A text-book of experimental cytology (1931) 16
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Gray J. A text-book of experimental cytology. (1931) Cambridge University Press, London.
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Chapter Sixteen The Germ
The function of the germ cells is the production of new individuals endowed with the hereditary characters of the parent organisms. Fortunately, our conceptions of this fundamental property is based on a relatively precise knowledge of both morphology and function. The morphology of the germ cells has been admirably described by Wilson (1925) and others, and rightly occupies a central place in the theory of inheritance. The behaviour of the cells as physiological units has also been summarised on more than one occasion. In the present chapter no attempt has been made to cover the whole held, but attention has been focussed on the theoretical significance of a comparatively small number of isolated phenomena. For a complete account of the activity of the germ cells, reference should be made to Wilson (1925), F. R. Lillie (1919), Lillie and Just (1924) and Dalcq (1928).
The spermatozoon
From a morphological point of view a spermatozoon is a unique type of cell. It consists largely of a nucleus together with a typical unit of locomotion — a flagellum. There is little or no cytoplasm, and no visible signs of metabolic reserves. The functions of the two main morphological constituents are beyond doubt — ^the nucleus is destined to play its peculiar r61e in the transmission of hereditary characters, while the flagellum endows the cell with the requisite powers of active locomotion.
It is not easy to follow the movement of a single spermatozoon in a highly active suspension, but as pointed out elsewhere (p. 472) it is highly probable that the mechanism of movement is essentially similar to that in the flagellum of the sponge (van Trigt, 1919); waves of contraction pass along the tail of the spermatozoon from the head end backwards. As a rule these waves follow a spiral course and pass round the long axis of the tail as well as along it; consequently, there are two forces acting on the medium — a backward thrust which drives the spermatozoon forward and a couple which rotates the cell about its long axis. If we are right in assuming that the nucleus is unlikely to contribute the requisite energy for movement, it follows that the life of the sperm must be strictly limited when moving in a medium which provides no nutrient substances.
From a teleological point of view it is desirable that a spermatozoon should conserve its energies until it reaches the medium in which fertilisation occurs, and in practice this can be accepted as a working rule although a precise formulation of the facts is not easy. A preliminary instance of the phenomena may be of use. The spermatozoa of the trout Salmofario are immobile in the testicular plasma, but become extremely active on the addition of water; the period of activity is, however, extremely short and at lO"" C. is limited to two or three minutes (Gray, 19206). Similarly, Kolliker (1856) concluded that the sperm of mammals is immobile within the male tract until it is activated by the secretions of the secondary male glands prior to insemination. More recently, however, Young (1929 a) has investigated the conditions in the guinea-pig and concludes that there is no specific activating principle present in the epididymal secretion; according to Moore (1928) if an activating element is present it is derived from the testis itself. It is by no means easy to determine whether spermatozoa are normally motile within the body of the male parent, for the necessary technique usually involves an alteration in the COg and Og tension in the neighbourhood of the cells. It is, however, significant to note that Hammond and Asdell (1926) have shown that the total effective life of rabbit sperm is not more than SO hours in the body of the female, although it may be as long as 38 days in the body of the male. That some spermatozoa can retain their vitality for prolonged periods is also clear in the case of bees and in some fish. Van Oordt (1928) reports that spermatozoa can live within the female tract of the fish XipJiophorus heller i for at least 10 months ; whether the same fact holds good for the bat is uncertain (Hartman and Cuyler, 1927). The point at issue is not, hoAvever, whether spermatozoa can retain their vitality for long periods but whether they can exist for long periods in a state of active locomotion.
Among invertebrates the period of active life can be studied with relative ease. Gemmill (1900) appears to have been the first to detect the important effect played by the density of the suspension on the period of active life. He showed clearly that dilution of the sperm of Echinus shortened the period of life. A cursory inspection of the contents of a ripe testis of Echinus miliaris reveals the fact that the spermatozoa are present in very great numbers, and that when a drop of this suspension is mixed with sea water the spermatozoa at once exhibit intense and active movement. In other species, notably Echinus esculentus, dilution of the sperm will not produce imnaediate movement unless the sperm is absolutely ‘ mature’ ; as a rule there is a variable period during which the cells remain more or less motionless in the diluted suspension. The nature of this activation by dilution is obscure, but it leads to GemmilFs important contribution — viz. that a concentrated sperm suspension will maintain its powers of fertilising eggs longer than one which has been diluted with sea water. If we assume that the metabolic reserves of the sperm are strictly limited and that dilution of a suspension stimulates mechanical movement, then the natural inference is that dilution shortens the effective life by allowing the cells to dissipate their available energy more rapidly than under conditions where movement is less active. This view was supported by the experiments of Cohn (1918). Working with comparatively dilute suspensions of Arbacia sperm, Cohn confirmed the fact that the more dilute the suspension the more rapidly did it lose its power of fertilising eggs (Table LXIV).
It is well known that acids inhibit all known types of vibratile movement (see p. 453) and, since Cohn found that the more concentrated is the sperm suspension the higher is its hydrogen ion concentration, it seemed feasible to suppose that the preservation of fertilising power in the concentrated condition is due to the effect of the COg produced by the sperm themselves. Cohn found that the rate of loss of fertilising power is decreased if the area of suspension ill contact with air is decreased or if the concentration of the CO 2 in the air is increased (Table LXV).
By estimating the output of COg from the changes observed in the of the suspensions, Cohn concluded that the total output of energy during the whole life of a spermatozoon is unaffected by the concentration of the suspension. The rate at which this energy is expended is, however, dependent on the hydrogen ion concentration. In normal sea water, or in a very dilute suspension, the spermatozoa are very active and, since they are thus expending their energy relatively quickly, they soon lose their power of active movement and of fertilising eggs. In more concentrated suspensions the CO 2 generated by the cells raises the hydrogen ion concentration and this slows down the rate of expenditure of energy, thereby increasing the length of their effective life.
That accumulated CO2 will depress the activity of spermatozoa there can be no doubt, but it would seem that the concentration effect
Table LXIV
Age of sperm in hours
Concentration of sperm suspension in percentage of undiluted sperm in 100 c.c. j
sea water I
4
1
0*5
0*25
Percentage of eggs fertilised when 1 drop of sperm was added to eggs in 10 c.c. sea water
14-2
100
98
h7
10
23-6
100
98
15
0 1
47-0
100
0
0
0 i
71-9
98
0
0
0 i
920
85
0
0
0 1
Table LXV
Approximate CO 2 tension of the air with | which the surface of the suspension is | in contact '
j
Age of sperm
0-3 mm.
0*1 mm. j
Percentage of eggs fertilised when 1 drop j of sperm is added to 10 drops of eggs
in 10 c.c. sea water 1
i
(hr.)
0
(min.)
