be — 0-64° while at the 8-12 blastomere stage it was — 0-42°.
From all these facts, and those mentioned on p. 334 in the section on Constitution, the conclusion is indicated that the factor which retains in the normal egg sufficient electrolytes to keep the ovoglobulin or ichthulin in solution is located in the protoplasmic membrane, | which, thickened at one part | to form the germinal disc, ex- l^^^^ tends right over the egg-surface ^ ^i underneath the thick external I200 membrane. The crucial experiment to test this view as against S the Moore-Osborne-Donnan I 'r theory was to dialyse the egg- p^g jge.
contents in a parchment thimble. If the electrolytes were retained in the egg by chemical affinities alone, they should not pass out into the dialysate. But experiment showed that that was just what they did, giving the curves shown in Fig. 187. The protoplasmic egg-membrane, therefore, cannot be merely impermeable to colloids and permeable to electrolytes, but must possess a certain degree of impermeability to the latter.
The actual process of elimination of electrolytes into the surrounding water by injured trout eggs was investigated by Gray in a subsequent paper, using Blackman's exosmosis apparatus. The electrical conductivity of the external medium was measured. The curve resulting was sigmoid, presumably because during the first period the cell-membrane was breaking down, and during the second period the electrolytes were diffusing away according to simple laws.
The independence of the environment which has been noticed in fresh-water teleosts extends also to marine teleosts, where there is a sharp contrast as against elasmobranchs. Dakin obtained the following values by direct freezing-point determinations :
Sea water
Egg-contents of Pleuronectes platessa Adult blood of Pleuronectes platessa Egg-contents of Scyllium canicula Adult blood of Scyllium canicula
AH
-1-91 -0-70
-0-75 -I -80 -I -go
Fig. 187.
This was in agreement with Bottazzi's well-known findings that the osmotic pressure and salinity of teleostean blood was different from the sea (though it varied with it), while the osmotic pressure and sahnity of elasmobranch blood was the same as the sea. Death of the plaice egg destroyed its osmotic independence. Thus the teleost egg, floating in sea water of salinity 35 per cent., had an osmotic pressure corresponding to a salinity of only 14 per cent.; correspondingly it will develop perfectly well in a mixture of 80 per cent, fresh and 20 per cent, salt water. With regard to Scyllium eggs, Dakin found the horny cases to be quite permeable to salt, by simple osmometer measurements (see also p. 331 in the section on Constitution). Jacobsen & Johansen made similar observations, finding that the A and salinity of plaice eggs varied somewhat with the environment, but was always much lower than that of sea water. Death permits the entrance of salt, and the eggs sink to the bottom. Certain observations have also been made on the adaptation of fish eggs to different saHnities; thus Amemiya found that the eggs of the anadromus "Ayu" fish, Plecoglossus altivelis, will not hatch in water of salinity above 20 per cent., 22 per cent, is very quickly lethal, and 15 per cent, optimum.
Osmotic pressure experiments with the eggs of Fundulus were done by Loeb & Cattell and by Loeb & Wasteneys. They studied the various antagonistic effects which electrolytes display on the stoppage of the heart-beat of the embryo. Embryos cannot recover from potassium chloride poisoning without the aid of other electrolytes, so Loeb & Cattell studied the efficiency of the different anions and cations. Loeb & Wasteneys concluded that the egg-membrane of Fundulus is almost impermeable to water and salt under normal conditions.
Fragmentary results on other fish eggs have been obtained by Ziegelmayer, who tested the effects of hormones and of various other substances on the size of Leuciscus eggs.
McClendon found, with Loeb & Wasteneys, that the egg-membranes of Fundulus were practically impermeable, and showed that, when by poisons this impermeability had been abolished, abnormalities occurred. Later, McClendon observed that the action of various toxic solutions markedly increased the permeability of the membranes to salts, but that their action was inhibited to some extent by anaesthetics. This last effect was confirmed in detail with the eggs of the pike, Esox. Anaesthetics that retarded development (2 to 3 per cent, alcohol or 0-5 per cent, ether) tended to inhibit the permeability-increasing action of a i/io molecular solution of sodium nitrate. Osterhout showed in 19 14 that plant cells were made more permeable with increase of temperature, and McClendon extended this finding to the eggs of Esox, which is interesting in view of Ephrussi's results on echinoderm eggs (see p. 806). Loeb used the egg-membranes of Fundulus in many experiments on ionic permeability, e.g. specific gravity tests
In 1928 Sumwalt pointed out that the Fundulus embryo was enclosed in two membranes, in the early stages, by the outer chorion or egg-membrane and the vitelline membrane, and, in the later stages, by the chorion and the skin. The importance of the skin factor in the ontogenesis of water regulation, etc., had already been emphasised in the case of amphibia by Adolph, but Sumwalt used a new method, measuring the permeability to ions of the various embryonic membranes o^ Fundulus in terms of concentration potentials across the membranes between solutions of jV/io and JV/ioo potassium chloride after Michaelis. By means of a capillary pipette in a micromanipulator, one electrode was introduced into the inside of the egg, and a similar electrode dipped into the solution in which the egg was placed. For a measurement of concentration potential through the chorion alone, the electrode was placed in the sub-chorionic or peri vitelline space; for a measurement across the chorion plus the embryonic skin, it was put in the yolk-sac of the embryo. The results showed that the compound membrane of skin plus chorion could produce much greater concentration potentials than the chorion alone, the average for the latter being 19-4 millivolts, and for the former 55-2. In both cases change to the dilute solution (sea water diluted 100 times) caused the inside of the egg to become negative to the outside, indicating a relatively greater impermeability of the membranes to anions than to cations. This is less pronounced in the chorion than in the skin. Measurements of electrical resistance confirmed this view, for the resistance of the chorion alone was about 45,000 ohms, but that of the embryonic skin about 208,000.
5-5. Osmotic Pressure and Electrical Conductivity in Worm and Echinoderm Eggs
Of the osmotic pressure of oligochaete worm eggs very little is known, but there is in this connection an interesting study by Svetlov of the eggs of the Lumbricidae, Bimastus constrictus, and Eiseniafoetida. The Terricolae, the sub-order to which the earthworms belong, lay their eggs in cocoons, which were formerly mistaken for the eggs themselves. These cocoons are brown and horny and vary in size according to the species; they contain ova and spermatozoa as well as a milky nutritive fluid in which the young worms float and by which they are nourished before they hatch out from the cocoons. Svetlov had morphological and cytological reasons for supposing that in the early stages of Bimastus the three micromeres acted as osmoregulators for the rest of the embryo by excreting water, but that this was not so in Eisenia, and when he came to estimate the osmotic pressure of the milky liquids in the cocoons he found that the two were indeed different. The cocoon liquid of Bimastus gave a freezing-point depression of— o-o6° corresponding to 0-78 atmosphere or o-io8 per cent, sodium chloride, while that of^ Eisenia gave one of — 0*39° corresponding to 4-8 atmospheres or o-68 per cent, sodium chloride. Svetlov found that the cocoon shells, although hard, were not impermeable, and showed, in fact, the properties of semipermeable membranes. Taking then samples of liquid from the normal habitat of the two worms, humus earth infusion for Bimastus and dunghill infusion for Eisenia, he found that the former was much less concentrated than the latter, the relation being the same as that found between the milky contents in the two cases. Svetlov constructed micro-osmometers with the cocoons from the two species and found that the permeability of Bimastus was not quite double that of Eisenia. Although the process was complicated in Eisenia, cocoons (of both species) placed in solutions of known osmotic pressure would come gradually into osmotic equilibrium with them. The osmotic pressure of the cell-interior of the eggs themselves was probably alike in both cases as the osmotic pressure of the adult blood was the same, and therefore, Eisenia, having, according to Svetlov's view, either abandoned or never evolved the osmoregulator micromeres of Bimastus, has to lay its eggs in the dungheap liquid or " Mistfliissigkeit " which has a high osmotic pressure.
The osmotic relations of the eggs of nematode worms have only once been investigated — by Szwejkovska. Between fertilisation and the first cleavage there occurs, she found, a great diminution in the size of the egg-cell while the egg as a whole remains unaltered in size. The egg swells only slightly in hypotonic solutions. By the plasmolysis method, she found the freezing-point depression of the egg-interior to be - 0-599° in the unfertiHsed egg, - 0-629° in the fertihsed egg before the eHmination of the first polar body, and — 0-636° after the elimination of the first polar body.
A great deal of work has been done on the permeabiUty and osmotic pressure of echinoderm eggs. The jellies surrounding them when unfertiHsed, and which can be made visible by the Indian ink method, have been found to take up salt from the water. Glaser noticed that the specific gravity of sea water in which Arbacia punctulata eggs had been standing for some time (i hour) was always lower than that of ordinary sea water (i-0229-i-02i9). This led him to ask whether the eggs or the jellies had abstracted any inorganic material from the water, and in fact experiments showed that there was always a deficit of from 2 to 7 tenths of a milligramme per cubic centimetre of chlorine in the eggwater after an hour when i part of eggs were suspended in from 7 to 10 parts of water. The absorption was real, for eggs which had taken up all the chlorine that they would in one vessel took up no more when
transferred to another; therefore the phenomenon was not due to an interference with the silver nitrate titration on the part of any egg-secretion. Eggs from which the jelHes had been removed did not show this behaviour, and, as histo-chemically the jelly was found to be rich in chlorine, everything pointed to the jelly being responsible (see Fig. 188).
