The cell in development and inheritance (1900) 4
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Wilson EB. The Cell in Development and Inheritance. Second edition (1900) New York, 1900.
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Chapter IV Fertilization of the Ovum
"It is conceivable, and indeed probable, that every part of the adult contains molecules derived both from the male and from the female parent; and that, regarded as a mass of molecules, the entire organism may be compared to a web of which the warp is derived from the female and the woof from the male."
Huxley.
- Evolution, in Science and Culture, p, 296, from Enc. Brit., 1878.
In mitototic cell-division we have become acquainted with the means by which, in all higher forms at least, not only the continuity of life, but also the maintenance of the species, is effected ; for through this beautiful mechanism the cell hands on to its descendants an^xact duplicate of the idioplasm by which its own organization is determined. As far as we can see from an a priori point of view, there is no reason why, barring accident, cell-division should not follow cell-division in endless succession in the stream of life. It is possible, indeed probable, that such may be the fact in some of the lower and simpler forms of life where no form of sexual reproduction is known to occur. In the vast majority of living forms, however, the series of cell-divisions tends to run in cycles in each of which the energy of division finally comes to an end and is only restored by an admixture of living matter derived from another celt. This operation, known as fertilisation or fecundation, is the essence of sexual reproduction ; and in it we behold a process by which on the one hand the energy of division is restored, and by which on the other hand two independent lines of descent are blended into one. Why this dual process should take place we are as yet unable to say, nor do we know which of its two elements is to be regarded as the primary and essential one.
Harvey and many other of the early embryologists regarded fertilization as a stimulus, given by the spermatozoon, through which the ovum was " animated " and thus rendered capable of development. In its modern form this conception appears in the " dynamic " theories of Herbert Spencer, Butschli, Hertwig, and others, which assume that protoplasm tends gradually to pass into a state of increasingly stable equilibrium in which its activity diminishes, and that fertilization restores it to a labile state, and hence to one of activity, through mixture with protoplasm that has been subjected to different conditions. Butschli ('76) pointed out that the life-cycle of the metazoon is comparable to that of a protozoan race, a long series of cell-divisions being in each case followed by a mixture of protoplasms through conjugation ; and he assumed that, in both cases, conjugation results in rejuvenescence through which the energy of growth and division is restored and a new cycle inaugurated. The same view has been advocated by Minot, Engelman, Hensen, and many others. Maupas ('88, '89), in his celebrated researches in the conjugation of Infusoria, attempted to test this conclusion by following out continuously the life-history of various species through the entire cycle of their existence. Though not yet adequately confirmed, and indeed opposed in some particulars by more recent work, 1 these researches have yielded very strong evidence that in these unicellular animals, even under normal conditions, the processes of growth and division sooner or later come to an end, undergoing a process of natural " senescence," which can only be counteracted by conjugation. That fertilization in higher plants and animals does in fact incite division and growth is a matter of undisputed observation. We know, however, that in parthenogenesis the egg may develop without fertilization, and we do not know whether the tendency to " senescence " and the need for fertilization are primary attributes of living matter.
The foregoing views may be classed together as the rejuvenescence theory. Parallel to that theory, and not necessarily opposed to or confirmatory of it, is the view that fertilization is in some way concerned with the process of variation. Long since suggested by Treviranus and more lately developed by Brooks 2 and Weismann 8 is the hypothesis that fertilization is a source of variation — a conclusion suggested by the experience of practical breeders of plants and animals. Weismann brings forward strong arguments against the rejuvenescencetheory, and regards the need for fertilization as a secondary acquisition, the mixture of protoplasms to which it leads producing variations — or rather insuring their "mingling and persistent renewal" 4 — which form the material on which selection operates. On the other hand, a considerable number of writers, including Darwin, Spencer, O. Hertwig, Hatschek, and others, believe that although crossing may lead to variability within certain limits, its effect in the long run tends to neutralize indefinite variability and thus to hold the species true to the type.