15
100
100
1
30
100
100 1
7
15
100
100
9
35
100
85
13
35
100
77
22
30
90
19
25
30
92
82
29
00
93
25
32
00
81
1
35
50
20
0
originally observed by Gemmill is more complicated in its action than would appear at first sight from Cohn’s observations. If accumulation of COgis the sole means whereby a concentrated suspension prolongs the effective life of a spermatozoon, then removal of CO2 from the suspension should reduce the effective life to the period characteristic of dilute suspensions. It was found by the author (Gray, 1928 a, h) that the uptake of oxygen by a suspension — and hence presumably the CO 2 production — was governed by a series of factors which does not appear in the analysis given by Cohn. If we take two suspensions of the sperm of Echinus miliariSf one of which contains 3 mg. of nitrogen equivalents of sperm (= approximately 0-25 c.c. undiluted
Fig. 162. Graph showing the effect of successive dilutions on the respiratory activity of a suspension of the spermatozoa of Echinus. (Gray, 1928 a.)
sperm) in 5 c.c. sea water and compare the rate of oxygen consumption per mg. nitrogen with that of the same amount of sperm in a more concentrated suspension (12 mg. nitrogen per 5 c.c.), the more dilute suspension is found to exhibit a much higher rate of oxygen consumption per unit quantity of sperm than does the more concentrated suspension (fig. 164). It is important to note that these observations were made on suspensions from which the COg was being constantly remoTed by agitation with CO 2 free air. Attempts to raise the level of the respiratory activity of concentrated suspensions to that characteristic of those which are more dilute have invariably failed — and it would appear that a complete activation of individual spermatozoa is only possible in a comparatively dilute suspension where each cell has an adequate space in which to execute its mechanical movements. It should be noted that this factor is not likely to operate in very dilute suspensions such as were investigated by Cohn, but is very obvious in suspensions such as are normally found in the cavities of the male genital organs. In the case of Echinus the greater mechanical activity of sperm which follows dilution can usually be detected under the microscope and the gradual loss of fertilising power after dilution is roughly parallel to the decline in mechanical activity. How far the dilution factor affects the duration of active life in other cases is difficult to say, but it may play a part in the conditions investigated by Walton (1927) in mammals. Walton found that there was a significant decline in the percentage fertility of rabbits after artificial insemination with diluted semen. This might conceivably be comparable to the state of affairs in the trout or Echinus where dilution causes premature activity and results in an exhaustion of locomotory powers before the sperm reaches the egg. As Walton points out, conditions in the mammalia are complicated; in dilute suspensions the total number of sperm reaching an egg is probably small and dilution has to be effected by the addition of an artificial medium which may injure the cells. Direct observations on the effect of dilution on the period of active life of mammalian sperm have recently been provided by Young (1929 b) for the guinea pig (see Table LXVI) ; it will be noted that the effect is substantially the same as on echinoid sperm.
Fig. 163. Graph showing the respiratory activity of a unit amount of the sperm of Echinus when diluted with sea water. The dotted line shows the activity plotted against the cube root of the dilution. (Gray, 19*28 a.)
Fig. 164. Graph showing the effect of dilution on the O 2 consximed by a unit quantity of the sperm of Echinus miliaris. (Gray, 1928 a.)
The survival of spermatozoa in dilute suspensions is clearly associated with the ability or non-ability of the cells to obtain energy from external sources. When spermatozoa are shed into an environment of sea or fresh water, they must presumably rely on intracellular sources of energy; as long as they are in the body fluids of the male or female parents they may, like other cells, be capable of obtaining energy from external sources. If such an absorption were possible from the medium in which fertilisation occurs, there is no obvious reason why spermatozoa should not possess an active life comparable to that of other cells; at any rate, the period of life would not be limited by the total energy available. Unfortunately, very little accurstte information appears to exist concerning tlie mobility of sperm within the male genital system. Hammond and Asdell (1926) have shown that as long as they are witliin the male tract the spermatozoa of the rabbit will retain their fertilising power for as long as 38 days, whereas after injection into the body of the female the power of fertilising eggs is lost after 30 hours. The loss of fertilising power in the female tract might be due to one or more causes. If the spermatozoa are more active in the female than in the male, they may exhaust their supplies of energy and so fail to be sufficiently active to fertilise the eggs; on the other hand, we can look upon the external conditions in the female tract as a relatively toxic environment which sooner or later destroys the sperm,
Table LXVI
Effect of dilution on duration of motion of guinea-pig sperm (46° C.) (From Young)
Exp.
no.
Parts of Locke’s solution to undiluted sperm suspension
Un diluted
1 ; 1
2 :1
3 : 1
6 :1
7 :1
13 :1
24:1
i
Min.
Min.
Min.
Min.
Min.
Min.
Min.
Min. :
1 1
105
105
—
105
—
70
—
— 1
2
100
120
—
110
—
60
—
25 1
3
105
—
85
—
55
—
—
—
4
90
—
60
__
28
—
25
— '
5
80
—
j 70
—
50
___
30
i
i
6
70
1
40
—
30
—
20
although they may still possess ample supplies of energy for movement. It is interesting to note that a similar analysis is applicable to fish spermatozoa (Huxley, 1930; Walton, 1930 b).
Reverting to the spermatozoa of invertebrates, it will be recalled that Cohn’s results suggested that motile life is limited by the amount of energy available within each individual cell. Attempts to confirm this conclusion by the author (Gray, 1928 b) failed to give clearly defined results. Using suspensions of Echinus sperm from which the CO^ generated was constantly removed, the rate of decay in the activity of the suspension (as measured by the rate of consumption of oxygen) failed to conform to any simple quantitative law. In the first place, it was found impossible to estimate the total oxygen used by any given suspension, since a small but measurable oxygen consumption persisted up to a point where other oxidative changes of a post-mortem nature obviously set in. This fact, recently confirmed by the data of Carter (1930), makes it impossible to give any clear experimental proof of Cohn’s main conclusion: presumably, the amount of energy expended by a spermatozoon is limited, but its amount has yet to be measured quantitatively. The natural and irreversible decay in the activity of a sperm suspension may be due to a variety of causes (Gray, 1928 b), and the facts are probably best described in terms of the statistical variability in the viability of individual units without any reference to the immediate causes which lead to the death of these units (Gray, 1931). It is of interest to note that Young (1929 6) concludes that mammalian spermatozoa actually undergo a process of irreversible decay whilst still within the epididymis.
Fig. 165 . Graph shomng the total O,, consumed during the life of a suspension of spermatozoa of Echinus miliaris. The dotted line shows c.c. Og plotted against the square root of the age of the suspension.
The survival time of sperm suspensions is more than an academic problem, for in the case of mammals and possibly also fish, it is often of economic importance to effect artificial insemination where a natural mating is either impossible or impracticable. Walton (1930 a) has shown that if the spermatozoa are removed from the vas deferens of a male rabbit under aseptic conditions in such a way as to prevent contamination with air, the survival time of the suspensions is largely determined by the temperature. At body temperature (40° C.) the maximum survival time (as estimated by fertilising power) is about 13 hours. Above this temperature, the spermatozoa are rapidly destroyed — whereas lowering the temperature to 15° C. prolongs the life to 7 days : below 15° C. the survival time again falls (see fig. 166). It is interesting to note that as drawn from the vas deferents the sperm was found to be motile prior to being injected into the female (see p. 409).
Fig. 166. Maximal survival times and velocity of destruction of the spermatozoa of the rabbit as functions of the temperature. (From Walton.)
Hammond’s (1930) experiments on the rabbit differed from those of Walton in that the original spermatozoa were taken from the vagina of a freshly impregnated doe and not from the vas deferens of a male. Under such conditions the periods of survival at different temperatures were lower (see Table LXVII).
It is significant that in Hammond’s experiments the percentage of sperm found to be motUe at room temperature after exposure for variable periods to higher or lower temperatures was found to be a fairly good index of the ability of the suspension to fertilise eggs in the normal way.
To some extent the reactions of spermatozoa to changes in their environment are precisely parallel to those exhibited by other types of cells. As ill the case of vibratile cells in general, prolonged movement is dependent on the presence of calcium ions and on a critical minimum concentration of hydroxyl ions (Gray, 1920 a, 1922 h\ and in respect to their orientation in an electric field spermatozoa behave as a system of negatively charged particles.