Most of what we know about the osmotic pressure of the eggcontents in echinoderms has to be deduced from what has been found to happen as regards the permeability of their membranes. McClendon in 191 o studied the electrical conductivity of masses of eggs before and after their fertilisation [Toxopneustes variegatus and Tripneustes esculentus) in a specially devised conductivity vessel. The following typical figures were obtained:
Fig.
Conductivity
Unfertilised Fertilised ..
0-01182 0-01537
0-01153 0-01277
There was undoubtedly an increase of electrical conductivity at the beginning of development. This might have been due {a) to increasing permeability of the egg-membranes, {b) diminution of fatty phase of the ^gg, [c] dissociation of protein-electrolyte complexes. The second of these possibilities was quickly ruled out by centrifugation experiments, which showed that just as much fat and oily matter was present after fertilisation as before. Cytolysis experiments showed that the first one was the most probable, and that as in Hober's erythrocytes, the chief factor producing electrical resistance in eggs was the membrane. This conclusion was supported by various other workers, such as Lyon & Shackell, who reported that salts would enter and leave the fertilised Arbacia Qgg more easily than the unfertilised. Their only exception was iodine, which seemed to show the re\'erse behaviour. Harvey found that eggs became more permeable to sodium hydroxide after fertilisation, and Lyon & Shackell made similar observations with vital dyes. McClendon concluded that there was probably no liberation of electrolytes after fertilisation, but rather an increase of permeability to outside electrolytes. Parthenogenesis gave identical results with fertilisation.
McClendon's results were in general confirmed by Gray^. Invariably there was a decrease in electrical resistance immediately after fertilisation, as the following figures show:
Percentage decrease of electrical resistance on fertilisation Echinus acutus ... ... ... ii-2
Echinus miliaris ... ... ... lo-g
Echinus esculentus ... ... ... I2"5
Asterias glacialis ... ... ... 7*4
Strongylocentrotus lividus ... ... 36-0
Sphaerechinus granulans ... ... 23-0
Arbacia pustulosa ... ... ... 15-0
but half-an-hour or more after fertilisation this decrease was not so apparent. Gray concluded that the entrance of the spermatozoon into the tgg causes an increase in electrical conductivity which attains its maximum within ten minutes, and is followed by a return to the value for the unfertilised G.gg. In later experiments he was unable to confirm this return to the original level, and published curves showing a gradual fall in resistance. Parthenogenesis, he found, did not give quite the same eflfect as normal fertilisation; thus by different methods, Sphaerechinus eggs gave increases of conducti\ity of 21-7, 6-8, and 6-o per cent., instead of the normal 23 per cent. Hypertonic sea water markedly increased the conductivity of normal unfertiHsed eggs, the increase actually taking place while the eggs are in the hypertonic solution, but after artificial membrane formation, the conductivity was unaltered by this treatment. Change in the internal pYL of the egg-cells, as found by exposing Arbacia eggs to ammonia solutions and noting the colour change of the pigment inside, produced no alteration in electrical conductivity. To support further his view that the properties of the membrane were the controlHng factors in egg permeabiHty, Gray put a list of haemolytic agents next to a Hst of parthenogenetic agents, and emphasised their general resemblance ; Dalcq has followed this relation further in his book on fertilisation, which should be consulted for further details.
- But see the criticisms of Cole regarding this (p. 829).
The work of Gray and McClendon on the conductivity of echinoderm eggs was extended by Bataillon to the eggs of amphibia. Bataillon found in their case also a regular decline in electrical resistance after fertiUsation, but his results differed from the final ones of Gray, in that with the frogs there was always a gradual return to a level slightly less than that of the unfertilised eggs. The percentage decreases were as follows :
Percentage decrease of
electrical resistance
on fertilisation
Ranafusca (parthenogenesis) Rana esculenta (fertilisation) Bufo calamita (fertilisation)
12-35 19-65
which agree well with those found by Gray, Bataillon's data are plotted in Fig. 189. He reported that the hepatopancreatic juice of the crab would destroy unfertilised amphibian eggs, but not fertilised developing ones (jelly ^ o ® having been removed in both cases), yet this immunity did not appearuntil after 30 minutes from fertilisation. He thought that the decreasing membrane permeability as measured in terms of electrical resistance might be connected with this fact.
Other investigations of permeability of marine egg-membranes were those of McCutcheon & Lucke, who found that in solutions of hydrochloric acid, sodium hydroxide, carbon dioxide and ammonia no swelling of Arbacia eggs occurred, and they decided that the membrane was normally almost impermeable to these substances. Lillie made similar experiments. Driesch and Konopacki studied the cytological effects of raising echinoderm eggs in hypotonic sea-water. Faure Fremiet investigated in some detail the osmotic relations of Sabellaria eggs. Placing them in solutions of glucose of A varying from — 0-566° to — o-686°, he measured their volume with a micrometer scale and found, as was expected, a regular change; thus at — 1-135° the calculated volume was 21-9. io~^ c.c, and at — 3-73° it was 9-95 . 10"^ c.c. Assuming that the membrane is impermeable to
Fig.
Urea solubion Sucrose solution Theorebical curve corrcspondinc) to -,
perfect semi-permeability of the egg membrane
NaCL CaCL2 Theorebical curve
2 3 4 5 6
Molarity of solution
— Zone of* normal development
Fig. 190.
)
2 3 4 5
Molarity of solution
Fig. 191.
anything except water, Faure-Fremiet calculated the imbibition at the different osmotic pressures. The normal Sabellaria ^gg contains 2-25 gm. of water for each gm. of solid, therefore a table was constructed showing the amount of water experimentally found in the eggs in the different sugar solutions, and the amount of water which should theoretically have been there. The observed and calculated values corresponded very well, as can be seen in Fig. 190, from which Faure-Fremiet concluded that it would be right to assume that the egg-membranes were permeable only to water. The difference between the theoretical and calculated figures were of the same sign, i.e. the eggs always contained slightly less water than on the complete impermeability hypothesis they should have done. Faure-Fremiet explained this by suggesting that a very small degree of permeability to electrolytes was present. Data for urea solutions gave the same results, and are shown plotted in the same figure. Fig. 191 shows similar experiments done with neutral salts, such as sodium chloride, magnesium chloride and calcium chloride. Here, however, at the higher osmotic pressures the calculated and observed values diverge to a significant extent, which indicates that the absolute impermeability of the membrane is not maintained under such conditions. Probably electrolytes then enter the cell, just as in sugar solutions they may to a slight extent come out of it. These effects were so slight, however, that Faure-Fremiet was inclined to see in them an adsorption of ions on the external surfaces of the egg-membrane rather than a true permeability. Acids and bases did not seem to penetrate at all into the eggs oiSabellaria. Temperature had a slight effect on the imbibition of water by the eggs; outside a constant range between 18° and 25°, the imbibition increased at low and decreased at higher temperatures. The margins of temperature between which the imbibition is at its normal value corresponded exactly to those between which perfectly normal development is possible. These effects have obviously an important bearing on the problem of egg viscosity, which has been handled (/'by so many workers. Again, there was a modification of imbibition by the eggs according to change in pH — at pYi 5 they contained sHghtly less water than normal, at pH 7, 6 per cent, more (/?H 8-4 normal), and at j^H 12, 10 per cent. more. These small differences were perhaps related, according to Faure-Fremiet, to the isoelectric point of the egg-proteins. The effects of these various agents on the imbibition, the water-content, and therefore the osmotic pressure of the egg-contents, were of the following comparative magnitudes :
Maximum °o variation External osmotic pressure ... ... i8o
Specific action of cations ... ... 70
Temperature ... ... ... ... 14
P^ 7
The heat factor was subsequently examined in more detail by Ephrussi, using the eggs of sea-urchins. He obtained an exactly similar curve to Faure-Fremiet's for the effect of heat on imbibition of water, and in collaboration with Neukomm for the effect of pH on imbibition (see Figs. 192 and 193). McCutcheon & Lucke, on the other hand, maintained that for Arbacia pH had no effect, but their measurements were rather few. The spermatozoa were, Ephrussi found, less susceptible to heat than the unfertilised eggs. The normal osmotic pressure of the egg-contents in Strongylocentrotus lividus was 25 atmospheres, i.e. practically isotonic with sea water. Heating at different temperatures for a short time led to [a) a rise in the internal osmotic pressure, and [b) a subsequent fall, the whole curve forming a peak, the angle of which was the more acute the higher the temperature. Ephrussi concluded that two irreversible processes were in operation.
Fig. 192. A. Strong^iocentrotus . B. Sabellaria.
