ABSTRACT
Chick blastoderms have been grown in vitro on pieces of vitelline membrane supported over albumen.
The blastoderms were found to remove fluid from the albumen and to secrete it from their endodermal surfaces.
It is concluded that this could be the mechanism by which the sub-blastodermic fluid is formed in ovo.
The importance of the sub-blastodermic fluid to the developing embryo is discussed.
INTRODUCTION
It is well established that during the first week of incubation of the hen’s egg the albumen loses far more water than can be accounted for by evaporation, and at the same time the yolk gains in water (see Needham, 1931, for detailed references). It is apparent, therefore, that water is transferred from the albumen to the yolk. This transferred water does not distribute itself evenly through the yolk, most of it accumulating immediately under the embryo (see Text-fig. 2) and giving rise to the sub-blastodermic fluid (this is the term employed by Murray (1933); Romanoff (1943 a and b) calls it liquefied yolk).
The sub-blastodermic fluid increases rapidly during early incubation and about 16 g. is present at the end of the first week; it then decreases in amount whilst the amniotic and allantoic fluids are forming and has almost disappeared by the end of the second week (Romanoff, 1943a). Until about the 9th day of incubation more than 90 per cent, of the sub-blastodermic fluid consists of water (Romanoff, 1943b).
Osmotic forces between yolk and albumen have sometimes been considered sufficient explanation of the mechanism governing this fluid formation (e.g. Yamada, 1933). Subsidiary mechanisms may also be present. Wladimiroff (1926) concluded from pH measurements that acid was released in the albumen, which, he argued, would bring the proteins closer to their isoelectric point and would therefore reduce their power of holding water.
It is the aim of this paper to show that whether or not such forces exist in the non-protoplasmic parts of the egg, a large part of the sub-blastodermic fluid is probably formed directly as a result of a secretory activity of the blastoderm.
METHOD
Blastoderms were grown in vitro by a method described in an earlier publication (New, 1955). Each blastoderm is grown on a piece of vitelline membrane stretched across a glass ring in a watch glass (Text-fig. 1A). Under the vitelline membrane is placed a little thin albumen. There is no yolk in this system other than a small amount ingested within the endoderm cells of the area opaca. If, therefore, any fluid is removed from the albumen and passed through the vitelline membrane and blastoderm, the forces controlling this transfer must be independent of osmotic or other forces between yolk and albumen.
A and B. Blastoderm explanted ectoderm downwards, A. Condition at the time of explantation, B. Condition after a total incubation time of 48 hours, c and D. Blastoderm explanted endoderm downwards, c. The edges curl under, D. Closed vesicle formed: the transferred fluid is mostly within the vesicle (a), though some is outside it (b). Ectoderm shown as a continuous thick line, endoderm as a dotted line.
A and B. Blastoderm explanted ectoderm downwards, A. Condition at the time of explantation, B. Condition after a total incubation time of 48 hours, c and D. Blastoderm explanted endoderm downwards, c. The edges curl under, D. Closed vesicle formed: the transferred fluid is mostly within the vesicle (a), though some is outside it (b). Ectoderm shown as a continuous thick line, endoderm as a dotted line.
The experiments fall into two groups, those in which the blastoderms were explanted ectoderm surface downwards (i.e. against the vitelline membrane) and those in which the blastoderms were explanted endoderm surface downwards. The quantities of transferred fluid in the first group of experiments were determined with a small measuring cylinder and are therefore given as volumes. In the second group, for greater accuracy, the quantities were determined by weighing the preparations before and after removal of the fluid.