In other respects, however, certain types of sperm show more specific characters. When a sample of sperm drawn from the testis of a ripe Echinus is placed in sea water, an immediate outburst of mechanical activity is observed. If the testis is not fully ripe, however, there may
Table LXVII
Temperature
Survival period in hours
Sperm from epididymis under paraffin
Sperm from vagina in contact with air
35
14
14
10
168
96
0
60
16
be a significant latent period after dilution before movement reaches its full level. If, however, such sperm is diluted not with fresh sea water but with sea water which has been in contact with ripe unfertilised eggs, the latent period of quiescence is either reduced or abolished altogether. This fact raises the question of how far the natural medium of fertilisation exerts a definite activating influence on the spermatozoa apart from the mechanical effects produced by dilution. Even on sperm which is fully activated in pure sea water (e.g. Echinus iniliaris), egg secretions exert a definite action, for they maintain the metabolic activity of the cells at its original level, whereas in normal sea water the latter rapidly declines (fig. 167). The nature of the sperm-activating principles in egg secretions of Echinus has been investigated by Carter (1930), who finds that to some extent their action is comparable to that exerted by thyroxin and other iodine compounds. On the other hand Clowes and Bachman (1921) found that the activating principle in the egg secretions of Asterias and Echinarachnius are volatile and that their effects are comparable to those induced by the higher alcohols.
When a suspension of spermatozoa is highly active, it is not uncommon to find that the cells no longer distribute themselves uniformly throughout the medium but tend to group themselves together to form microscopic or macroscopic clusters. This is the
Fig. 167. Graph illustrating the effect of egg secretions on Og consumption of Echinus miliaris sperm. The unbroken line shows the Oo consumed by a suspension in the presence of egg secretions; the broken line shows the Oo consumption of an identical suspension without egg secretions. (After Gray.)
case with the spermatozoa of Nereis (F. R. Lillie, 1918). When fresh spermatozoa of Nereis are mixed with sea water they rapidly aggregate into clumps of actively moving cells whose size gradually decreases again as the activity of the spermatozoa declines. More than one explanation has been advanced for this phenomenon; Cohn (1918) regards the process of aggregation as the direct result of CO 2 inhibition, whereas Lillie rejects this suggestion. Lillie’s explanation is that each spermatozoon exerts a chemotactic effect on its neigh, hours by virtue of the COg which it liberates; such chemotactic effects are not at all uncommon in flagellate or ciliate protozoa (Fox, 1920; Saunders, 1924). It seems reasonable to suppose that in each case the phenomena are due to an active orientation of the cells to a specific concentration of hydrogen ions (see Gray, 1922 a).
The formation of clusters or aggregates of active spermatozoa readily occurs when the sperm of certain echinoderms is exposed to seawater containing egg secretions (Buller, 1902 ; F. R. Lillie, 1918). If the sperm are such that they are not highly motile in normal sea water, the first effect of the egg water is the production of a state of intense mechanical activity; within a few seconds clusters of highly motile sperm begin to form in a way which closely resembles the effect of CO 2 on Nereis sperm. In some cases the aggregated condition is temporary, for as the sperm begin to be less active the size of the clusters is reduced and the number of isolated feebly moving cells is increased. In other cases, however, aggregation is followed by true agglutination in which the cells adhere firmly to each other after they have ceased to move. The significance to be attached to these reactions must be deferred to a later stage (see p. 433), but it is interesting to note that the reactions of a given type of spermatozoon and egg water are seldom if ever entirely specific. Loeb (1914, 1915) found that the egg water of Strongylocentrotus franciscanus will not cause aggregation of the sperm of S. purpuratus, whereas the egg water of S, purpuratus will agglutinate the sperm of S. franciscanus. Just (1919) found that egg water of Arbacia will agglutinate the sperm of Echinarachniiis, whereas the reverse combination is without effect.
The agglutinating principle in egg secretions is a colourless substance of high molecular weight, for it will not pass through a Berkfeld filter and is non-dialysable. According to Glaser (1914 a), it fails to give positive tests for proteins, although Woodward (1918) claims that it is removed from solution by the salting-out action of ammonium sulphate. Whatever be its nature, it appears to differ materially from the activating principle already described. The immediate interest of the sperm agglutinant lies in its possible rdle in the process of fertilisation, for F. R. Lillie’s conception of the union bet^veen a spermatozoon and an egg is based on the assumption that this union is effected by the mechanism which in some cases can be shown to be responsible for the union between one spermatozoon and another. For this reason Lillie ascribed the name of ‘fertilizin’ to sperm-agglutinating principles. For a full discussion reference should be made to the original papers or to Lillie and Just (1924).
The egg cell
The egg cells of nearly all the higher animals differ morphologically and physiologically from all the other cells in the body. Typically, the egg is much larger than a somatic cell, and both c}i:oplasm and nucleus exhibit specific characteristics. The primitive egg cells or oogonia can often be recognised at a very early stage in the life history and, as in the case of the spermatogonia, their subsequent development has been studied in great detail (see Wilson, 1925, Chapter iv). For present purposes, it is unnecessary to recapitulate even a summary of the morphological facts, and we may look upon the egg almost exclusively as a physiological unit.
From this point of view an egg is peculiar in two respects. Firstly, it is essentially a totipotent system capable, after fertilisation, of giving rise to a complete new individual. This property is not absolutely specific, for we have seen that in sponges, coelenterates and other forms, aggregates of somatic cells are capable of forming new individuals: as far as is known, however, the ovum (in the metazoa) is the only type of isolated cell which can normally produce a complete organism. Secondly, as the developing egg is often isolated from all external sources of organic material, it must contain within itself all the ingredients requisite for the production of new tissue : we find, in fact, that the cytoplasm of a typical egg contains a much larger percentage of nitrogenous and other deposits than does any other type of cell.
When a young oogonium begins its development, it is usually possible to recognise three distinct phases before the production of a new organism begins. Firstly the cell grows rapidly in size — often reaching a bulk many thousands of times that of typical somatic units; it is during this growth phase that the egg accumulates the yolk reserves which will afterwards be used for the manufacture of new cells and tissues. The growth phase is clearly one of great metabolic activity, for there is an increase in the size of the nucleus, and in the bulk of the cytoplasm, as well as a deposition of the yolk. So far our knowledge of these processes is limited to morphological observations, and it is by such means that nuclear growth and yolk formation have been extensively investigated. On the completion of growth, a pause may or may not be interposed in the developmental history before the onset of the second main phase of maturation. The most obvious changes effected by maturation are concerned with the nucleus, but there may also be simultaneous changes in the cytoplasm (see p. 95). In some cases maturation is complete before fertilisation, whereas in others maturation is only completed after the spermatozoa has entered the egg.
Nothing is known of the cause which incites the young oogonium to grow or to deposit yolk. In every ease, however, there must be an active secretion of nitrogenous bodies. The most usual form of nitrogen storage is the deposition of proteins — which may either be in the solid form (as in amphibian, or elasmobranch eggs) or in the fluid state (many teleostean eggs, e.g. Salmo). Gatenby (1922) and Nath (1925) have shown that nitrogenous yolk is not infrequently associated with the extrusion of substances from the nucleus into the cytoplasm. Non-nitrogenous yolk may exist as carbohydrates or as fat. In the eggs of Ascaris and some molluscs glycogen is abundant, whereas in other forms {Nereis, fish) large oil globules of a fatty nature are very distinct constituents of the cytoplasmic secretions. The chemical changes whereby these secretions subserve the requirements of the developing egg hardly enter into the field of cytology — but reference may be made to Faure-Fremiet (1925) and to Needham (1931).