Perhaps the most interesting result obtained by Faure-Fremiet on Sabellaria eggs in this connection was the finding that the eggs obeyed the Boyle-Mariotte law. Ephrussi & Neukomm could not show that this was so for Strongylocentrotus eggs. If j&F = K holds good, the eggs should swell in hypotonic solutions and shrink in hypertonic solutions to an extent just sufficient to keep the product of the equation constant, but the sea-urchin's eggs did not do this. Fig, 194 shows on curve A the actual behaviour of the eggs, volume being plotted against osmotic pressure of external medium, and B the theoretical curve which should have resulted if the Boyle-Mariotte law had been followed. It is evident that the divergence is greatest in very hypotonic solutions, nil in normal sea water, and again
Fig. 193. A. Strongylocentrotus. B. Sabellaria.
marked in hypertonic solutions. In other words, the sea-urchin's egg opposes a certain amount of resistance to extreme hydration on the one hand, and to extreme dehydration on the other. Ephrussi & Neukomm offered no explanation for these facts, but thought it as difficult to picture any considerable amount of glucose getting into the Qgg from hypertonic solutions as to picture any electrolytes passing out in hypotonic solutions. If Fig. 194 be compared with Fig. 190, the difference between the polychaete and the echinoderm egg as regards the Boyle-Marriotte law will easily be seen.
A certain number of investigators have occupied themselves with the examination of the effects produced on developing eggs by environments of varying osmotic pressure. Morphological studies of this kind are fairly numerous, but the present summary is confined to those with a physiological bearing. Perhaps the most complete is that of Vies & Dragoiu, who introduced the conception of "pression
Fig. 194. A, experimental; B. theoretical.
osmotique d'arret". They thought that, if one could determine the osmotic concentration at which development ceased, it might be possible to calculate the "energy of segmentation" or of development itself. I shall return to this notion in the section on Energetics; here only that part of the work which relates to osmotic pressure will be considered. Placing the fertilised eggs of Strongylocentrotus lividus in solutions of glucose in sea water, they made a statistical study of the effects produced. From 25 to 30 atmospheres, the effect was almost inappreciable, perturbations of development being almost absent, and a delay in cleavage not exceeding 15 minutes occurred. From 30 to 55 atmospheres there was a critical zone ; the percentage of eggs achieving complete first division rapidly diminished until it reached a figure between 5 and 10. From 55 to 100 atmospheres, the state of affairs changed, for now, not only would cleavage not take place, but a great number of abnormal and teratological forms appeared. Later experiments gave greater regularity, and the same process was gone through for each of the early stages, i.e. second cleavage, third cleavage, fourth cleavage, etc. The result was that for all the stages averaged, it took an osmotic pressure of 33 atmospheres to stop 10 per cent, of the eggs developing, and 39 atmospheres to stop 90 per cent, of them. Therefore an osmotic pressure of about 1 1 atmospheres more than that of sea water was required to stop development. Vies & Dragoiu went on to calculate the work done in elevating the osmotic pressure to this point, and hence "the external osmotic work of cytoplasmic division", where W is the work, co and wq the normal and stoppage osmotic pressures, and V and Vq the normal and stoppage egg-volumes. The values for W fell with development in a straight line, as follows :
Ergs First cleavage ... ... ... 4'09
Second cleavage ... ... 2-05
' Third cleavage ... ... 0-85
Fourth cleavage ... ... 0-29
Vies & Dragoiu pointed out that the "travail d'arret" diminished as the volume of the blastomere diminished, but also tended to diminish when related to the volume of the egg as a whole. Chevroton & Vies had previously accurately measured the volumes of the blastomeres, finding, for instance, that the two were not quite halves and the four not quite quarters. The following figures were obtained:
Volumes "Travail d'arret"
(Chevroton & Vies) (Vies & Dragoiu) ( X io-« c.c.) (ergs)
Unfertilised egg ... ... ... 52-3 —
Fertilised egg (membrane) (egg only) ., i blastomere J blastomere ^ blastomere
109-0 —
47-7 4-02
17-3 1-66
7-78 o-8i
2-64 0-28
Vies & Dragoiu also made a cytological examination of the material, and plotted the time taken to produce a definite morphological event such as formation of accessory aster, or "paquet chromatique".
against the osmotic pressure of the solution. The resulting graph showed the usual descending curves, for the higher the osmotic pressure the more quickly the abnormalities appeared, but in one case a complete curve was drawn through only two points, and in two cases through only one point — happily an unusual way of presenting facts.
Spaulding's work on the energy of segmentation, though it was earlier and based on excellent theoretical principles, was very similar to that of Vies & Dragoiu. By immersing echinoderm eggs in solutions of differing osmotic pressure, he was able to find the solution which just stopped cleavage, thence the internal osmotic pressure, and thence the energy in ergs required to stop cell-division. The osmotic pressure sufficient to inhibit the first segmentation, and therefore equal to the resultant internal pressure, was 7-32 atmospheres, for the second segmentation 6-53, and for the third 6-40. From this he calculated that the energy required to stop the first division was 1-567 ergs, and that required to stop the second one was 1-399 ^^S^. 'This means", he said, "that, as involved in or as identical with the first segmentation, there has been a resulting energy decrease, therefore, of 0-168 erg, or that it has taken this amount of energy, about 1/9 of the total increase resulting from fertilisation, etc., to bring about this cleavage." Similarly, to bring about the second cleavage, 0-028 erg was involved.
The conclusions of Spaulding and of Vies & Dragoiu were for a time generally accepted, but the conception of "travail d'arret" has now, largely owing to the criticisms of Rapkine, fallen into disrepute, for it rests on a fallacy, assuming as it does that it would be possible to find the work done by a man in walking a mile by measuring the work done in stunning him with a stick or a stone at the end of it. " In order to use the 'travail d'arret' as a measure "of the 'travail de division' the phenomena would have to be reversible, i.e. capable of coming to equilibrium, but that is just what they are not." (Rapkine.)
Bialascewicz approached the problem in another way, estimating the relative speed of development of embryos in solutions of different osmotic pressures. Working with Echinus microtuberculatus , Strongylocentrotus lividus and Ranafusca, and using hypotonic and hypertonic sea water in the two first cases, and glucose in the third, he found that the maximum speed of development took place in a medium isotonic with the normal medium, and on both sides of which the rapidity of development fell off sharply. These bell-shaped curves are shown in Fig. 195. In the case of the frog, the curve appears to drop only on one side. The zone within which normal segmentation would proceed also differed markedly for the two echinoderms; for instance, it was A — 0-63° in the case oi Echinus and only —0-57° in the case of Strongylocentrotus, while in the case of Rana it was only — 0-17°. On the other hand, the zone between the osmotic pressures which caused death was relatively wider in the case of the amphibian than in the case of the two echinoderms. The effect of osmotic pressure on the embryos, moreover, was not the same at all stages. As regards mortality, the sensitivity of frog embryos to hypertonic solutions augmented with age; thus at the 8-cell .stage they were killed in 24 hours by solutions of A -^.0-95°, but at the time of hatching they we^ killed in 24 hours by a solution of only — 0-43°. On the other hand, the embryos of Strongylocentrotus lividus supported changes of osmotic pressure better the^older they were. As regards speed of development between given stages, this also was affected differently. As is seen from Fig. 195, between the 4-cell stage and the appearance of the first mesenchyme cells, the speed of development falls off distinctly less slowly from the optimum with change of osmotic pressure than it does between the appearance of the first mesenchyme cells and the three spicules of the pluteus.
StrongylocentroUiS whole period □ • » between 4 c
stage S^blastula (primitive
mesenchyme) B ■Strongyjocentrotus between blastula&, 3 spicule stage
Strongylocentrotus &.Echinu8 p®
•9 2-0
2-2 2-3 2-4 2-5 2'G
38 -40 '42 -44 -46 -48 -50 -52 '54 -56 '58 -60 -62 Rana •
Faure-Fremiet made parallel experiments on the eggs of Sabellaria alveolata, and obtained precisely similar results. Fig. 196 taken from his paper shows the usual bell-shaped curves, resulting from the plot of active osmotic concentration against the time required to reach certain stages of development. There is a certain optimum osmotic pressure, from which the speed of development rapidly falls away on both sides; thus, although the processes of division will go on between the limits of 0-85 and 1-48 mol. per litre, the optimum is at 0-956 mol. per litre. Mortality also shows a bellshaped curve, with an optimum at 1-024. Faure-Fremiet concluded that as the Boyle-Mariotte law j&F = K holds for the egg of Sabellaria alveolata, the method of calculation of Vies & Dragoiu is not applicable to it. He noted finally that, at external osmotic pressures which greatly slowed down the speed of development, the ABC
Fig. 195
1-41 F
A, 1st polar body. D, Stage II.
Fig. 196.
B, 2nd polar body. E, Stage IV.
C, Cordiform stage. F, Stage VI.
processes of division, etc., were not appreciably modified. In another work he stated that the outer egg-membranes of Ascaris were absolutely impermeable, the surface of the tgg cytoplasm only permeable to water, and that the egg-contents was isotonic with a 0-7 per cent, sodium chloride solution.
5-6. The Osmotic Pressure of Terrestrial Eggs
The eggs of terrestrial animals have not received as much attention from this point of view as have those of aquatic animals. A little work has, however, been done on the silkworm embryo. Polimanti in 1915 and Pigorini, Tonon, Tona & de Ziller in 1927 obtained the following figures :
Ovarian eggs
Newly laid eggs (yellow) ...