RESULTS
Explantation ectoderm downwards
Forty-five cultures of this type were made. These cultures contained blastoderms explanted at various stages between 24 and 48 hours of incubation. Expansion of the blastoderm continued in vitro until the whole available area of vitelline membrane (about 28 mm. diam.) was covered (Text-fig. 1B); this occurred soon after 48 hours of total incubation time. The cultures were examined 2 or 3 days after explantation and frequently fluid was found to have accumulated above the blastoderm. This was pipetted off and measured. In those cases where the blastoderm died particularly early the volume of fluid obtained was usually very small. When development continued to 25 somites or more, however, considerable quantities of fluid appeared. The average value from 30 such blastoderms was 0·75 c.c. with a maximum of 1·4 c.c.
In order to be certain that this fluid was not just albumen that had seeped through damaged parts of the vitelline membrane, a quantity of it was collected from several cultures and boiled in a test-tube. It behaved exactly like sub-blastodermic fluid and quite unlike albumen. A loose floccular precipitate formed, which gradually settled into the bottom of the test-tube leaving a clear fluid above. Albumen treated in the same way forms a rigid mass.
But there can be no doubt that although this fluid is not albumen, it has arisen from the albumen. Each culture was initially supplied with 1-2 c.c. of albumen below the vitelline membrane. If no fluid formation occurs a similar quantity of albumen can be recovered after a few days’ incubation. When fluid is formed above the blastoderm, however, the volume of albumen remaining drops to low values. Instances have been common in which over 0·8 c.c. of fluid has been obtained above the blastoderm whilst less than 0·5 c.c. of albumen was left.
The absence of ‘free’ yolk in these preparations excludes the possibility of forces between the yolk and albumen being the cause of the fluid formation. They suggest, on the contrary, that the fluid is produced as a result of secretion by the blastoderm. As a check on this interpretation eight controls were set up in which the blastoderm was removed; in four of them the blastoderm was replaced by a drop of yolk. After 2 to 3 days of incubation none of these controls showed more than negligible quantities (< 0·1 c.c.) of fluid above the vitelline membrane.
There remains the possibility, however, that the weight of the blastoderm and vitelline membrane might set up a pressure, itself sufficient to force fluid out of the albumen and into a position above the blastoderm. It was found that when blastoderms at their maximum stage of development in vitro were replaced by small glass rods of two to three times the weight, small quantities of fluid (up to 0·25 c.c.) appeared above the vitelline membrane after a further 2 days’ incubation. Although these quantities are much smaller than those found when a blastoderm is present they are sufficient to raise doubts as to whether or not the blastoderm is secreting. However, the experiments described in the next section have removed this uncertainty.
Explantation endoderm downwards
The chick blastoderm normally expands with its ectoderm surface applied to the vitelline membrane. In making these preparations, therefore, the blastoderm had to be separated from the vitelline membrane and then inverted on it. Under such conditions it does not expand normally, but instead the edges curl under, bringing their ectoderm surface again in contact with the vitelline membrane (Text-fig. 1c). Expansion then occurs, but from the geometry of the system the edges are now forced to grow inwards instead of outwards. Eventually they meet, fuse, and convert the blastoderm into a hollow vesicle (Text-fig. 1D).
When cultures of this kind were incubated for 2 or 3 days each blastodermic vesicle appeared as though ‘blown up’ from within, and if punctured a quantity of fluid flowed out of it. This was observed in 27 cultures, the maximum quantity of fluid recovered from a single vesicle being 0 69 g. This fluid reacted to boiling in the same way as sub-blastodermic fluid: it separated into a small precipitate with clear fluid above. Only very small quantities of fluid were found above the vitelline membrane outside the blastodermic vesicle. Details of measurements made on four of these cultures are shown in Table 1. It can be seen that the loss in weight of the albumen increases almost proportionately with the amount of fluid formed, leaving little doubt that the fluid is formed from albumen. The greater part of the fluid formed in each case was recovered from within the blastodermic vesicle. (As might be expected the loss in weight of the albumen is in fact greater than the weight of fluid formed since small quantities of material are consumed by the developing embryo.)
