Talk:Waddington1956 3

From Embryology


THE Moment of fertilisation is the conventional point of origin from which to date the existence of a new individual. We have seen in the last chapter that in fact many processes which are most important for the developing embryo occur before fertilisation, during the maturation of the egg. It cannot be denied, however, that fertilisation is, normally at least, the most crucial event within the continuous series of changes by which the new creature comes into being. It is not a simple occurrence, at which there is only one happening of importance; but its two important phases succeed one another quite quickly, and although they can be dissociated from one another in experiments, they are normally closely bound up with each other, so that fertilisation appears as a single, though complex, event (Fig. 3.1).

It would be out of place to discuss here the many and various mechan WS

Fix N





Fertilisation in the annelid Urechis. In 1 a sperm is entering the egg at the

bottom left. In 2 the egg has formed a fertilisation cone and the germinal

vesicle is breaking down; 3, second polar body division and sperm aster;

4, egg and sperm nuclei approaching one another—polar bodies at animal

pole; 5, union (conjugation) of male and female nuclei; 6, first cleavage division. (After Belar.)


isms which have been evolved to assist the male and female in bringing their gametes into proximity; for details of copulatory and other devices, reference must be made to works on the general biology of sex. We may take up the tale of fertilisation from the point when eggs and sperm are in each other’s presence. The first reactions between them are, in many cases, chemical. It was shown by Lillie in 1913 that the water in which eggs of sea-urchins had been lying is able to induce a reaction in sperm of the same species, which are caused to agelutinate together into clumps. This is due to a substance given off by the eggs, to which the name ‘fertilizin’ was given. It has since turned out that this substance is none other than the jelly (or some constituent of it) by which the eggs are surrounded. It reacts with a substance in the sperm, which has been named ‘anti-fertilizin’. Very little is known about the nature of these substances, except that they are both proteins; the reaction between them is probably similar to that between antibodies and antigens. Such substances have only been definitely proved in some of the marine invertebrates, but they may perhaps be present in all animals, since the agglutination reaction by which they are recognised is really due to an excessive performance of their real task; a fertilizin which reacted with sperm, but did not go so far as to immobilise it by agglutination would easily escape detection.

It has also been claimed that sea-urchin eggs secrete a substance which attracts sperm, so that the latter move by chemotaxis towards the eggs. There seems, however, to be no conclusive evidence of this, and opinion seems to be crystallising against it. On the other hand, there do appear to be sperm secretions which affect the egg, assisting the sperm in the task of penetrating the egg surface or the Jelly which surrounds it. (In mammals, enzymes having this function occur in the secretions of glands which contribute to the semen, and it is not clear that the spermatozoa themselves produce anything of the kind.)

All active substances produced by either eggs or sperm and acting on gametes of the opposite sort are sometimes collectively known as Gamones, the egg secretions being Gynogamones and the sperm secretions Androgamones. In this terminology, fertilizin becomes Gynogamone II and anti-fertilizin Androgamone II. The subject has recently been reviewed by Tyler (1948, 1949), Runnstrém (1949) and Rothschild (19514, b), and further details may be found in their papers.

The actual process of fertilisation starts when the sperm first touches the egg surface. As mentioned above, the ensuing processes fall into two phases. These are:

(i) The activation of the egg. (ii) The union of the two haploid nuclei. FERTILISATION 47

(1) Activation

By ‘activation’ we mean the setting in train of a series of changes which bring the egg out of the quiescent state in which it awaited the arrival of a spermatozoan and start it off on the course of development. These changes take somewhat different forms in different groups, but there are certain common elements which are nearly always found. First in point of time, there may occur, in eggs surrounded by thick jelly, a reaction of the egg surface which assists the sperm in penetrating these outer coverings. For instance, in some echinoderm eggs, a conical projection pushes out from the egg surface and, as it were, catches hold of a sperm and draws it inwards through the jelly. Such happenings are, however, not found in all eggs.