Usually the life of a ripe unfertilised egg is considerably longer than that of an active spermatozoon, particularly if the temperature is low or if the egg is deprived of oxygen (Loeb, 1913). Cases are known, however, where the effective life of the ripe egg is very short. Just (1915 b) showed that in order to effect successful artificial insemination of Platynereis megalops the eggs and spermatozoa, like those of the trout, must be mixed without previous contamination with sea water. In Platynereis, however, it is the eggs which suffer by washing and not the sperm ; for if the former are washed in sea water and the water be then removed, no fertilisation is effected by the addition of fresh dry sperm. If the reverse experiment is performed, the previously diluted spermatozoa will fertilise fresh dry eggs after removal of the excess of water. Just regards this loss of fertilising power as due to the loss of an essential ‘fertilizin’ from the eggs (see p. 421).
There is no evidence which suggests that an animal egg invariably secretes a substance which attracts neighbouring spermatozoa, although in those cases in which the eggs produce a substance which stimulates the male gametes to a higher degree of mechanical activity, a limited amount of chemotactic movement may occur (Dakin and Fordham, 1924; Just, 1930). As far as one can see, contact- between sperm and egg is usually fortuitous, so that the chance of fertilisation will depend on the number of spermatozoa and eggs present. This does not imply that the egg is necessarily fertilised by the first spermatozoon with which it comes into contact : probably several collisions occur before the effective spermatozoon reaches the egg. Glaser (1915) observed that this is often the case in Arbacia eggs. Quite clearly if a spermatozoon is to fertilise an egg, the two cells must remain in contact with each other after collision. It is generally supposed that the sperm head is adhesive in the sense that after contact the sperm can only be separated from the egg surface by the application of a force which is greater than that which can be exerted by the tail of the spermatozoon. Subsequently, of course, the sperm head is engulfed into the body of the egg cell. The general series of events is not unlike the process of phagocytosis — in which the inanimate particle is replaced by an active spermatozoon. There are, however, two features peculiar to fertilisation; (i) there is a high degree of specificity, (ii) one and only one spermatozoon is usually incorporated into the egg.
If we look upon the opening phase of fertilisation as equivalent to physical adhesion, the problem of specificity resolves itself into a study of specific adhesion between surfaces : at present, this point of view has not led to any very hopeful results, but it is interesting to note that specific adhesion has been observed in the case of red blood-corpuscles (Tait, 1918). At the same time, the attachment of the sperm to the egg initiates far-reaching changes at the egg surface, for within a very short space of time the whole surface is altered in such a way as to prevent adhesion and penetration by other spermatozoa. The physiological nature of these changes is obscure, although they are followed by visible alterations at the periphery of the egg (see below).
In many cases a sperm is able to enter the egg from any point on the egg surface (sea urchins, starfish), even if the egg is surrounded by a relatively tough membrane. Although spermatozoa appear to burrow through such external membranes in considerable numbers, only one effective sperm usually enters the egg cell itself. In other cases, spermatozoa can only enter the egg at one point (the micropyle) where there is a definite opening in the external egg membrane (insects, fish). It is interesting to note that in the nemertine Cerebratulus the spermatozoon can reach the egg at any point, although a well-defined micropyle is present (Wilson, 1925). The ' burrowing’ powers of spermatozoa are often very remarkable; Gragg (1920) describes the migration of the sperm of Cimecc through chitin. An anomalous method of penetration through egg membranes has recently been described by Chambers (1923) in Asterias — ^where the sperm adheres to one of the fine protoplasmic filaments which protrude from the egg surface, and is passively drawn into the egg by the active contraction of this filament. The description given by Chambers has recently been criticised by Just (1929), who attributes the formation of protoplasmic filaments on the egg surface to abnormal conditions of fertilisation or to moribund egg cells ; this conclusion is not accepted by Chambers (1930).
Visible phenomena of fertilisation
The visible phenomena of fertilisation have often been described. Little can be added to the accounts given by Wilson (1925) and by F. R. Lillie (1919), and the following paragraphs are therefore largely restricted to points of theoretical interpretation.
Many invertebrate eggs on contact with an effective spermatozoon exhibit two simultaneous changes in surface structure: (i) at the point of contact with the sperm, the egg surface is protruded to form a small conical projection — the fertilisation cone; (2) starting at the point of contact a definite fertilisation membrane is rapidly pushed outwards over the whole egg surface. Although these two phenomena occur together they can be regarded both morphologically and physiologically as separate processes.
As far as the fertilisation cone is concerned, it is important to note that, in certain cases, it is formed before the egg cortex is in actual contact with the sperm head (see, however, Just, 1929). Thus in Nereis, ' a transparent fertilisation cone arises from the inner wall of the perivitelline space opposite the attached spermatozoon and extends across the space until it touches the membrane at the point of attachment of the spermatozoon. The perforatorium of the spermatozoqn pierces the vitelline membrane and becomes imbedded in the cone.' These phenomena occupy about fifteen minutes ’ (F. R. Lillie. 1919, p. 53). It is not easy to visualise the type of physical change responsible for the protrusion of the fertilisation cone, although the behaviour of the egg surface recalls to same extent the formation of food-cups in phagocytosis. Even when the fertilisation cone seems to form after the egg surface and the sperm head are in contact, it is hardly possible to believe that a localised disturbance of surface energy is alone responsible for the formation of the cone — ^for it must be remembered that the surface of the egg and probably also that of the sperm is of a rigid nature— and this rigidity must be lost before the sperm can be drawn into the fluid interior of the ego' (Gray, 1922 a).
Concerning the formation of the fertilisation membrane, a considerable divergence of opinion has existed. There can be no doubt that the membrane in its final form has different properties to any membrane present prior to fertilisation: the fertilisation membrane of a sea urchin’s egg is a relatively tough and elastic structure which cannot be identified as such prior to fertilisation. There remain two alternatives : (i) the membrane is an entirely new structure formed subsequently to fertilisation, (ii) it is a modification of a membrane which is present before fertilisation. The first view was held by Harvey (1910, 1914), and is supported by the fact that if ripe sea urchin eggs are shaken into pieces, and the pieces subsequently fertilised — they will all form normal fertilisation membranes. This conception was supportedbyLoeb (1913), McClendon{1914), and Gray (1922a). It was at one time suggested that the fertilisation membrane is a precipitation membrane due to the interaction of the gelatinous envelope with an oppositely charged exudation from the egg surface. Both McClendon and Gray believed that no fertilisation membrane would form if the gelatinous membrane was removed prior to fertilisation. In point of fact, this conclusion was based on an error in experimental technique, for Hobson (1927) has shown conclusive^ that the failure to form fertilisation membranes observed by Gray wms due, not to the removal of the gelatinous envelope, but to the presence of hydrogen ions which had been used to dissolve this envelope prior to fertilisation. It is clear that the formation of a fertilisation membrane is not dependent on the presence of the gelatinous capsule. According to Kite (1912) and Chambers (1921), it is possible to dissect aw'ay from the surface of the unfertilised egg a definite membrane, and if the egg is then fertilised, no fertilisation membrane forms. It looks as though membrane formation represents the elevation of an existing membrane with accompanying changes in its mechanical properties. The extent to which the fertilisation membrane is elevated from the egg surface depends on more than one factor. Loeb (1908 d) showed that the membrane will collapse if proteins are present in the external medium, and from this he concluded that it was impermeable to large molecules and ions. Since the osmotic pressure exerted by solutions of albumen in sea water must be comparatively low, the force required to distort the membrane even in its final tough state must be comparatively small. If, before elevation, there is set free between the membrane and the egg surface a colloid possessing an adequate osmotic pressure, the membrane would be pushed out from the egg surface. Direct evidence of the existence of such a colloid is lacking, but it is significant to note that its existence would account for the fact that the degree of elevation depends on the concentration of hydrogen ions present in the external medium, since the osmotic pressure of a colloid on the alkaline side of its isoelectric point would be depressed by the addition of hydrogen ions. The phenomena observed in Nereis (F. R. Lillie, 1911) are somewhat different from those in the sea urchin egg, but are not detrimental to the above interpretation. In Nereis the unfertilised egg is surrounded by two layers : (i) a delicate vitelline membrane, (ii) an alveolar layer or zona radiata. On fertilisation, fluid from the alveoli of the zona radiata passes through the vitelline membrane, and swells up to form a layer of jelly outside the egg. In this way the zona radiata is destroyed and remains as the perivitelline space. It looks as though the vitelline membrane were permeable to the colloids liberated by the zona radiata, and consequently the vitelline membrane is not lifted away from the egg surface as is the case in sea urchins.