It is remarkable that we should still remain uncertain as to the physiological meaning of a process so general and one that has been the subject of such prolonged research. Both the foregoing general views are in harmony with the results of Darwin's work on variation and with the experience of practical breeders, which have shown that crossing produces both greater vigour and greater variability. In view of all the facts, however, we are constrained to the admission that the essential nature of sexual reproduction must remain undetermined until the subject shall have been far more thoroughly investigated, especially in the unicellular forms, where the key to the ultimate problem is undoubtedly to be sought.
1 Cf. Joukowsky, '99. 8 Amphimixis, 1 89 1.
2 The Imw of Heredity, 1883. 4 '99, p. 326.
A. Preliminary General Sketch
Among the unicellular plants and animals, fertilization is effected by means of conjugation, a process in which two individuals either fuse together permanently or unite temporarily and effect an exchange of nuclear matter, after which they separate. In all the higher forms fertilisation consists in the permanent fusion of two germ-cells, one of paternal and one of maternal origin. We may first consider the fertilization of the animal egg, which appears to take place in essentially the same manner throughout the animal kingdom, and to be closely paralleled by the corresponding process in plants.
Fig. - B9. — Fertilization of the egg of the snail. Piy sa. £Kost*necki and WlERZEJSKI.) A. The entire sperm at oroon lies in the egg, its nucleus at the right, fiagellum at the left, while the minute sperm-am|)hiaster occupies the position of the middle-piece. The first polar hody has been formed, (he second is forming. B. The enlarged sperm-nucleus and sperm-amphiaster he near the centre; second polar body forming and the first dividing. The egg-centrosornes and asters afterward disappear, their place being taken by those of the spermatozoon.
Leeuwenhoek, whose pupil Hamm discovered the spermatozoa (1677), put forth the conjecture that the spermatozoon must penetrate into the egg; and the classical experiments of Spallanzani on the frog's egg (1786) proved that the fertilizing element must be the spermatozoa and not the liquid in which they swim. The penetration of the ovum was, however, not actually seen until 1854, when Newport observed it in the case of the frog's egg; and it was described by Pringsheim a year later in one of the lower plants, CEdigonium. The first adequate description of the process was given by Hermann Fol, in 1879, 1 though many earlier observers, from the time of Martin Barry (43) onward, had seen the spermatozoon inside the egg-envelopes, or asserted its entrance into the egg.
In many cases the entire spermatozoon enters the egg (mollusks, insects, nematodes, some annelids, Petromyzon t axolotl, etc.), and in such cases the long flagellum may sometimes be seen coiled within the egg (Fig. 89). Only the nucleus and middle-piece, however, are concerned in the actual fertilization ; and there are some cases (echinoderms) in which the tail is left outside the egg. At or near the time of fertilization, the egg successively segments off at the upper pole two minute cells, known as the polar bodies (Figs. 89, 90, 116) or directive corpuscles, which degenerate and take no part in the subsequent development. This phenomenon takes place, as a rule, immediately after entrance of the spermatozoon. It may, however, occur before the spermatozoon enters, and it forms no part of the process of fertilization proper. It is merely the final act in the process of maturation, by which the egg is prepared for fertilization, and we may defer its consideration to the following chapter.
1. The Germ-nuclei in Fertilization
The modern era in the study of fertilization may be said to begin with Oscar Hertwig s discovery, in 1875, of the fate of the spermatozoon within the egg. Earlier observers had, it is true, paved the way by showing that, at the time of fertilization, the egg contains two nuclei that fuse together or become closely associated before development begins. (Warneck, Biitschli, Auerbach, Van Beneden, Strasburger.) Hertwig discovered, in the egg of the sea-urchin (Toxopneustes lividus), that one of these nuclei belongs to the egg, while the other is derived from the spermatozoon. This result was speedily confirmed in a number of other animals, and has since been extended to every species that has been carefully investigated. The researches of Strasburger, De Bary, Schmitz, Guignard, and others have shown that the same is true of plants. In every known case an essential phenomenon of fertilization is the union of a sperm-nucleus, of paternal origin, with an egg-nucleus, of maternal origin, to form the primary nucleus of the embryo. This nucleus, known as the cleavageor segmentation-nucleus, gives rise by division to all the nuclei of the body, and hence every nucleus of the child may contain nuclear substance derived from both parents. And thus Hertwig was led to the conclusion ('84), independently reached at the same time by Strasburger, Kolliker, and Weismann, that the nucleus is the most essential element concerned in hereditary transmission.