Some kind of surface reaction of the egg is nearly always produced by the sperm. The most important and widespread form of the reaction is a change by which the first sperm which penetrates renders the egg surface impenetrable to later sperm. The exact nature of this change is still unknown; it may even differ in different groups. In many of the naked marine eggs, it is made visible by the formation at the surface of the egg of a new membrane, the ‘fertilisation membrane’, which lifts a little way off the egg immediately after activation (Runnstrém, 19524, b). This is very well seen in echinoderms; and in them it appears to be formed by the swelling and breaking up of a thin layer of colourless granules which can be found just below the surface of the ripe, unfertilised egg (Fig. 3.2). It seems, however, that in some species of echinoderms if not in all, the

Fertilization membrane {1


Perivitelline space”

Unfertilized egg Fertilized egg


Diagram showing the elevation of the fertilisation membrane in the sea urchin egg. In the unfertilised egg there is an inner layer of pigment granules

(pig. gr.) and an outer layer of cortical granules (‘Janus Green granules’,

JGG). The perivitelline space appears between these two layers. (From Runnstrém 1952, after Motomura.) 48 PRINCIPLES OF EMBRYOLOGY

impact of the first spermatozoan causes a cortical change in the egg which spreads over the whole surface in a much shorter time than it takes for the fertilisation membrane to appear. Rothschild and Swann (1949) were able to reveal this change by the use of dark ground illumination, and they present some reasons for thinking that it is this almost immediate effect, rather than the relatively slow elevation of the fertilisation membrane, which constitutes the block to polyspermy.

A surface change which guards the egg from the entry of more than one sperm can, however, occur without any visible sign of a fertilisation membrane. In fact, a change of this kind seems to be a quite general part of the activation process, excepting only in some of the very large, extremely yolky eggs, such as those of reptiles and birds and some insects. In these, the entry of considerably more than one sperm is a normal occurrence; only one sperm nucleus fuses with the egg nucleus, and the remainder gradually disappear after remaining for a time in the region where the cytoplasm mingles with the yolk, in the digestion and assimilation of which they may play a part (and see p. 62).

Changes of the egg surface are not always the only visible signs of activation. In many eggs, the penetration of the sperm initiates a more or less complete rearrangement of the internal constituents (Fig. 3.3). Other examples are described in detail later (see ascidians, p. 106, Amphibia p. 146). In these cases the pattern of the egg before activation bears little obvious relation to that of the embryo which will develop from it, while after activation a clear connection can be traced; thus we may say that in these forms, activation is the final stage in preparing the egg for the series of foldings and bendings by which the embryonic body will be shaped.

. These internal results of activation can only be discovered at all easily if there are differences in colour or texture between the various regions of the egg which make it possible to follow their movements after the sperm penetrates. It is therefore only in certain favourable types of eggs that they have been described, and it is still somewhat uncertain how generally they occur. The evidence suggests that there may always be some internal rearrangement, but that it is often quite small in extent.

One aspect of activation which may be specially mentioned is the determination of the plane of bilateral symmetry. Most eggs, as has been stated, have before fertilisation an axis of symmetry running from the animal pole (where the polar bodies are formed) to the vegetative (yolky) pole. Some eggs (e.g. of insects) are bilaterally symmetrical, but in most types there is no sign in the unfertilised egg of anything corresponding to a “Greenwich meridian’; and it may turn out in later development that the plane of bilateral symmetry is related to the point of entry of the sperm. FERTILISATION 49

Considerable controversy has raged about whether the entry of the sperm fixes the position of the dorso-ventral plane, or whether this is already determined in the egg and some mechanism ensures that the sperm always enters on it. The evidence is still somewhat conflicting, but seems to indicate that there is something in both ideas. It is certainly true that if eggs (e.g. of frogs) are artificially fertilised by sperm placed on the surface, the point of entry of the sperm determines the plane of bilateral symmetry;

mocrom. +


Movements of odplasms following fertilisation in Limnea. (a) Shows the

vegetative plasm (dots) before fertilisation. The entry of the sperm is fol lowed (b) by amovement of this plasm towards the animal pole. A little later

(c) the nuclei (pron.) conjugate near the animal pole and a new odplasm (close

dots) appears there. During the cleavage divisions, this extends towards the

vegetative pole (d and c), the original vegetative plasm becoming less easily recognisable. (After Raven 1948.)

but it is also probable that there is a predisposition of a fixed plane in the egg, which controls the point of entry in normal unforced fertilisation. (For a full account of the rather complicated events following fertilisation in frogs, see Ancel and Vintemberger 1948 and p. 146.)