Considerable light was thrown on the mechanism of membrane formation in echinoderm eggs by the discovery that it can be evoked in unfertilised eggs by cytolytic agents such as saponin, soaps, xylol, or chloroform. We know that the elevation of the membrane only occurs in the absence of a critical concentration of hydrogen ions (Gray, 1922 a; Hobson, 1927) and that it will not occur if proteins are present in the external medium. The simplest picture of these facts is provided by the hypothesis that an active spermatozoon or a cytolytic ag^ent alters the cortical layers of the egg in such a way as to liberate a negatively charged colloid whose osmotic pressure is sufficient to overcome the elastic resistance of the membrane lying at the egg surface. It must not be forgotten, however, that during this process the membrane itself undergoes a physical change and becomes thicker and tougher as long as free calcium ions are present (Gray, 1922 a).
The fate of the male nucleus after its absorption into the substance of the egg has been described in detail by F. R. Lillie (1919), and by Wilson (1925), and the essential facts are summarised by Doncaster (1924). From a physiological point of view the movement of the sperm head within the cytoplasm of the egg presents a perplexing problem. The automatic locomotory activity of the sperm is undoubtedly abolished before engulfment, and in most cases the tail does not enter the egg. In order to move against the viscous resistance of the cytoplasm the nucleus must be provided with, a definite amount of energy. Various theories concerning the source of this energy have been propounded. Roux (1887) suggested that the two pronuclei attracted each other, whilst others have postulated an amoeboid activity on the part of the nuclei. Alternatively, the male pronucleus has been denied any power of active movement. Chambers (1917) holds that the propulsive unit is the male aster which mechanically forces the two pronuclei into mutual contact (see p. 160).
Physiological effects of fertilisation
In 1895 Loeb showed that fertilised marine eggs fail to develop if deprived of oxygen; and subsequent observations have substantiated his conclusion. Seven years later, the same author (see Loeb, 1913) found that if unfertilised eggs are deprived of oxygen or exposed to a 0-0005M solution of KCN in sea water the eggs remained healthy and capable of fertilisation for a much longer time than is the case in normal sea water. Both before fertilisation, and after, the activity of the egg is therefore closely associated with its ability to absorb oxygen. That the nature of this association is altered by the act of fertilisation was first shown by Warburg (1908), who measured the oxygen consumption of Arbacia eggs before and after fertilisation; he found that after insemination the oxygen absorbed was six or seven times that prior to fertilisation. Essentially similar results were recorded by Loeb and Wasteneys (1911 a), and more recently Shearer (1922 a) has shown that this significant increase occurs immediately the sperm has effected the visible
Interspecific hybrids of echinoderms have been described by Shearer, de Morgan, and Fuchs (1918). By using a suitable concentration of sperm of Echinus esculentus^ E. acutus and E. miliaris it was found possible to fertilise eggs of any of these forms by the spermatozoa of any other and in every case larvae were obtained. Many of these hybrid larvae, however, failed to metamorphose.
The most complete breakdown of specificity appears to occur in teleost fishes. According to Moenkliaus (1904, 1910), any teleostean egg can be fertilised by the speinn of any other species and Newman (1908-15) described widespread cross fertilisation between different species and genera of these fish. As is general in such experiments, a given species of egg was found to be more readily fertilisable by homogenic sperm than by sperm of a foreign species and the larvae so obtained were more viable.
The ability of two species to cross fertilise is often more or less irreciprocal; for example, the sperm of E, miliaris enters the eggs of E. acutus with a greater frequency than does the sperm of E, acutus into the eggs of E. miliaris. Similar observations are well known not only among the generic crosses of echinoderms (Fischel, 1906; Vernon, 1900; Baltzer, 1910; Tennent, 1910) but also in Amphibia (Born, 1888, 1886). Fischel (1906) found that the sperm of Arhacia will fertilise Strongylocentrotus eggs, but the sperm of Sirongylocentrotus will not fertilise Arbacia eggs.
It is significant to note, however, that the block to fertilisation is often not absolute, but can be overcome by a variety of artificial means. Loeb (1903) found that the sperm of Asterias will not fertilise the eggs of Strongylocentrotus in ordinary sea water, but will do so if the sea water is made slightly hyperalkaline. Similar means of effecting fertilisation have often been employed in other cases with success (see Baltzer, 1910). The ability of a spermatozoon to enter the egg of a different species may also be influenced by the time which has elapsed after the egg has been laid. Thus the fresh eggs of Hipponoe or Toxopneustes are not fertilisable by the sperm of Ophiocoma or Pentaceros, whereas cross, fertilisation occurs fairly regularly after the eggs have remained in sea water for two or three hours (Tennent, 1910). Similarly the eggs of Strongylocentrotus can be fertilised by the sperm of Mytilus (Kupelwieser, 1906, 1909), if the eggs are treated with a high concentration of sperm for a prolonged period.
Although ripe sperm will always fertilise the eggs of the same species if the form used has separate sexes, there are a number of cases known where no fertilisation occurs if both gametes are derived from a hermaphrodite individual. The best known case is that of the ascidian dona. As originally investigated by Castle (1896) and by Morgan (1904) the eggs of C. intestinalis appeared to be unfertilised by the sperm of the same individual, although readily fertilisable by sperm from another individual. Fuchs (1914, 1915) working at Naples found, however, that the degree of self-sterility in dona varied with the quantity of sperm used (see Table LXVIII).
Self-fertilised eggs seldom developed normally. More recently Morgan (1923) found that the barrier to self-fertilisation is located in the membrane which surrounds the egg rather than at the surface of the egg itself. The mature egg is surrounded by a number of
Table LXVIII
Percentage of eggs fertilised
Self-fertilisation
Cross fertilisation
5 drops sperm
4 c.c. sperm
5 drops sperm
4 c.c. sperm
Specimen A
0
58
100
100
B
0
22
100
100
„ c
12
100
100
100
0
56
100 1
100
follicle cells which are bounded on the outer side by a thick membrane. After the egg is laid, it shrinks in size, so that the follicle cells lie freely in a space between the egg and the outer membrane. Morgan found that if the membrane be removed together with the follicle cells, the naked egg is readily fertilisable by the sperm from the same individual and concluded that the barrier to fertilisation is possibly due to the depressant effect of the perivitelline fluid on the activity of the sperm.
The phenomena of hybridisation are of considerable interest in the interpretation of the facts of normal fertilisation. As far as can be seen, there are no general principles which can be used to indicate whether or not a particular cross fertilisation will succeed or not; consequently, it is not easy to see how the union between an egg and a spermatozoon can depend upon the presence or absence of a specific type of substance or molecule. If, on the other hand, a spermatozoon activates an egg by setting up at its surface a critical but non-specific degree of physical potential between the point of contact and the remainder of the egg surface, it is not so difficult to understand how the sperm of a totally different species or phylum can at times activate a foreign egg, and how the possibility of activation may be influenced by changes in external environment (see Gray, 1922 a).