The most likely explanation for the accumulation of fluid within these blastodermic vesicles is that the blastoderm actively absorbs fluid on its ectoderm surface and secretes it from its endoderm surface. Alternative explanations involving fluid formation as a result of the weight of the blastoderm or vitelline membrane (as might have been possible when the blastoderms were explanted ectoderm surface downwards) are here avoided since they would be inadequate to account for the appearance of fluid inside the vesicles.
DISCUSSION
The experiments that have been described strongly suggest that the blastoderm has the ability during early incubation to remove fluid from the albumen and to secrete fluid from its endoderm surface. Just how far this secretory property is responsible for sub-blastodermic fluid formation in ovo is an open question. Table 1 suggests that at least 60 per cent, of the albumen can be secreted as fluid—a figure high enough to account for the whole of the fluid formed in ovo. However, the present work has not excluded the possibility that in the egg other mechanisms (e.g. osmotic) may also operate, and the safest conclusion would seem to be that since the blastoderm exhibits secretory activity so markedly in vitro, it is highly probable that this activity accounts for at least a major part of the fluid formed in ovo.
Why is the sub-blastodermic fluid formed at all? The transfer of such a large volume of fluid—nearly one-third of the whole egg contents—is a remarkable achievement at a time when the amount of living matter in the egg is relatively small.
Probably the most important function of the sub-blastodermic fluid is to assist respiration. It follows from the facts that the fluid is lighter than albumen, is formed from albumen, and increases the contents of the yolk sac, that a large area of chorion and yolk sac must reach a position very close to the shell membranes (Text-fig. 2). This means that the area vasculosa of the yolk sac, in addition to having a nutritive function, is also in a position greatly to assist respiration at a time when the allantois is still small or undeveloped. Furthermore, when the allantois does develop, it finds the chorion immediately under the shell membranes (the vitelline membrane having broken down after the 4th day) and the vascular chorio-allantoic membrane is established as close as possible to the source of oxygen.
Sagittal and transverse sections of an egg incubated 4 days (yolk and albumen fixed by boiling). At this stage the blastoderm encloses the sub-blastodermic (S-B) fluid and most of the yolk.
The fact that the sub-blastodermic fluid is of lower density than the yolk is probably itself a feature of some value. It is known that eggs require turning over from time to time during incubation, otherwise the percentage hatchability is greatly reduced (Eycleshymer, 1907, and others). The division of the yolk sac contents into lighter sub-blastodermic fluid and heavier yolk means that they come under a turning couple as soon as the egg is rotated to a new position. As a result, both constituents return to their former position carrying with them the blastoderm. (At the start of incubation the yolk itself seems to be polarized and returns to its former position when the egg is turned.) The whole mechanism probably serves to break down harmful adhesions which might otherwise occur (Dareste, 1891; Eycleshymer, 1907).
Finally it may be suggested that the sub-blastodermic fluid provides a better environment for the developing embryo than the yolk. It is known that the chick embryo excretes quantities of ammonia and urea during the first half of incubation (see Needham, 1931, p. 1089). Since the yolk appears to be highly resistant to diffusion of substances through it (Maurice, 1952; Maurice & Fidanza, 1952) a yolky environment might create a dangerous concentration of these substances close to the embryo; little is known of the chemistry of the sub-blastodermic fluid but during the first week of incubation it contains about 95 per cent, water (Romanoff, 1943b) and presumably offers little resistance to diffusion. Work by Spratt (1947) has suggested that the low pH of yolk (4-5-6 0) is harmful to embryonic development: his embryos developed well, however, when the pH was raised to 7 5. The pH of sub-blastodermic fluid is 7*7 (Shklyar, 1937). Possibly the sub-blastodermic fluid even creates less mechanical resistance to development than the denser yolk.
ACKNOWLEDGEMENTS
I should like to thank Mr. M. Abercrombie for the very helpful advice and criticism he has given me during the course of this work. I am also grateful to Miss D. Isenstein for technical assistance and to the Agricultural Research Council for financial support.