Accompanying the visible structural changes produced by fertilisation, there are almost certainly associated alterations in the biochemical processes proceeding in the egg. Many years ago Warburg showed that the oxygen uptake of fertilised sea-urchin eggs is very considerably higher 50 PRINCIPLES OF EMBRYOLOGY

than that of unfertilised eggs, and it was thought that fertilisation brought about a great increase in respiration. However, more recent work has shown that the difference he described is due rather to a decrease in oxygen uptake by the stale unfertilised eggs than to an increase in the fertilised ones. Rothschild (19516), in a recent discussion of the metabolic changes produced by fertilisation, is very cautious about the extent of our knowledge on the subject. He suggests that the production of an acid of unknown nature is one of the most certain and striking phenomena, and lists a number of other changes, such as a reduction in glycogen content and a fall in respiratory quotient, without feeling justified in deciding which if any of these are of major importance.

(2) The union of the nuclei

The union of the two haploid nuclei of the egg and sperm is, from the long-term point of view, the most important phase of fertilisation. It is an essential part of the system of reproducing diploid adults by means of haploid gametes which has proved itself most efficient as an evolutionary mechanism and has therefore been perpetuated in the vast majority of animals and plants. The genetical and evolutionary aspects of the phenomenon fall outside our immediate field of interest. We must, however, give some account of the actual process by which the two nuclei come together.

The essential parts of the sperm in this connection are the head, which contains the nucleus, and the middle-piece, which contains a centrosome or spindle-body. The tail is primarily a locomotor organ, and its functions _ are finished when the sperm head becomes attached to the surface of the egg. It is often discarded at this point, only the head and middle-piece penetrating; and in those cases in which the tail goes in too (e.g. in mammals), it soon degencrates and plays no known part in later events.

The actual coming together of the two nuclei is simple, though mysterious enough if one tries to imagine how it works. The sperm nucleus after penetration always moves off towards the egg nucleus, while the latter sometimes moves to meet it; what moves them, and how the movements take the correct directions, are quite unknown. The sperm head (or as one may now call it the male nucleus, often called the male pronucleus), is accompanied by the middle-piece or centrosome. This body has a strong tendency to cause the cytoplasm around it to form an ‘aster’, which is a spherical aggregation of radiating fibres. If often starts to do so soon after getting inside the egg; but it soon divides, and an aster begins to form round cach of the daughter centromeres. Where these two asters come together, a still more strongly fibrous body is FERTILISATION sli

formed, known, because of its shape, as the ‘spindle’. This is the first cleavage spindle of the egg, and provides the mechanism for the first division of the chromosomes. It will be seen that the egg centrosome normally takes no part in it, though there are certain exceptions to this rule.

Meanwhile, changes have been taking place in the male pronucleus. The originally compact sperm head swells and takes on a normal nuclear appearance; probably this swelling, which must involve the imbibition of water from the cytoplasm, is largely responsible for the formation of the sperm aster, which is much more highly developed than such structures are in the later cleavages. The two pronuclei, as has been said, move together, often to some rather definite position in the egg. They rarely fuse entirely before the nuclear membranes break down and the chromosomes arrange themselves on the metaphase plate of the first cleavage spindle. It is, in fact, only at the first cleavage that the essential union of the two haploid nuclei is finally consummated, and fertilisation can finally be said to be complete.

The two aspects of fertilisation, which we have distinguished as activation and the nuclear events, are not in fact completely separate from one another, but have certain interactions. An interesting example of this has been described by Allen (1954). He sucked an echinoderm egg into a narrow tube, so that it became considerably elongated. If, in such an egg in which the nucleus is located at one end, the fertilising sperm is introduced at the other end, only this latter end becomes activated; that is, it is only at this end that the cortical granules break down and the fertilisation membranes form. From a variety of experiments of this kind, the conclusion could be drawn that the nucleus tends to inhibit the breakdown of the cortical granules in its neighbourhood. At the same time, the events in the cortex have a reciprocal influence on the nucleus. If the germinal vesicle lies in a region in which the cortical granules remain intact, it seems unable to migrate towards the sperm nucleus, and does not divide, although its nuclear membrane disappears at the same time as that of the sperm nucleus.

3. Artificial parthenogenesis

The activation effect of the sperm can be separated in experiment from its action in bringing the gamete nuclei together. Thus Hertwig, in 1916, showed that sperm are still effective as activating agents after they have been given a dose of x-rays which entirely puts out of action their nuclear component. The sperm nucleus can also be rendered inviable by other means; for instance by ultra-violet or by certain chemicals such as trypaflavines. Eggs fertilised by such sperm can develop normally, but they will §2 PRINCIPLES OF EMBRYOLOGY

contain only the maternal chromosomes and hereditary factors. If the sperm used belonged to a different species to the egg, apparent hybrids appear which, however, have only maternal characteristics. They are known as gynogenetic hybrids. They often survive better than true hybrids between the two species concerned, since their development is not complicated by the presence of the paternal genes which may be incompatible with the egg cytoplasm.