After 2 or 3 days (ash-yellow)
After 3 br 4 days (quite ash-coloured)
After some months, i.e. in the torpid state
Caterpillar newly hatched
Adult imago
Polimanti
A(°)
-0-650 -o-66o -o-68o -0-690 -0-665 -0-750
Pigorini et al. A(°) -0-630 -0-580 -0-700 -0-700
There would thus seem to be a gradually increasing concentration of osmotically active substances in the silkworm egg as it develops. Polimanti did not make any remark on it, but some possibility evidently exists that the fundamental mechanisms here are like those we have already seen to hold in the case of other arthropod embryos. It must be remembered, however, that the water-content of the silkworm egg is not constant, but decreases by evaporation from the time of laying onwards.
The investigations of the osmotic pressure of the constituents of the bird's egg before and during its development have been few in number, and not very complete. The first study was that of Atkins, who found that the osmotic pressure of the blood of the adult hen was two atmospheres greater than that of the fresh egg (mixed white and yolk).
AH
Callus (hen) Anas (duck) Anser (goose)
Adult blood -0-607 -0-574 -0-552
Egg -0-454 -0-452 -0-420
During incubation the osmotic pressure of the white and yolk mixed rose to about that of blood, a phenomenon at first sight Hke that seen in the amphibian and cladoceran egg, but probably partly due to the loss of water by evaporation, and partly to the inorganic salts entering the egg from the shell. Bialascewicz later went into the question in detail. His results are shown plotted in Figs. 197 and 198. It may be noted at a glance that the osmotic pressure of the embryonic body rises steadily as development goes on, that of the amniotic liquid stands more or less stationary, and that of the allantoic liquid greatly declines. What is the significance of these changes?
To begin with, the fact that the yolk has a distinctly higher osmotic pressure than the white may at any rate partially explain the passage of water from the white into the yolk, which has been noticed by so
Fig. 197.
many workers (Aggazzotti; Greenlee) during the first week of incubation. That it does not entirely account for it will be apparent when the work of Vladimirov and his collaborators is considered (see (p. 88 1 ) . Then it must be remembered that all the points on the graph in Fig. 198 are at a lower level ^(O) than the A of the blood of the adult hen, which Bialascewicz found to be — 0-635°. The yolk of ovarial eggs he found to be
— 0-613°, and the yolk of eggs from halfway down the oviduct
— 0-585°, so that the falling curve for the yolk seen during the early days of incubation is simply the continuation of a curve which could be constructed for all stages after the yolk leaves the ovary. Thus, by the time the yolk has arrived at the stage of being laid, it is distinctly hypotonic to the parent blood, as is also the white, which has surrounded it. Processes of some sort, therefore, must be taking place, tending to diminish the concentration of osmotically active substances in
the yolk. Bialascewicz supposed the yolk to absorb water as soon as it came into contact with the white in the oviduct, and he regarded the state of affairs at laying as an equilibrium condition, in which the osmotic pressure value was regulated by the degree of stretching which the elastic vitelline membrane could stand^. During incubation, the membrane became more extensible, and further dilution of the yolk by water from the white proceeded. Bialascewicz confirmed the old observation of Harvey that the white almost entirely disappears before the end of incubation, and gave a few figures,^ but the most complete data showing this are some which I collected in
Fig. 198.
^ The equilibrium may also, however, be chemical in origin (see p. 819). Bialascewicz had no evidence for his views on the elasticity of the vitelline membrane. 2 See also Fangauf's data.
alascewiC3 Curtis Komori / O Sendju l^ ID Needham • -Weight of yolk (Sendju) A White] Pre'vostS^Morin XT Yolk j 1846 This was first observed by W.Proutinl822 ^Water absorption by yolk (current yolkwards)
1927, and which are shown plotted in Fig. 199. The sharp descent of this curve between the 15th and 20th day must no doubt be due to the formation of what Duval called "the avian placenta". As the two ends of the allantois fuse at the sharp end of the egg they enclose in a vascular bag what remains of the egg-white together with a little yolk squeezed out through the incomplete closure of the yolk-sac. Absorption of the contents of the bag is thus greatly facilitated, so that by the time of hatching the only reserve of food left is the yolk itself As the contents may be said to consist mainly of protein we should expect to find a marked peak on the curve relating absorption-intensity of protein to time, at this point, and as Fig. 250 shows, we do in fact find such a peak.
It is next important to note that the amniotic fluid is first of all hypertonic, then isotonic, and lastly hypotonic with respect to the embryo, which holds on its way steadily throughout development, until ^^' ^^^'
at the close of incubation it has almost reached the adult level. The embryo would seem to be independent of its surroundings, and to possess osmotically active substances in constantly increasing amount. It is interesting that chick embryo cells in tissue culture require an isotonic medium for long-continued growth (Ebehng) though they can support hypertonic and hypotonic media for some time. Hogue and Willmer found that osmotic pressure affected cellmigration but according to Lambert and Ebeling it has no effect on cell-multiplication. As for the allantoic fluid, the sudden fall of osmotic pressure towards the end of incubation was brought about, Bialascewicz suggested, by the functioning of the embryonic kidneys, excreting into it dilute urine of low osmotic pressure. Possibly the fall is an index of secretory activity of the metanephros. In all stages the allantoic fluid is strongly hypotonic to the amniotic fluid, so that the embryo and amnion form a quite isolated system of high osmotic concentration, surrounded by a hypotonic allantois and yolk. In a certain sense, then, the amniotic fluid in the chick might be said to correspond with the perivitelline fluid in the frog embryo. The slight falling off' in the osmotic pressure of the amniotic fluid which Bialascewicz found in the case of the chick, is paralleled by a similar fall in the case of the sheep (Jacque), the cow (Griinbaum), the dog and the rabbit (PoHti), and man (Ubbels). Again, Jacque and Griinbaum found in mammals a rise, not a fall, of osmotic pressure of the allantoic fluid during development. Consideration of the mass of literature dealing with the osmotic pressure of the mammalian amniotic fluid, placenta, and foetal blood, will be deferred to the sections specially devoted to those subjects. However, the allantoic liquid in mammals also is always hypotonic to the foetal blood.
Water ^ absorption / by chick &. jjj>^evaporation
Bialascewicz pointed out that, in the period when the chick embryo was hypotonic to the amniotic fluid, it was not only not losing water, but rather was actually gaining it. His determinations of embryo water-content, however (see Fig. 220), were in a region which has been very little studied (i.e. before the 5th day) and it has been classical to believe that the embryo steadily loses water from the very beginning. As the discussion on p. 871 shows, however, Schmalhausen's data support those of Bialascewicz. The embryo becomes in turn isotonic and hypertonic with respect to the amniotic liquid, the water-content of which is indeed unknown, but must remain uniformly very high compared to the embryo.
Straub & Hoogerduyn were impressed by the very large difference existing between the osmotic pressures of the yolk (nearly — o-6° A) and the white (about — 0-45° A) atthe timeof laying^. They devoted a long paper to the explanation of this fact, which is certainly remarkable considering how tenuous and fragile the yolk-membrane is. After considering all the possible systems which might be involved (Donnan equilibrium, etc.) they made some estimations of the constituents of the yolk and white (see Table 27) and drew up the following:
- At room temperature, this difference can be maintained for at least two months, i.e. quite as long as normal viability is retained (Moran).
K
Na
CI
Lactate
Other monovalent anions ...
Free glucose
Unknown neutral substances
white
yolk
o-o6 0-04 o-o8
o-og 0-15 0-13 o-o6 o-o6
o-oo
0-02
0-05 0-17
o-oo 008
0-42 0-57
Although their figures for ash, especially in the yolk, do not agree altogether with those of other workers, yet their contention, that each dissolved substance varies quite independently in yolk and white, must be admitted. It is not merely that the total osmotic concentration of each phase is distinct but also that practically no agreement exists between the constituent items in that concentration. The difference between yolk and white is as much as i-8 atmospheres, and if this was all due to the osmotic properties of the membrane alone, it would have to support, Straub & Hoogerduyn calculated, a pressure of 2 kilos per square centimetre. They considered that the Schreinemaker equations were therefore inapplicable, and as for those of Donnan, the distribution of ions in yolk and white was so different from what would be expected according to Donnan's theory that it was very unlikely it could hold in this case.