1 See Vllenogenu, pp. 124 ft, for a full historical account.
This conclusion received a strong support in the year 1883, through the splendid discoveries of Van Beneden on the fertilization of the thread-worm, Ascaris megalocephala, the egg of which has since ranked with that of the echinoderm as a classical object for the study of cellproblems. Van Beneden's researches especially elucidated the structure and transformations of the germ-nuclei, and carried the analysis of fertilization far beyond that of Hertwig. In Ascaris, as in all other animals, the sperm-nucleus is extremely minute, so that at first sight a marked inequality between the two sexes appears to exist in this respect. Van Beneden showed not only that the inequality in size totally disappears during fertilization, but that the two nuclei undergo a parallel series of structural changes which demonstrate their precise morphological equivalence down to the minutest detail ; and here, again, later researches, foremost among them those of Boveri, Strasburger, and Guignard, have shown that, essentially, the same is true of the germ-cells of other animals and of plants. The facts in Ascaris (variety bivalcns) are essentially as follows (Fig. 90): After the entrance of the spermatozoon, and during the formation of the polar bodies, the sperm-nucleus rapidly enlarges and finally forms a typical nucleus exactly similar to the egg-nucleus. The chromatin in each nucleus now resolves itself into two long, worm-like chromosomes, which are exactly similar in form, size, and staining-reaction in the two nuclei. Next, the nuclear membrane fades away, and the four chromosomes lie naked in the egg-substance. Every trace of sexual difference has now disappeared, and it is impossible to distinguish the paternal from the maternal chromosomes (Fig. 90, D, E). Meanwhile an amphiaster has been developed which, with the four chromosomes, forms the mitotic figure for the first cleavage of the ovum, the chromatic portion of which has been synthetically formed by the union of two equal genu -nuclei. The later phases follow the usual course of mitosis. Each chromosome splits lengthwise into equal halves, the daughter-chromosomes are transported to the spindle-poles, and here they give rise, in the usual manner, to the nuclei of the two-celled stage. Each of these nuclei, therefore, receives exactly equal amounts of paternal and maternal chromatin.
Fig. 91). — Fertilitation of the egg of Asatris megahiefiala, var. bivalins, [I later stages see Figs. 31. 145)
A. The spermatozoon has entered the egg. its nucleus is shown at -f ; beside i tar mass of "archoplasm " (attraction-sphere); above of the second polar body (two chromosomes in each reticular stage; the attraction-sphere (a) contains the forming in the germ-nuclei; the ccntrosome divided, chromosomes; attraction-sphere (a) double. E. Mil! the chromosomes (c) already split. F. First cleavage ir -daughier-chromoaoxnes toward the spindle-poles (on'
are the
closing phases in
the formation
B. Ger
rmuele
(9. J)
n the
lindinj
ne. C.
Chromosomes
0. Eac
germ-nu
tic figure forming
for the first cleavage;
in progress, shoi
of the
reechrt
mosomes
shown:
These discoveries were confirmed and extended in the case of Ascaris by Boveri and by Van Beneden himself in 1887 and 1888 and in several other nematodes by Carnoy in 1887. Carnoy found the number of chromosomes derived from each sex to be in Coronilla 4, in Ophiostomum 6, and in Filaroides 8. A little later Boveri ('90) showed that the law of numerical equality of the paternal and maternal chromosomes held good for other groups of animals, being in the sea-urchin Echinus 9, in the worm Sagitta 9, in the medusa Tiara 14, and in the mollusk Pterotrackea 16 from each sex. Similar results were obtained in other animals and in plants, as first shown by Guignard in the lily ('91 ), where each sex contributes 1 2 chromosomes.
Fig. 91. — Germ -nuclei and chromosomes in the eggs of nematodes. [Carnoy.] A. Egg of nematode parasitic in Scyltium ; the two germ-nuclei in apposition, each containing four chromosomes; the two polar bodies above. B. Egg of Filaroidts ; each germ-nucleus with eight chromosomes ; polar bodies above, deuto plasm -spheres below.