It is possible to go further than this, and eliminate not merely the sperm nucleus but the sperm as a whole. Several authors in the cighteen-nineties (Morgan, Hertwig, Loeb) found that ripe eggs of various species could be activated and started on a course of development by purely chemical or physical treatments. In the early years of this century a great deal of work was done on the subject and many different treatments were worked out for different types of eggs. A very large number of agents were found to be effective. For instance temperature shocks, both hot and cold, the action of acids, changes in osmotic pressure, ultra-violet irradiation, physical puncture with the tip of a needle, etc. No one procedure works satisfactorily over the whole range of animal species.

The development of an egg without fertilisation is known as parthenogenesis. The procedures for artificial parthenogenesis have in the first place been worked out empirically, as a series of “cookery book recipes’ which experience has shown to be effective in the particular species of animal being studied. There have, of course, been many attempts to formulate a theory which will account satisfactorily for the effectiveness of the various agents. The most important of these are the following:

(t) Loeb argued that the most effective procedures involve two stages of treatment. He suggested that the first step is to produce a superficial cytolysis of the egg cortex, which he thought was associated with an increase in respiration. In many species this step can be produced by the action of acids or a temperature shock. The second step is to apply a protective treatment which prevents the cytolysis going too far. This may often be done by treatment with a hypotonic solution. The difficulty with this theory is that the notion of cytolysis is so ill defined as to have little definite meaning.

(2) F. R. Lillie ascribed the main role to the operation of fertilizin produced by the egg itself.

(3) Dalcq, Heilbrunn and Pasteels, emphasised the importance of calcium in the medium.

(4) R. S. Lillie considers that the parthenogenesis is brought about by an activating agent (A) which is produced from two other substances, a FERTILISATION 53

product of hydrolysis (B) whose formation is stimulated by acid, and a synthesised substance (S), the concentration of which is increased by hypotonicity.

(s) Bataillon, like Loeb, emphasised the double nature of the process. In his view one element is the production of a cortical change, and the second the production of the cleavage apparatus in the interior of the


Recent discussions of the pros and cons of these various theories may be found in Dalcq (1928), Tyler (1941) and Brachet (1944). The fact that there are so many and such different theories still in the field indicates that none of them is very satisfactory. The process of activation must almost certainly be complex and involve at least the two factors emphasised by Bataillon; that is to say, an action on the cortex and an action on the internal cytoplasm. Little more need be said at present about the cortical effect than has been given above in the discussion of normal fertilisation. It may be mentioned, however, that it is not uncommon for parthenogenetically activated eggs to fail to produce a plane of bilateral symmetry, but to develop into radially symmetrical forms. This is another demonstration that, in the species in which this happens, the point of entry of the sperm plays an essential role in determining the dorso-ventral plane. The failure of this plane to appear in parthenogenesis is presumably a consequence of the fact that the activating agent in this case operates simultaneously over the whole surface of the egg.

Several very interesting facts have emerged in recent years about the internal cytoplasmic events in parthenogenetically activated eggs. In the normal development of almost all types of animal eggs the spindles for the cleavage divisions arise from centrosomes which have been brought in by the sperm. The centrosome remaining in the egg from the last maturation division normally degenerates and takes no part in the later cleavages. We have to inquire, therefore, whence the centrosomes for the cleavage spindles come in cases of parthenogenesis when no sperm centrosome is available.

In some forms there is no doubt that the spindles which arise in connection with the formation of the polar bodies can in these circumstances take over the control of the cleavage division. A case which thas been studied in detail is that of the echiuroid worm Urechis (Tyler 1941, Fig. 3.4). The egg of this form when laid contains a large germinal vesicle, and at the animal pole there is a deep indentation of the surface. Treatments either with hypotonic solutions or with ammoniacal seawater can bring about activation, which is normally made visible by the rounding-up of 54 PRINCIPLES OF EMBRYOLOGY

the egg and the elevation of the fertilisation membrane. After short exposures to activating agents these two changes are delayed and there is no sign of any extrusion of polar bodies. Eventually, however, the egg rounds up, the membrane rises and the egg proceeds to cleave, the cleavage spindle being, in fact, that which would normally have given rise to the first polar body. Slightly longer exposure results in what appears to be normal activation. The egg rounds up, the membrane rises and the two polar bodies are formed in normal sequence. The eggs, however, then usually fail to undergo any further cleavage, apparently because they