Straub & Hoogerduyn entered a quite new field when they suggested that the osmotic difference between yolk and white was a " Lebenswirkung " in the sense that the vitelline membrane might be physiologically bound up with the egg-cell or pre-gastrula. If this was so then after long storage the infertile egg should show a disappearance of the special membrane properties which characterise it when it is fresh. They mentioned, in this connection, the experiments of L. K. Wolff who had found that in the fresh egg there is a trace of zinc in the yolk and a trace of copper in the white while after storage for some time these metals are equally distributed throughout the egg. Their own experiments showed that eggs stored for a long time tend to acquire equal osmotic pressures on both sides of the membrane :
A n A n
white yolk
Fresh eggs ... ... ... ... 0-45 o-6o
Conserved eggs ... ... ... 0-50 0-52
Frozen eggs ... ... ... ... 0-49 0-50
A(=) white
A(°) yolk
-0-46
-0-58
-0-2I
-0-23 -0-46 -0-44
-0-58 -0-38 -0-38
-0-51
They also found that if morphine, cocaine, or potassium cyanide was added to the egg-white in extremely small amounts (2-5 mgm. per egg) the difference in osmotic concentration between yolk and white could be much reduced, as if the membrane was no longer performing its function. Similarly, if yolk was put into a parchment capsule with egg-white outside, the system rapidly attained a state in which there was only a difference of 0-01° between the inside and outside freezing-points, instead of the 0-15° of the fresh egg. In another interesting experiment the yolk of a fresh egg was placed in diluted egg-white.
The yolk and white of the egg were at the beginning of
the experiment ... The yolk was then placed in egg-white which had been
diluted with an equal quantity of water. Result After 48 hours the system was
The yolk was then put back into natural egg-white. Result After 48 hours the system was
Thus in all the conditions the yolk maintained its hypertonicity, even when it had sunk to 0-38 and, at the beginning of the second period, was actually hypotonic to the natural egg-white. The "living" vitelline membrane must therefore tend- to encourage the exit of water from the yolk or to impede its entry, on the one hand, and tend to encourage the entry of salts or to impede their exit, on the other hand.
If, then, the large difference m osmotic pressure between yolk and white was a " Lebenserscheinung " some energy-expenditure on the part of the egg-cell would be expected, and Straub & Hoogerduyn calculated the " Konzentrationsarbeit " required, to be o-oi cal. per egg per day. Now there exist in the literature one or two papers which give the gas exchange and heat production of infertile eggs. A. J. M. Smith found that one unfertilised egg gave off 0-2 mgm. of carbon dioxide per day at 10° and Langworthy&Barott found that unfertilised eggs produced o-oi cal. per kilo per hour at 12°, 0-02 at 15°, and o-o6 at 19°. Pucher studied the changes which take place in the incubated infertile egg during 20 days from laying. He found that the total glucose of the white fell by 90 mgm. per cent, and the total glucose of the yolk rose by 40 mgm. per cent. For purposes of rough calculation, the white may be taken as 33 gm. and the yolk as 16 gm., in which case the former loses 30 mgm. in 20 days, and the latter gains 6 mgm., so that the loss from the egg as a whole would be about 24 mgm., or 1-2 mgm. per egg per day. Infertile eggs show, therefore, the following effects within 20 days after laying:
Loss of 0-2 mgm. carbon dioxide per egg per day (Smith). Loss of 0-072 cal. per egg per day (Langworthy & Barott). Loss of I -2 mgm. glucose per egg per day (Pucher) .
Smith's figure requires comment in view of the fact that eggs after leaving the hen, give off carbon dioxide to the environment, which is less saturated with that gas than the maternal body. Earlier work by Stepanek and by Atwood & Weakley (see Fig, 152), had resulted in values of the order of 10 mgms. of CO2 per egg per day, but these were for the first week after laying. Smith's figure for the same period was about 3 mgms., but calculation shows that even this amount cannot be accounted for on the view that the egg is giving off the CO2 which has been physically dissolved in it. It is necessary to postulate some acid in the shell liberating carbon dioxide from the carbonates there.
A steady level of 0-2 mgms. per egg per day, then, is reached by about a month after laying. The heat produced with this would be of the order of 0-37 cal., which would be more than ample to provide {a) for the heat output found by Langworthy & Barott, and (b) for the "Konzentrationsarbeit" of Straub & Hoogerduyn. The latter authors showed that their heat requirement could be satisfied by the daily combustion of 0-0025 mgm. of glucose. They also gave a theoretical excursus suggesting physical mechanisms by means of which this energy could be used at the membrane surface in the way they postulated. They regarded the vitelline membrane as a "galvanic combustion-element" for glucose with oxygen to carbon dioxide and water, so that the maintenance of specific concentration difference between the exterior and the interior would be a complicated case of concentration polarisation^. (For the histology of the membrane see Lecaillon.)
The egg of the pigeon (Riddle & Reinhart) and that of the mackerel (Alsterberg & Hakansson) show a very strong positive Manoilov reaction, which is probably to be interpreted as indicating a very weak metabolic intensity in the ovum before fertilisation.
1 It was later shown by Hill, however, that infertile eggs kept in pure hydrogen for a month still retained the normal difference in osmotic pressure between their yolk and white. Whatever the mechanism of osmotic work may be, it can function anaerobically. In this connection the glycolytic power of the yolk, studied by Stepanek and by Tomita, should be remembered (see Sections 8-13 and 14-6).
The frequent occurrence of broken yolks in stored eggs shows that the water current eventually overcomes the resistance. Rice & Young estimated the osmotic pressure and the refractive index in the eggs of various kinds of hen, in order to assess the relative intensity of the water current in different eggs, but there were no perceptible variations from the mean. Kamei's data are in good agreement with those of Bialascewicz.
Table 92.
Osmotic pressure Refractive index at
A (°) 20° C.
White Yolk White Yolk Investigators
White Leghorn pullet -0-435 -0-580 1-3565 i-4i75 Rice & Young
White Leghorn hen -0-442 -o-6oi 1*3560 1-4188 „
White Wyandotte -0-428 -0-576 1-3546 i'4i83 "
Barred Plymouth Rock -0-436 -0-602 i-3550 1-4192
Rhode Island Red -0-446 -0-575 i'3568 1-4185 ,,
Various attempts have been made to gain some further information about the nature of the osmotically active substances in the yolk, as, for instance, Bialascewicz's own work on the electrolyte content of bird and fish egg-yolks, already mentioned in Section i • 1 6. In 1 902 Stewart showed that hen's egg-yolk is a very much poorer conductor of electricity than a solution of its salts made up to the same volume. McClendon in 19 10 examined the electrical conductivity of centrifuged suspensions of yolk, one poor in lipoid-protein yolk granules, the other rich in them. There was a sHght difference between the two, the granule-poor suspension conducting rather better than the granule-rich one. Dilution, which breaks up ion-colloid compounds, made the conductivity of both suspensions decrease, a paradoxical result which McClendon was unable to explain.
5-7. Specific Gravity
The subject of osmotic pressure during embryonic life leads naturally to the discussion of specific gravity, many measurements of which have been made by marine biologists interested in the eggs of plankton. Here the question is complicated by the fact that the relative quantity of fats and oils, substances which have Httle or no osmotic activity, has importance in deciding whether an egg shall float or sink. The facts have been reviewed by Strodtmann and by Russell.
A good deal of our information about the specific gravity of marine eggs is inexact, for it is derived from the reports of those who have studied the frequency of the occurrence of forms of different developmental stages at different depths. Thus we know from the work of Holt that the eggs of the turbot Rhombus maximus sink rather quickly after the 7th day of development, and, according to Ehrenbaum, the eggs in the plankton sink as a general rule, for the lower the catch the more advanced the embryos. Raffaele, again, found that nearly all pelagic fish eggs get heavier as development proceeds, presumably because of loss of buoyant oily substances by combustion. Most eggs begin to sink at once, but one (that of Labrax) very slowly, and one {Trachinus vipera) only when development is half completed. Similarly Jespersen & Taning observed that the larvae of the small oceanic fish, Vinciguerria attenuata, move down into deeper water as they develop (see also Yagle).
Indeed, as far back as 1897 Hensen & Apstein affirmed that the eggs of the cod, Gadus morrhua, sank regularly during development, for at the lower levels only the more advanced stages were found. This was denied by Hjort & Dahl, and by Kramp, but Jacobsen & Johansen (for Gadus and Pleuronectes) and Bowman confirmed it. The most recent investigations, those of Russell, show that eggs of Gadus morrhua and of Sardina pilchardus certainly sink, but those of Clupea sprattus seem to be equally distributed at all depths, and those of Onos are at all stages most abundant just under the surface of the water. Franz in 191 1 supplied some quantitative data as shown in the following table, which demonstrated that at any rate for many eggs the specific gravity was higher at the end of the development than at the beginning.
Table 93.
Mackerel {Scomber scomber)
Gurnard ( Trigla gurnardus)
Lemon dab (Pleuronectes microcephalus)
Turbot {Rhombus maximus)
Sprat {Clupea sprattus)
Tadpole fish {Raniceps raninus) ...
Gunner {Ctenolabrus rupestris)
Rockling {Motella)
Dragonet {Callionymus lyra) Sole {Solea lutea) ... Dab {Pleuronectes limanea)
Specific
gravity
ginning
End
031 1
1-0317
0307
1-0320
02q8
1-0306
•0307
1-0315
•0309
1-0312
•0291
1-0309
•0296
1-0310
0297
1-0307
•0320
—
•0313 •0308
1-0341
I -0339
In 1926 Sparta showed once more that most teleostean eggs have a rising specific gravity during development, but found that Gobius jozo was an exception, for the sp.g. of its eggs fell from 1-093 to 1-061.