Monospermy and polyspermy
Although a large number of active spermatozoa may burrow through the gelatinous coat of a sea urchin’s egg, only one of them enters the cell under normal circumstances. Monospermic fertilisation of this type is characteristic of all small ‘alecithal’ eggs (sea urchins, worms, molluscs, and mammals). In the case of large and yolky eggs, how’-ever, a considerable number of spermatozoa may enter the ovum, although only one is incorporated into the zygote nucleus. The fate of the accessory spermatozoa varies (see Wilson 1925): they may simply degenerate (insects, urodeles), or as in elasmobranch fish and birds, the accessory sperm nuclei may undergo active mitoses in the peripheral regions of the blastoderm before final resorption.
In sea urchin eggs, polyspermy can readily be effected by experimental means. 0. and R. Hertwig (1887) showed that if these eggs are treated with dilute solutions of various drugs (nicotine, strychnine, morphine), or if the temperature is abnormally high, a number of spermatozoa will enter each egg. It is well known that polyspermy frequently occurs if the eggs are allowed to stand in sea w^ater for some hours, or if they are in any way unhealthy. These facts are not only of significance in respect to the physiology of fertilisation, but also illustrate a point of very general significance. We can hardly believe that nicotine, high temperature, and hydrogen ions (Smith and Clowes, 1924) all act on the egg in precisely the same manner — yet as far as polyspermy is concerned their effects are identical: in other words, the response of the cell to an abnormal environment is not necessarily specific — but may be of a generalised nature. We have to look upon the cell as a system whose equilibrium is very easily upset in a variety of ways. Whatever be the point at which a disturbance starts, the end result may sometimes be the same. It is possible to postulate a series of linked or consecutive reactions in which each member is eventually dependent on the activities of the others, but we must be careful not to correlate too readilv the re spouse of a cell with the specific nature of an applied reagent
It is important to note that when more than one spermatozoon enters a sea urchin’s egg, each male nucleus is capable of mitotic division (see fig. 53). The same phenomenon is observed in fro<^s’ eggs (Brachet, 1911; Herlant, 1911).
It is by no means clear why only one spermatozoon will normallv eiiter small alecithal eggs. Earlier workers suggested that an effecbt-P block to other sperm was to be found in the fertilisation membrane but this can hardly be the case, since Driesch showed that mechanical removal of the fertilisation membrane did not allow additional sperm to enter. More recently Just (1919) has observed that after a sperm has attached itself to the surface of an egg of Echinarachnius no other sperm will enter even although it reaches a region of the egg from which the fertilisation membrane has not vet been elevated It is certain that whatever be the nature of the monospermie mechanism it must come into operation within a very short space of time after effective fertilisation has occurred.
It will be recalled that F. R. Lillie has demonstrated the production of ‘fertilizin’ from ripe unfertilised eggs of sea urchins and of Nereis and has shown that this substance is not produced by fertilised eggs. Lillie regards this substance as an essential requirement for fertilisation, so that if it ceases to be formed as soon as an effective sperm touches the egg, no other sperm Avill enter if fertilizin has been removed from the sphere of action. From this point of view, monospermy is due to a sudden cessation in the production of fertilizin. For this reaction Lillie put forAvard a model based on Ehrlich’s immunity chain reactions (see fig. 168), in Avhich the removal of fertilizin is effected by an occupation of the spermophile receptor by ‘anti-fertilizin’ present in the egg, the anti -fertilizin being inactiA'e until one sperm has linked itself to one unit of fertilizin. Lillie’s conception has been criticised by Godlewski (1926), AA^ho questions the validity of regarding fertilizin as an amboceptor. It AA-ill be noted that Lillie’s scheme is probably intended to be little more than a pictorial representation of the facts, and is helpful in so far as it leads to a more complete understanding of experimental data. Similar principles to those suggested by Lillie haAm been applied by Godlewski to sperm antagonism. In 1911 Godlewski found that if the sperm of Chaetopterus is mixed Avith a suspension of spermatozoa of Sphaer echinus, the mixture fails to fertilise the eggs of Sphaerechinus. As far as could be determined, one Chaetopterus sperm will totally inhibit the fertilising power of four Sphaerechinus spermatozoa, but the effect in part depends on the absolute concentration of both constituents. Godlewski (1926) regards these phenomena as
Symbols
0
sperm
receptor
foreign
sperm
combining
group
spermophile
n group fertilizin
UJ
.ovophile
group
^ anti-fertiiizin
Jl egg ^ receptor
blood
inhibitor
■piV 168 Diagram illustrating Lillie’s analysis of the mechanism of fertilisation. Sector ^the arrangement of substances immediately prior to fertilisation Sector 2, the mechanism of normal fertilisation. The sperm receptor unites with the spermonhiinroup of the fertilizin and the egg receptors with the ovophile group of the fertmzin owinvto the activation of the latter by the sperm (a). Molecules of the Sthfertilizin combine with the spermophile group of the adjacent fertilizin (6 and c) Sd thS present polvspermy. Sector 3, inhibition of fertilisation by loss of fertilizin. Seetm 4 antagonistic sperm action. The sperm receptors are occupied by groups cSt off by the foreign spermatozoa. Sector 5, hypothetical blocking of egg receptors srctoi 6 /inhibitor; acLn of blood. The ovophile group is occupied by derivatives from the blood. (From F. R, Lillie, 1914.)
analogous to the agglutinating effect of foreign sera on blood corpuscles, but it is not altogether clear that mutual narcosis by bUj plays no part, since when dead Dentalium sperm is added to Sphaerechinus sperm the inhibitory effect is very markedly reduced.
The production of fertilizin from unfertilised eggs can be clearly demonstrated, as is also the fact that it is not produced bv fertilised eggs. If we admit that this substance is essential for fertilisation, the facts can be described in chemical terms without reference to immunological symbols. We can postulate that if one unit of fertilizin reacts with a spermatozoon it will induce a secondary reaction which destroys all the remaining fertilizin in the egg. Since the egg is fertilisable at any point we must postulate a surface layer of fertilizin which is constantly changing by the outward diffusion of fertilizin into the water. Polyspermy can only be prevented by destroying this layer over the whole surface of the egg and there must therefore be a propagated disturbance which starts at the point of entry of the sperm and spreads over the whole egg surface at a rate which is sufficient to complete the destruction of fertilizin before another sperm can touch the egg surface. We can judge this conception in two ways: (i) we can use it as a definite working hypothesis and attempt to isolate the process of fertilizin destruction, and to follow experimentally the propagated wave over the egg surface, or (ii) ^ve can accept it or discard it according to the extent to which it will, as a picture, faithfully portray the whole of the available facts. So long as we are dealing solely with the phenomenon of monospermy the facts can be expressed in at least three ways. We can use the nomenclature of immunology, of chemistry or of physics. In every case we are describing the same facts. In 1922 the author pointed out the difficulty of extending Lillie’s original conceptions to the facts of cross fertilisation and artificial parthenogenesis, and urged that these difficulties are diminished if we replace immunological or chemical conceptions by those which are primarily concerned with the distribution of free energy. For example, if we regard the surface of an unfertilised egg as an equipotential surface which is excited at the point of entry of the spermatozoon, then a wave of negative potential will pass over the surface of the egg, if this surface is at all comparable to those of other excitable tissues. If we take a very moderate estimate of the speed of propagation from smooth muscle, then such a wave will complete its circuit over a sea urchin’s egg in 0*00001 second. If, on the other hand, the egg is large or if it is subjected to those reagents which reduce the rate of propagation of bioelectric disturbances, the chance of polyspermy is increased. Similarly, if the essential act of fertilisation is to be traced to a localised change in distribution of free energy, it is not surprising to find that artificial fertilisation can be induced by mechanical puncture (see p. 443). Quite recently this conception has been criticised by Just (1928), who prefers to describe the facts in chemical terms. It is obvious that the essential difference between the physical and chemical conceptions of egg activation lies in how far we believe that specific substances must be present at the point of activation; if we ai'e inclined to attribute activation to a particular distribution of specific molecules, then the redistribution of energy becomes of secondary significance. All chemical changes probably involve such a redistribution but a purely chemical conception of fertilisation is not intimately concerned with this fact. On the other hand, if we attribute primary significance to the distribution of energy at the point of contact between the egg and spermatozoon, then the chemical mechanism responsible for this distribution need no longer be specific. We must choose between a physical and a chemical nomenclature solely according to the relative significance which we are inclined to attribute to energy changes as such. If we regard the existence of a critical and localised potential as the essential element of activation, the means whereby this is established are immaterial and egg activation becomes allied to the stimulation of a muscle or a nerve as R. S. Lillie suggested twenty years ago.