Parthenogenesis in the echiuroid worm Urechis. The upper figure shows the unfertilised egg, in which the animal pole is depressed. Columns A, B and C represent the behaviour following increasing exposures to ammoniacal seawater. Row I, time of first polar body formation; row II, time of second polar body formation; row III, time of first cleavage. (From Tyler 1941.)

contain only a single aster and not a bipolar spindle. With still longer exposure the rounding up of the egg and the membrane elevation proceed normally, but the first polar body division takes place inside the cell,

which is thus provided with two nuclei at each of which a spindle appears ready for the second polar body division. At this second division two, one, or no polar bodies may be extruded, leaving two, three or four nuclei still inside the egg. The eggs then divide into the corresponding number of cells and continue their development to give normal larvae. It is clear in this case that although in some ways the activation is most nearly normal with that treatment which allows the polar bodies to be formed in the FERTILISATION 55

usual manner, yet this gives the worst results in later development, because the eggs are left without a proper spindle mechanism to control the cleavages.

In other types of parthenogenesis new cleavage spindles may arise independently of the polar body spindles. For instance, the most effective treatment for the parthenogenesis of frogs’ eggs consists of pricking the egg with a sharp needle. But this is effective only if the needle carries into the egg some foreign protein. Normally sufficient such material is present in the form of the cellular debris adhering to egg jelly. It was originally thought, e.g. by Bataillon, that it was necessary to inject a complete nucleated cell. Recent studies by Shaver (1953), however, have shown that the effect is actually brought about by ribo-nucleo-protein granules. He made the interesting observation that the granules of this kind present in the unfertilised egg are without effect, but they rapidly acquire effectiveness just at the time when the blastula is developing into the gastrula. This is interpreted by Brachet (19522) as another indication that the synthesis of new proteins begins to occur in the embryo at that time. The action of these foreign proteins on the egg cytoplasm has not been followed in detail, but there is no doubt that their main effect is to cause an ‘aster’ or cleavage spindle to arise, possibly by some action rather like that of coagulation.

In some cases cleavage spindles appear to arise quite spontancously without any connection with polar body spindles or with introduced foreign proteins. For instance, the eggs of some echinoderms can be broken by strong centrifugation into a number of fragments of different specific gravity. Only one type of fragment contains a nucleus; the others are completely non-nucleated. They can, however, respond to activation treatments similar to those which are effective on normal eggs. Asters then appear in the cytoplasm and the fragments become divided up into smaller lumps of cytoplasm which can apparently be regarded as cells, except that they do not contain any nucleus (Harvey 1936, 1940b). This absence of the nucleus is presumably responsible for the fact that the cleavage figures remain as single asters and do not unite in pairs to form spindles. Nevertheless the ‘cleavages’ to which they give rise continue for a considerable time and occur with some regularity. It seems that the centrosomes around which the asters are organised are fully normal, and are therefore endowed with the property of genetic continuity. As Tyler (1941) has pointed out, this means that bodies endowed with the capacities for identical self-duplication and division can arise spontancously in the cytoplasm. It might be said, indeed, that we have here an instance of the appearance of new plasmagenes (cf. Chapter XVIII).


A final point of interest in connection with parthenogenesis concerns the number of chromosomes found in the resulting embryos. The treatments which bring about artificial parthenogenesis do not usually cause a complete suppression of the maturation divisions, which would produce diploid eggs, such as those characteristic of the diploid parthenogenesis which is a normal means of reproduction of many species in nature. On the contrary, the activated egg as a rule contains a haploid nucleus or nuclei, and might be expected to develop into a haploid individual. This is indeed what very often happens. There is, however, a well-marked tendency for the diploid chromosome number to be restored. This would happen if the first cleavage division of the nucleus were not accompanied by a division of the cytoplasm, and the two daughter nuclei reunited. The imperfections of the spindle mechanisms developed in artificially parthenogenetic eggs seem often to bring this result about. In fact, in some forms such as frogs, similar irregularities in division often occur at later stages of parthenogenetic embryos, and isolated cells or regions of tissue may arise with many different multiples of the basic chromosome number.


Tyler 1941, 1948, Rothschild and Swann 1949, Runnstrém 1952a. a