The course of the specific gravity has been charted out for a great variety of fishes by Remotti, some of whose curves are shown in Fig. 200. No satisfactory explanation of these phenomena has so far been given, but the experiments of PoHmanti are rather suggestive. He estimated the fatty acid content of various fishes, getting the following results: „ , ,
Table 94.
^^ Fatty acids in % of
dry weight
Pilchard {Clupea pilchardus) 20-447
Mullet (Mugil chelo) 12-609
Anchovy {Engraulis encrasicholus) ... ... ... 9'i33
Scorpion hsh (Scorpaena scrof a) ... ... ... 7-2 11
Mediterranean eel {Congromuraena balearica) ... 6-768
Blenny {Blennius gattorugine) ... ... ... 6-422
Torpedo [Torpedo ocellata) ... ... ... ... 6- 100
Sole [Solea ocellata) ... ... ... ... ... 5*448
Dogfish (Scyllium canicula) ... ... ... ... 5'3i5
Weever {Trachinus draco) ... ... ... ... 4'730
Eel {Conger vulgaris)... ... ... ... ... 3'774
Star-gazer (JJranoscopm scaber) ... ... ... 2-600
Sole {Rhomboidjctis podas) ... ... ... ... 1-474
Goby {Gobius paganellus) ... ... ... ... 1-115
He then noted that this was also practically the order in which the fishes would be arranged, beginning with surface and descending to bottom fishes. He therefore suggested that the rise and fall of fish eggs during their incubation period was probably correlated directly with their changing content of fat. The influence of light on these ontogenetic vertical migrations must also be considered; indeed, according to Russell, it is the most important factor in them. The eggs of fishes are not the only ones which move upwards and downwards in this way. Similar migrations have been observed in the cases of the chaetognath Krohnia hamata (Fowler), various copepods (Farran and Kraeff't), the stomatopod Squilla (Santucci), and the siphonophore Velella spirans (Woltereck). Zahony, who investigated the ontogenetic migrations of the chaetognath Sagitta serratodentata, regarded temperature and not specific gravity as the determining factor, but in support of Russell's view there are the laboratory experiments of Groom & Loeb (on the acorn barnacle, Balanus perforatus), and of Mast and Grave (on the tunicate Amaroucium), who all noted a changing reaction to light during the course of embryonic development.
Some curious experiments which may have a relation to these facts were made by Remotti, who caused the eggs of Salmo lacustris to develop, some in darkness, some in the illumination of a very powerful electric light. At the end of development the serum of the embryos which had developed in the light reacted more slowly and feebly with quinol than that from those which had developed in the
Fig. 200. Each curve begins at fertilisation.
dark. By the continuation of similar experiments Remotti hoped to identify in some way the factors responsible for the effect of light on developing embryos. For further discussion of this subject see Section 2-17.
Various other attempts have been made to unravel the mechanism responsible for ontogenetic vertical migrations. Sanzo reported, though without giving any figures, that the perivitelline liquid of murenoid eggs has a lower freezing-point at the end of development than at the beginning, i.e. that probably the membrane becomes permeable to the salt of the environment as the embryo develops. This finding requires confirmation, Remotti has also studied the thermal expansion coefficient of teleostean eggs, which he claims differs slightly from that of sea water. He found that it definitely augmented during development.
The specific gravity of the amphibian embryo received a careful examination at the hands of Williams, whose data are plotted in Fig. 20 1. In all cases, there is a fall during development which is not interrupted at hatching. No doubt this is due to the absorption of water which is going on at that time (shown in Fig. 230). It must be remembered that the frog embryo is not separated from the yolk, so that the water absorption is not necessarily into the embryonic tissues. Williams also studied the centre of gravity in the frog embryo, finding that in the pre-hatching stages it was always at the cephaHc end of the body, but afterwards, as the yolk was disappearing, it moved to the caudal end. Bialascewicz's figures for the specific gravity of frog larvae are in complete correspondence with those of Williams.
Hardly any investigations have been made of the specific gravity of the constituents of the hen's egg, except the very early one of Baudrimont & Martin de St Ange, who reported in their famous memoir of 1 846 the specific gravity of various samples of yolk. The figures were as follows :
Table 95.
mm. length Fig. 201.
Specific gravity
Investigators
External albumen
Internal albumen
Whole yolk well mixed
Yolk taken from under the cica tricula, i.e. from the latebra Yolk taken from the opposite
side ... Egg-white (all, well mixed) ...
1-0399-1-0421 1-0421-1-0432 1-0288-1-0299
I -0266-1 -0277
1-0310-1-0321
Average
1-0410 1-0426 1-0293
1-0271
i-03i5„ 1-04028
Baudrimont & M. de
St Ange
Do.
Do.
Do. Rakusin & Flieher
They felt, therefore, that they had explained why the yolk always floats with the germinal disc upwards, and they suggested that the oily substances were more concentrated there. This explanation is difficult to accept, in view of what we know about the relations between the white yolk and the yellow yolk. Baudrimont & Martin de St Ange drew a tentative parallel between the oriented floating of the hen's egg-yolk and the animal and vegetal poles of the frog's egg. Among the few pieces of work on this subject is that of Mussehl & Halbersleben and Dinslage & Windhausen who found that the specific gravity of different individual batches of eggs bore no relation to the percentage hatch. Groebbels has shown in the case of a number of wild birds' eggs that the specific gravity decreases during the course of development: in some instances this process goes exactly inversely to the weight of the embryo.
5-8. Potential Differences, Electrical Resistance, Blaze Currents, and Cataphoresis
Some further remarks must now be made on the subject of the electrical properties of embryos. A certain number of investigators have found a constant potential difference between the head and the tail ends of various embryos, and their work was reviewed by Hyman & Bellamy in 1922. Hyde's work on the potential of sea-urchin embryos may be mentioned — her experiments on frog embryos were confirmed by Viale.
More recently work on these lines has been carried on by Gayda. Leading off through electrodes placed at the poles of the embryos, he found the potential differences to be as follows :
Gayda's
figures
Hyde's figures
Bufo vulgaris
(volts)
(volts)
(amperes)
Fertilised egg
<3-o . 10-*
3 • io~®
Embryo ready to hatch
2 . 10-5
<5-o . 10-5
3 • 10-9
Tadpole a week after hatch:
[ng
head-tail end
21-9 . IO~*
—
head-rump
14-5 . 10-^
—
rump-tail end
5-1 . 10-3
—
After metamorphosis
head-rump
—
13-6 . 10-^
—
More detailed figures are shown plotted in Fig. 202. It is very striking to notice the way in which the electrical resistance rose continuously. More examples of this phenomenon will shortly be given, and it seems to be, indeed, a general rule that the electrical resistance of a tissue or of a whole organism increases with age. Whether this has any relation to the salt- or ash-content will be discussed later, but Gayda's figures for electrical resistance in Fig. 202 should certainly be compared with the figures for salt-content of frog embryos and tadpoles obtained in the work of Schaper, and plotted in Fig. 230.
Fig. 202.
The time relations are astonishingly concordant, for, whereas according to Gayda the electrical resistance rises steadily until the 1 6th day from fertilisation, so according to Schaper the ash-content falls steadily until the i6th day after fertilisation. After that point the ash-content slowly rises again, and conversely the electrical resistance slowly falls, or remains constant. In Fig. 202, the potential difference between head and tail end of the embryos in volts and amperes should be observed, passing steadily upward out the same curve. I have not figured Gayda's points for the period after the 30th day from fertilisation, for, with the initiation of metamorphosis, the embryological sphere is transgressed, but it may be said that the electrical resistance is gradually lowered, until at the time of completion of the posterior legs it has attained a practically constant value. This equates extremely well with the behaviour of the total ash as shown in Fig, 230. The amperage and voltage of the current between head and tail continue to increase more or less regularly through metamorphosis. The difference of potential between the tail end and the head end suffers an extremely rapid change over at about the 95th day from fertilisation, i.e. just before the completion of metamorphosis ; before that time the tail end was always about 10. lO"^ volts higher potential than the head end, but after it the reverse relation held. Gayda concluded that the embryo could be pictured as a series of solutions of diverse concentration and constitution, separated by semipermeable membranes^, and that changes in morphology in such a system would have the effect of setting up small currents such as he had measured.
Mendeleef has concerned herself much with the comparative electrical resistance of tissues. Using PhiHppson's method for determining the electrical resistance of tissues, which consists in measuring the resistance a known amount of tissue opposes to the passage of an alternating current of frequency varying from 1000 to 3,000,000 periods per second, produced by a thermionic valve, she investigated the magnitude of PhiHppson's constants for embryonic and adult tissue. These constants are (a) R, the specific resistance of the cell-contents, i.e. the reciprocal of the electrolyte-content of the cytoplasm, (b) r, the resistance per cubic centimetre of tissue, i.e. inclusive of intercellular spaces, membranes, etc., and (c) p, the resistance in ohms per cubic centimetre of tissue corresponding to polarisation at a frequency of zero. For the liver of the normal guinea-pig, these were as follows:
Ohms
R 195
r ... ... 1790
P 4-56 . lo^
1 Responsibility for the resistance must probably be placed more on the membranes than on the ash-content.
During pregnancy, R was lowered by not more than 5 per cent., r by about 15 per cent. a,nd p by 50 per cent. Mendeleef concluded
that the resistance of the cell-interior was hardly affected, but that the resistance of the cell-membranes was definitely less than normal. When these experiments were extended so as to include embryos, the interesting figures shown in Table 96 were obtained. Throughout embryonic life, the resistance of the embryonic tissues is obviously less than that of the tissues of the maternal organism but, by the time that birth is reached, the two are at an equality, or the reverse relation may even be present. After the birth of the embryo the
Table 96. Electrical resistance of liver cells. Mendeleef 's figures.