The diphasic nature of natural fertilisation
When an egg is fertilised in the ordinary way, a series of events can be traced from the moment the effective spermatozoon reaches the egg until the whole egg embarks on the cleavage cycles which ultimately yield a new organism. The first changes occur at the egg surface and only at a later stage is the whole cell engaged in the processes of nuclear division and cytoplasmic cleavage.
By purely experimental methods it is possible to show that this long cycle of events can be divided into two natural groups, each of which is, to some extent, independent of the other. We know, for ' example, that the increase in respiratory activity characteristic of the fertilised echinoid egg reaches its full level soon after contact is established between egg and sperm (Shearer, 1922 a); similarly the cortical changes at the egg surface are completed long before the first cleavage cycle begins. By centrifuging the sperm away from the surface of a> Nereis egg F. R. Lillie (1912a) enabled us to distinguish those rapid and cortical changes which are the immediate result of fertilisation from those slower and subsequent changes which, are dependent on the penetration of the male elements into the cytoplasm of the egg. In Nereis the sperm head does not penetrate into the egg until about forty-five minutes after the cortical changes are complete. By applying centrifugal force, Lillie found it possible to dislodge the spermatozoon from the egg surface. After return to normal conditions such eggs complete their maturation, but they do not segment. At the time when the zygote nucleus normally forms its first cleavage spindle, the female pronucleus of the centrifuged egg is clearly visible and appears to be of normal size; the nuclear membrane breaks down and chromosomes are formed in the normal manner; there is, however, in the absence of the sperm nucleus, no sign of spindle or of asters — and the chromosomes lie in a clear area of cytoplasm. Each chromosome splits longitudinally but the halves do not separate from each other. Finally, the chromosomes become scattered and degenerate as chromatic granules. As Lillie says: ‘This experiment shows that the fertilisation process may be divided physiologically as well as morphologically into two main phases of external and internal phenomena. The external action is adequate to produce the cortical changes alone, but not the entire series of developmental events A similar conclusion may be drawn from the observations of Loeb (1913), who found that the sperm of Asterias can excite membrane formation on the eggs of Strongijlocentrotus purpuratus in hyperalkaline water, although the sperm may not enter the eggs. Such eggs do not segment: if, by chance, the sperm does enter, normal segmentation follows. We can therefore assume that fertilisation effects two changes in the egg: (i) by a modification which starts at the cortex, the metabolism of the egg is increased to its full level: the egg is, in fact, activated; (ii) by penetration into the egg, the sperm nucleus with its attendant aster enables the egg to form the requisite machinery for normal cleavage. Further discussion of these two phases must be deferred until the facts of artificial parthenogenesis have been considered.
Artificial parthenogenesis
Although the main phenomena of artificial parthenogenesis have been known for thirty years they are none the less spectacular and none the less significant. Starting with Mead’s observations in 1895 and culminating perhaps in Loeb’s later work, we can trace step by step the discovery of a method whereby certain eggs can be induced to Lvelop normally without the intervention of the male gamete. The history of the problem has been given by Loeb (1918) and by Morgan (1927), and is an interesting example of the way in which biological discoveries may be made, although the primary effects of the reagents employed are shrouded in complete mystery and where the methods used are entirely empirical.
The story starts with an attempt by Mead (1895) to excite cell division in unfertilised Chaetoptems eggs by treatment with those electrolytes which were known to affect the activity of a muscle fibre. Under normal circumstances the eggs of Chaetoptems give off their polar bodies only after fertilisation; Mead found that if the eggs are placed in sea water containing per cent, of potassium chloride the unfertilised eggs not only form their polar bodies but also form a polar lobe which is the natural preliminary to the first cleavage. The addition of potassium chloride to sea water not only altered the concentration of potassium but also altered the osmotic pressure. Shortly afterwards Morgan (1896) showed that if the unfertilised eggs of Arbacia are exposed to hypertonic sea water for a limited period, then on transference to normal sea water artificial asters are formed in the cytoplasm and these may result in the formation of a functional amphiaster (see p. 162). In the same year Loeb succeeded in obtaining swimming plutei by exposing the unfertilised eggs of Arbacia to 50 c.c. sea water -t- 50 c.c. MgClj for li to 2 hours and shortly afterwards the same author (Loeb, 1900)”showed that the nature of the salt used is of minor importance; the essential factor concerned is osmotic pressure. In two respects the parthenogenetic development of such eggs was abnormal: the eggs developed without the formation of a fertilisation membrane and the larvae failed to swim freely.
During their extensive studies of the effect of drugs on echinoderm mitosis, 0. and R. Hertwig (1887) found that when unfertilised sea urchin eggs are exposed to sea water containing chloroform, atypical fertilisation membrane is formed, and Herbst (1893) showed that benzol, toluol, and xylol have a similar effect. Loeb (1905) extended this list by using ethyl acetate, although the ester was found to exhibit a specific difference to chloroform or xylol in that the fertilisation membranes only formed after the eggs had been transferred to normal sea wmter ; when so transferred, the eggs showed incipient signs of segmentation, although no larvae were obtained. Clearly the next step towards effective parthenogenesis consisted in effecting membrane formation and afterwards exposing the same eggs to hypertonic sea water. The eggs so treated developed normally and free swimming larvae were obtained. In all his later observations Loeb replaced the esters of the fatty acids by the acids themselves, so that his final method is as follows. The eggs are placed in 50 c.c. sea water + 2*8 c.c. NjlO butyric acid for l|-4 minutes and are then transferred to 200 c.c. of sea water. After 15-20 minutes they are placed in 50 c.c. sea water -f 8 c.c. 2^M NaCL After 30-60 minutes they are transferred to clean sea water, and normal development (under favourable circumstances) ensues. The precise details of this technique vary for different species of egg and for different batches of eggs. For British species reference should be made to Shearer and Lloyd (1913).
As already mentioned (see p. 426), membrane formation can be effected by a variety of reagents; saponin, soaps, chloroform, fatty acids, salicylic aldehyde, foreign blood sera, metallic silver (Herbst, 1904), but on the whole saponin or the lower fatty acids have proved to be the most convenient. As far as is known, there are only three substitutes for hypertonic sea water in the second phase of Loeb’s technique. These are (i) treatment with cyanides (Loeb, 1913), (ii) anaesthetics (R. S. Lillie, 1913), and (hi) low temperature (Loeb, 1906; Hindle, 1910).
The application of similar methods to eggs, other than those of sea urchins, has led to the production of artificially parthenogenetic larvae in a number of other groups (annelids, molluscs, sipunculids). Curiously enough, if such eggs respond at all, they do so with less difficulty than those of sea urchins. For example, when membranes are artificially produced in such forms as Poly me (Loeb, 1913), Thalassema or Asterias, normal larvae are obtained without subsequent treatment with hypertonic sea water. Similarly, parthenogenetic larvae can be obtained from the eggs of the molluscs Lottia or Acmaea by treatment with hypertonic sea water alone. It is quite clear that the two phases of Loeb’s final method are by no means universally necessary. An account of parthenogenesis in the various phyla of the animal kingdom will be found in the books of Loeb (1913) and Morgan (1927), and we may confine our attention to the general interpretation of the more outstanding facts.
We have already seen that normal fertilisation can be divided into two series of events: those which result from adhesion between the egg and the spermatozoon, and those which follow the penetration of the sperm into the substance of the eggs. It is at once significant that Loeb’s final method of effecting artificial parthenogenesis in sea urchin eggs also involves two processes one of which induces changes at the surface of the egg precisely similar to those induced by the contact of the egg with an effective spermatozoon. That this morphological parallel has real physiological significance is shown by the fact that in both cases the respiratory activity of the eggs is raised to the same high level (Warburg, 1910), and in both cases there is a marked increase in the ability of the eggs to absorb water from hypotonic solutions (Lillie, R. S., 1916, 1917). It will be remembered that F. R. Lillie succeeded in removing the spermatozoon from the surface of a Nereis egg after the cortical changes were completed and showed that the nucleus formed a number of chromosomes but failed to form a normal amphiaster. Much the same phenomena are observed when the eggs of a sea urchin are treated wdth a membrane-forming substance such as
butjnic acid. Using Strongylocentrotus purjjuratus Hindle (1910) showed that V” c
eggs exposed to membrane treatment do iol r°;A,o° ° °
not divide (unless at low temperatures), ^ “■
but the nucleus undergoes a typical pro- ,■
phase, and forms a large monaster on
which lie the scattered chromosomes ""v
(fig. 169). More recently, Herlant (1918, VX ■■
1919). has confirmed this fact in Para- .X'
centrotus lividus, where the nuclear cycle „ o » - ■ V
of the female pronucleus may be re peated several times. It seems reason able to conclude that just as normal
^ . -T , • 1 1 j Fiff. 169. Monaster in unfertilised
fertilisation can be regarded as a com- oi Strongylocentrotus 2^ hours
bination of two series of events, so after treatment with butyric acid
artificial parthenogenesis is divisible into (i) a process operating on the egg
surface (which is best induced by treatment with membrane-forming substances), and into (ii) a subsequent process whose distinctive effect is the provision of a normal amphiaster in the place of the single ineffective aster w^hich is the result of the activation process acting by itself. In some way or other hypertonic sea water must provide this amphiaster. It will be recalled that in 1900 Morgan showed that cytasters will form in fertilised eggs if these are subjected to hypertonic sea water and this process was clearly described in Sphaerechmus eggs by Wilson (1901). All authors are agreed that hypertonic sea water provides the astral machinery for normal cleavage, but two distinct views are held concerning the way in which this is effected. According to Herlant (1918-19) the final amphiaster is composed of two separate entities — the aster normally associated with the female pronucleus and a second aster which is an artificial product produced denovo in the cytoplasm by treatment with hypertonic sea water; this latter aster is comparable to the ‘ cytasters ’ originally described bv Morgan. If treatment with hypertonic sea water is too severe or too prolonged, more than one effective cytaster may co-operate with the female aster to form a polypolar spindle, and the resultant cleavage is abnormal. Herlant’s conclusions have, however, been criticised by Fry (1925). According to Fry, the amphiaster of artificially parthenogenetic eggs is the result of cleavage on the part of the single female aster, while artificial cytasters play no essential r61e ; Fry claims that when a cytaster is incorporated into the spindle, the eggs invariably develop abnormally. The present position is therefore uncertain, but it is interesting to note that in normal fertilisation it is undoubtedly the male aster which possesses the power of spontaneous division, whereas the female aster is unable to do so; that the first amphiaster is entirely derived from the male element by no means follows (see p. 1 61).
A study of the sea urchin’s egg might justify the conclusion that artificial parthenogenesis can be effected when two reagents are used — one of which activates the egg, and the other completes the machinery for normal cleavage. We must, however, remember that the earlier experiments of Loeb and others show that it is possible to obtain activation without the formation of a normal fertilisation membrane, and in the case of other eggs — notably those of the starfish and Thalasse^na (Lefevre, 1907) — normal development may result from treatment with activating agents only. Further it will be recalled that a limited number of sea urchin eggs can be activated by hypertonic sea water alone. The great variety of effective agents is, in fact, one of the most striking features of artificial parthenogenesis: mechanical agitation {Asterias, Mathews, 1901), {Nereis, Just, 1915 a), saponin (Loeb, 1913), alkalies (Loeb, 1913), and foreign blood sera {Dendrostoma, Loeb, 1907, 1908 a, 5). As far as we know, the final results are always the same, viz. activation and the production of a normal amphiaster — but the precise chain of events leading up to this result may be different in different cases and it is dangerous to generalise. For example, according to Buchner (1911) in Asterias glacialis the amphiaster is derived from the female aster together with that of the second polar body whose nucleus sinks back into the egg and acts as a male pronucleus. Whether this is always the case is doubtful, for Tennent and Hague (1906) previously described the haploid condition of the parthenogenetic eggs of A. Forbesii,
Theories concerning the underlying causes of artificial parthenogenesis have been numerous. As is well known, Loeb (1913) looked upon the process of membrane formation in the sea urchin egg as the incipient stages of a cytolysis which, if unchecked, would lead to the destruction of the whole egg. According to Loeb the activation of an unfertilised egg is effected by the introduction into the egg of two substances: (i) a specific cytolysin, which brings about the destruction of the surface layer of the egg, and (ii) a substance which limits or controls the destructive influence of the cytolysin. This scheme is clearly based on the phenomena of artificial parthenogenesis of sea urchin eggs, and encounters difficulties when applied to the facts of normal and cross fertilisation. It is not easy to see why the sperm of Myiilus or a shark should be able to introduce a specific lysin into sea urchin eggs, whereas that of a neighbouring species of sea urchin cannot do so — further it is by no means clear that membrane formation and subsequent cytolysis are essentially of the same nature. If sea urchin eggs are placed in a suitable concentration of saponin in sea water, membranes are extruded in the normal way within a few minutes, but there is a long latent period before any visible cytolysis occurs. Further, if hypertonic sea water antagonises cytolysis, it is curious that its effect on the egg can be induced before the egg is subjected to membrane -forming substances. As Loeb himself showed, the ' corrective ’ treatment with hypertonic sea water is in some cases unnecessary. Although Loeb advocated the conception of specific lysins which destroyed the surface layer of the unfertilised egg, there can be little doubt that he regarded such an effect essentially as a profound change in the physical state of the egg cortex. We have already suggested the advantages to be gained from this point of view : it is more flexible than the hypothesis of specific chemical reactions. Unless we regard the effect of mechanical puncture of an egg as specifically different to that produced by the localised injury of other excitable tissues, there seems some a priori evidence in support of the physical hypothesis. Bataillon (1910—11) found that if eggs of the frog are punctiu’ed by a fine glass needle in the presence of lymph, a limited number will develop into normal tadpoles. Bataillon regarded this treatment as essentially diphasic— and comparable to Loeb’s technique; the puncture was equivalent to surface cytolysis and the action of the lymph to the corrective treatment of hypertonic sea water. Herlant (1911, 1918) regarded the amphiaster (found after puncture in lymph) as a product of two cytasters, which do not develop unless lymph is present. Whatever be the origin of the cleavage centres, it is difficult to resist the conclusion that the initial effect of mechanical puncture is of a physical nature.
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