Length of
R (ohms)
r((
jhms)
p (ohms
X lO^)
embryo guinea-pig
(cm.)
Foetal
Maternal
Foetal
Maternal
Foetal
Maternal
2
123
185
870
1515
1-37
2-21
3
70
180
880
1310
1-37
2-04
4
"5
180
1175
1710
1-69
2-45
1
160
180
815
1710
1-27
2-45
120
185
1200
2065
1-87
2-65
7
175
200
985
1670
1-68
2*00
8
155
175
1320
1585
2-04
2-32
Term
147
180
1380
1300
2-01
I -go
maternal liver tissue returns within 48 hours to its normal level. Mendeleef concluded from all this work that the membrane permeability as well as the electrolyte concentration in the cytoplasm were higher in the embryo than in the adult, and probably higher the younger the embryo. We have had already a good example of the increase of electrical resistance with age in the work of Gayda (see Fig. 202). Evidently the electrolyte-content of a tissue or an embryo is not measured by its ash-content, yet it is not without significance, perhaps, that the ash-content of the chick embryo (see Fig. 402) and of the frog embryo (see Fig. 230) decreases with age. A striking illustration of this is provided by Fig. 249, which shows the behaviour of the inorganic substance/organic substance ratio for the chick embryo, a ratio which falls steadily from the beginning of incubation till the end. Mendeleef laid special emphasis on the importance of the placenta in separating two organisms with very different cellular permeabilities. She went on to study the effect produced by keeping the extirpated tissues in vitro for some time. If the measurement is made three-quarters of an hour after the removal of the cells from the body, a much higher resistance is found than if it is made immediately after removal. This augmentation in vitro she found to be due only to r, not to R or p; in other words, to an increasing ionic membrane impermeability. The cells do not vary in electrolyte-content, or in membrane polarisation. The augmentation is stopped by cold, and is therefore probably chemical; some tissues show it more intensely than others. In the case of the embryo, the following results were obtained :
Length
of embryo
(cm.)
3 5 6 8 Term
Table 97. Electrical resistance of liver cells.
r (ohms) at the time of removal of the material
Foetal
909 1240
1425 1 140 1252
Maternal
1457 i860 1071 1840 1233
45 minutes afterwards
Foetal Maternal
1453 1986 1756 1300 2602
3702 4217 3590 3232 2790
Percentage augmentation
Foetal 60 60
24
14
108
Maternal
I2D
Evidently the electrical resistance of the embryonic tissues does not augment in vitro to the same extent as that of the maternal tissues, at least until birth, by which time the two react very similarly. Mendeleef concluded that there was a relation between decrease of membrane permeability in vitro, and its absolute level in growing and stationary tissues.
Philippson's methods (much modified) have also been used by Cole to determine the impedance of suspensions of eggs to various frequencies of alternating current. Operating on the eggs of Arbacia punctulata Cole found that before fertilisation the values of the impedance for any given frequency were quite variable, and there were similar variations in the average specific resistance of the ^gg at both high and low frequencies. Immediately after fertilisation, however, these quantities became quite constant and did not change noticeably thereafter. Cole did not regard the work of Gray and of McClendon, mentioned above, as very satisfactory, for it was done at low frequencies and in such conditions that almost all the current would flow through the intercellular sea water. In this way a very small change in the size of the eggs would cause a considerable change in the overall conductivity. Cole concluded, for his part, that the specific resistance of the interior of the egg was about 90 ohms per c.c. or 3-6 times that of sea water, and that the impedance of the surface of the egg is probably similar to that of a "polarisation capacity". On these values membrane formation and cell division seemed to be without effect.
The electrical resistance of the constituents of the hen's egg has been studied by Bellini. The figures he obtained are shown in Fig. 203. If the egg is not incubated at all, the resistance of the yolk and the white remains quite constant even over a much longer period than that represented on the graph.
If, however, the egg is placed at 37°, the resistance of the yolk augments until the 6th day, after which it declines to its initial value. That of the white was not followed by Bellini after the 8th day, but rose quickly up to that point. It is not easy to interpret these results, but presumably they indicate a demobilisation of free electrolytes throughout the extra-embryonic part of the egg during the I St week of development. Practically nothing is known about the behaviour of the inorganic constituents of the yolk and the white during this time^. The fact that the embryonic body is at this stage more rich in ash, and therefore probably in electrolytes, than at any other stage, though unknown to Bellini, may have some bearing on the question, but the embryo is now so small in relation to the yolk and white that it is doubtful whether its absorption of electrolytes could account for the changes in electrical conductivity of the rest of the egg.
According to Fiirth the dielectric constant of yolk in the hen's egg is 60 and that of white 68.
Other phenomena were discovered by Hermann & von Gendre, who found in 1885 that the developing chick embryo is always positive to the yolk and the white. At 80 hours' development, there
^ But see Fig. 405 and Section 13' i.
Fig. 204.
was a maximum e.m.f., but they were not able to offer any explanation of this, nor have any more recent workers gone into the matter anew. Their figures, which are presumably to be explained on a concentration-cell basis, are plotted in Fig. 204. Waller investigated the "blaze currents" of the developing hen's egg. He had previously defined a blaze current as an electrical response to some kind of stimulus, whether electrical, chemical or photic. In its most characteristic form it occurred in the same direction as the current by which it had been excited, and this was important, for it could therefore not merely be a polarisation counter-current. The blaze current, according to Waller, is precisely analogous with the discharge of an electrical organ, excited by an electrical current in the homonymous direction. Having studied it in the eyeball and the crystalline lens, he turned his attention to the hen's egg, and, thinking that its presence or absence might be an indication of whether the embryo was living or not, he investigated a number of incubated eggs. As he had expected, the blaze reaction made its appearance as development progressed. Electrodes were brought into contact with the shell-membrane, a small piece of the shell having been removed, and whenever a blaze current appeared on stimulation, there a Hving embryo was found when the egg was opened, and vice versa. Waller confirmed the observation of Hermann & von Gendre that there is a small current normally passing from egg-contents to embryo, and observed also that by repeated excitations the embryo can be killed or exhausted. Fig. 205 shows the decreasing blaze current obtained from an egg which was stimulated several times after 48 hours' incubation. Waller found that in the early stages, before the blastoderm membrane had folded to form a tubular embryo, the blaze currents were always positive, no matter whether the excitation was positive or negative, but later the blaze currents were always homodrome with the direction of excitation, positive if the excitation was positive, negative if it was negative. He extended these observations to frog's eggs, and reported that for the most part definite blaze currents had been given by them.
Not a few researches have been made in which the effect of electrical energy applied in various ways has been tested as a stimulus for embryonic growth or differentiation. The results have been uniformly negative, except from a teratological point of view, and even then very little illumination has resulted from such experiments. In 1840 Rusconi initiated this type of work by exposing hen's eggs to the action of an electrical current, constantly flowing, but his results were quite negative. Fasola in 1887 and Roux in 1889 both found nothing but a few malformations, which any treatment might have produced. In the last decade of the last century, there were several
Fig. 205.
papers on this subject; thus Windle stated that a shght retardation of growth was noticeable when the embryo was subjected to a continually flowing electric current, but that incubation in a strong magnetic field was favourable. His experiments were not done statistically. Dareste obtained teratological effects, but was contradicted by PieralHni, and Rossi stated that development was undoubtedly modified by electric currents, but probably only indirectly on account of interference with other factors. The only recent investigations of the matter are those of Gianferrari & Pugno- Vanoni, who in 1923 reported the results of subjecting salmon eggs to high frequency currents during their development. Duplications and various terata were produced. On the whole, this seems to be a very unpromising line of research, for, to begin with, it will always be a matter of extreme difficulty to hit on the right strength of current or of magnetic field to influence the embryonic processes without causing pathological states to arise. It is interesting, however, that Brown found that the eggs of Fundulus were as immune to electrical stimulation as to other influences, such as osmotic pressure.
Among the various electrical properties of eggs and embryos which are to be discussed in this section the charge on the egg has so far not been mentioned. Cataphoresis experiments with echinoderm eggs were made by McClendon in 19 14, who found that, when the jelly had been removed from them, they were transported to the anode, and possessed therefore a negative charge. The surrounding jelly, however, went the other way, and had a positive charge; thus McClendon suggested that the fertilisation membrane might be the result of mutual precipitation by antagonistically charged colloids. Tomita, again, working with nematode eggs [Ascaris, Oxyuris, Ankylostomum), trematode eggs {Distoma) and cestode eggs {Taenia and Bothriocephalus) , found in all cases a migration from cathode to anode, and so a negative charge. Szent-Gyorgyi, however, found no cataphoretic movement at all in the case of Labrus rupestris and Echinus vulgaris eggs. Vies & Nouel, who made some experiments involving the degree of agglutination of echinoderm eggs at different j&H, agreed with the conclusions of McClendon, although experimentally their Strongplocentrotus eggs moved towards the cathode. In view of the fact that three different workers have obtained the three possible results on this material, it is clear that further observations are to be desired. Vies, Achard & Prikelmaier, working on the pounded and cytolysed protoplasm of Strongylocentrotus eggs, found cataphoresis towards the cathode below />H 5-8 and towards the anode above it, from which they concluded, as has already been mentioned, that the iso-electric point of the egg-proteins was about 5-5.
5-9. Refractive Index, Surface Tension and Viscosity
Vies has studied a number of the biophysical properties of the seaurchin's egg, such as refractive index. He regarded the refringent spherical or ellipsoidal ^gg as a lens, from which the refractive index could be calculated, knowing the focal distance. The necessary measurements were (i) the curvature of the egg, ascertained by micrometric measurements along different diameters, (2) the focal distance, measured optically. In a second paper he studied the change in refractive index during the period from fertilisation to first cleavage in the sea-urchin's egg. A small fall after fertilisation led to a large rise after the appearance of the diaster, and a rapid fall immediately before cleavage, the outside limits of refractive index within which these changes took place being 1-381 to 1-405. Their significance was doubtful.
The effect of heat on the physical properties of egg-cells has been studied by Achard. The volume, calculated from the diameter, attained a definite maximum at 35°, being 44-5 . io~^ c.c. at 20°, 54-3 at 35°, and 46-0 at 41°. The specific gravity measured by immersion of the eggs in solutions of equal .osmotic concentration but different density varied little, but had a slight peak at 36°. The electrical charge had a maximum at 35°. The oblateness (calculated from measurements of maximum and minimum diameter) had a maximum at 35°, and the surface tension (calculated from the oblateness) had a minimum at the same temperature. Achard concluded that the physical properties of these egg-cells could be interfered with to a slight degree apparently without preventing normal fertilisation and cleavage. The curves she obtained are given in Fig. 206, and show how 35° is in nearly all cases a critical point. The important point is that it is also a critical point biologically, for below it the percentage of eggs forming normal fertilisation membranes is uniformly 100, and above it falls off, while just the same applies to the percentage of eggs successfully accomplishing their first cleavage.
Vies himself did a good deal of work on the surface tension of echinoderm eggs, treating them from the point of view of the physics of semiliquid drops, and examining their departure from the form of a perfect sphere under different experimental conditions. The surface forces acting on an egg can be evaluated, according to Vies, from the degree of oblateness or flattening which the egg suffers when it rests on a horizontal surface under the influence of gravity. Unfertilised sea-urchin's eggs, devoid of jelly, show minima of flattening between pH 3 & 5 and 8 & 10. The tensions involved are of the order of 10 to 25 dynes per centimetre. ^ After fertilisation the surface tension is reduced ; it rises again before the fusion of male
^ A much lower value (1-3 dynes per centimetre) is obtained for Chaetopterus eggs by observing their fragmentation in the microscope-centrifuge (Harvey & Loomis; Harvey).
and female nuclei, and falls when that process is complete. It rises again to the diaster stage, and diminishes markedly immediately before cleavage. The second augmentation would correspond with the semipermeable phase of Herlant and the period of augmentation of
Fig. 206.
refractive index found by Vies. The curves with which this paper is illustrated seem to indicate an unwarranted confidence in the accuracy of individual points, and the number of observations is hardly sufficient to establish some of the conclusions.
The subject of protoplasmic viscosity has attracted a very great number of investigators, and cannot be reviewed here, but certain work of special embryological importance must be mentioned. In the first place, Bellini has studied the viscosity of the yolk and the white in the hen's egg during the early part of its development, using Fano & Rossi's viscosimeter. The values he obtained are shown in Fig. 207. Evidently if the eggs are not incubated the viscosity of the white increases steadily but slowly, and that of the yolk decreases at much the same rate. If the eggs are incubated, the viscosity of the white increases more rapidly, and that of the yolk decreases far more so, descending from 30 units to less than I by the 6th day of development. This reciprocal interchange of viscosity between the yolk and white evidently goes on irrespective of the embryo, though it is much accelerated by the presence of the latter, and Bellini rightly concluded that it was the result of a continuous dilution of the yolk from the water of the white. I shall return to this point in the succeeding section, when the whole question of water metabolism in embryos is being discussed, and though, as we have already seen, the osmotic pressure of the yolk is much superior to that of the white, Vladimirov
Fig. 207.
work has made it very unlikely that this can be the sole cause of the flow of water. Again, the viscosity of the white increases by only 4 units in the first 10 days, and that of the yolk decreases by as much as 30 units, so Bellini envisaged other processes than the dilution of the yolk by the water from the white as playing some considerable part in the decline of yolk- viscosity. It is known that the yolk contains an abundance of enzymes, which the incubation at 37° might allow to act, e.g. proteases, lipases; diastases (see Plate XI). Experiments that Bellini did with incubated infertile eggs showed that the great decline of yolkviscosity did not go so far as with, developing ones, but on the other hand there was little difference between the viscosity of the white in incubated fertile and infertile eggs. The whole question merits more study, in view of the obvious importance possessed by the mechanism of preparation of the first pabulum of the embryo. For the frog's egg, rhythms of viscosity have been reported by Odquist, who, however, used a centrifugation method followed by observation on pigment distribution.
YOLK OF HEN'S EGG AT THE ELEVENTH DAY OF INCUBATION
Stained with iodine, showing its very heterogeneous character half-way through development. Note especially the large vacuoles filled with fluid contents. Magnification 6 X A, prepared and microphotographed by Dr V. Marza.
As regards the protoplasmic viscosity of alecithic eggs, Heilbrunn has worked on those of the clam, Cumingia, and the sea-urchin, Arbacia punctulata, using the centrifuge method. In both cases there was a maximum viscosity at 15°, from which the values fell away on both sides. Pigorini subsequently obtained very similar results for the expressed juice of silkworm's eggs.
Heilbrunn in 1920 also studied the effect of anaesthetics upon viscosity in eggs, and in 1 9 1 5 the effect of hypertonic solutions ; the original papers must be consulted for the details,
Seifriz has also done much along these lines, using for the most part the method of microdissection. In many cases this method has given diametrically opposite results to those obtained by other techniques, especially the centrifuge, and has occasioned some polemics which need not be described in detail. Seifriz maintained that the protoplasm of Echinarachnius eggs was immiscible with water, and allotted a definite degree of viscosity to the cytoplasm at different stages during cleavage, etc. A large element of the subjective must have entered into the determination of these values, and where they have conflicted with those obtained by the centrifuge method they have been abandoned by most biologists, who prefer the more objective technique. Seifriz's viscosity values were as follows:
Substances of
equivalent
vv
% gelatine
viscosity
I
o-o
Water
2
0-05
—
3
0-2
—
4
0-4
—
0-5
Paraffin oil
6
0-6
7
0-7
Glycerol
8
0-8
Bread-dough
9
i-o
Vaseline
10
2-0
Finn gelatine gel
On this scale the vv of mature unfertiUsed eggs of Echinarachnius and Tripneustes was 7. This agreed with the microdissection estimates of Kite and of Chambers, who had stated that the ooplasm was just viscous enough to put a stop to Brownian movement. During mitosis the marked changes in viscosity led to a local lowering of viscosity to vv 3 or thereabouts, Seifriz's later experiments were very ingenious. He introduced by the aid of a micromanipulator a minute nickel ball about 7jLt in diameter into the cytoplasm of the Ggg of the sand-dollar, Echinarachnius parma (a principle that had been adopted in the work of Freundlich & Seifriz on inorganic colloids) . The particle having been introduced, it was attracted by a powerful electromagnet, until the colloid was stretched a certain amount. If this amount was not exceeded the particle would return to its original position when the current was cut off. The distance over which the particle travelled furnished a measure of the stretching capacity of the colloidal substance, and the force necessary to produce the stretching was a measure of the elasticity. It was found that a particle introduced into the protoplasm of an Echinarachnius egg could be moved backwards and forwards through the inner parts by the electromagnet, apparently without any injury being done, but that, when it came up against the cortical parts of the egg, its motion was much slower and might completely cease. Obviously their viscosity was greater. Quantitative calculations showed that the vv of the inner part was just the same as that which Seifriz had previously allotted to other marine eggs, i.e. barely that of concentrated glycerine (sp.g. 1-2500). The stretching distance of the cortex protoplasm was 9 /a. Nothing has since been done to follow up this interesting type of investigation, but various investigations of viscosity in echinoderm eggs have been made (Barth ; Jacobs ; etc.) and for further information the monograph of Heilbrunn should be consulted.
Cite this page: Hill, M.A. (2024, April 28) Embryology Book - Chemical embryology 2-5 (1900). Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Book_-_Chemical_embryology_2-5_(1900)
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