In the onion the number is 8 (Strasburger) ; in the annelid Ophryotrocha it is only 2 from each sex (Korschelt). In all these cases the number contributed by each is one-half the number characteristic of the body-cells. The union of two germ-cells thus restores the normal number, and here we find the explanation of the remarkable fact commented on at page 67 that the number of chromosomes in sexually produced organisms is always even. 1
These remarkable facts demonstrate the two germ-nuclei to be in a morphological sense precisely equivalent, and they not only lend very strong support to Hertwig's identification of the nucleus as the bearer of hereditary qualities, but indicate further that these qualities must be carried by the Template:Chromosomes ; for their precise equivalence in number, shape, and size is the physical correlative of the fact that the two sexes play, on the whole, equal parts in hereditary transmission.
' Cf, P . 67.
2. The Achromatic Structures in Fertilization
It is generally agreed that the amphiaster of the primary mitotic figure of the fertilized ovum arises from the egg-substance precisely
A. The ovarian egg still surrou lucida] ; the polar spindle formed (sperm-nucleus at rf). C. The ti D. Germ-nuclei approaching, of e cleavage spindle in the centre; on 1
ded by the follicle-cell; B. Egg immediately > germ-nuclei (rf. ?) lal siie. E. The chi her side the pater
as in the ordinary mitosis of tissue-cells, and its mode of origin therefore involves the same questions as those already discussed at page 72. It is quite otherwise with the centrosomes at the astral centres, the origin of which still remains one of the most difficult, as it is one of the most interesting, problems relating to fertilization.
After the formation of the polar bodies, the egg-nucleus is reconstituted near the upper pole of the egg, and the entire polar mitotic apparatus disappears. In the meantime a new astral system (sperm aster or amphiaster) is developed in the neighbourhood of the spermnucleus, and this in a large number of cases gives rise or is definitely related to the cleavageamphiaster (crelenterates, flat- worms, echinoderms, ' nematodes, annelids, arthropods, mollusks, tunicates, vertebrates). In many of these cases the spermaster, which by division gives rise to the amphiaster, has been found to arise in intimate relation with the middle - piece of the spermatozoon ; e.g. in ec h inoder m s ( Fl e m min g, Hertwig, Boveri, Wilson, Mathews, Hill, etc.), in the axolotl(Fick) and salamander (Michselis), in the tunicates (Hill), annelids (Foot, Vejdovsky), insects (Henking), nematodes (Meyer, Erlanger), and mollusks (Henking, Kostanecki, and Wierzejski). . The agreement between forms so diverse is very strong evidence that this is a very general phenomenon, and it is one of great interest, owing to the fact that the middlepiece is itself derived from or contains the centrosome of the spermatid. 1
"fr!U- — Fertilization of the egg of the gasteropod, Pftrolracira. [BOVRRI.] A. The egg-nueleus (£) and sperm.nucleus (S) approaching after formation of the polar bodies; the latter shown above (P. B.)\ each germ-nucleus contains sixteen chromosomes; the sperm-am phi aster fully developed. 8. The mitotic figure lor the first cleavage nearly established ; the nuclear membranes have disappeared, leaving the maternal group of chromosomes above the spindle, the paternal below it.
Fig. 94.-EM malion of Ihe sperm(A-F, X 1600; G. H, X 800).
A. Sperm-head before entrance; die-piece and part of Ihe UageHum. after entrance, showing entrance-cone. D, Rotation of the sperm-head, formation of the sperm-aster about the middlepiece. £. Casting off of middle-piece; centrosome at focus of the rays {cf. big. la). The changes figured occupy about eight minutes. F. G. Approach of the germ-nuclei ; growth of the aster.
The facts may be illustrated by a brief description of the phenomena in the sea-urchin Toxopneustes (Fig. 94). As described at page 197, the tail is in this case left outside, and only the head and middle-piece enter the egg. Within a few minutes after its entrance, and while still very near the periphery, the lance-shaped sperm-head, carrying the middle-piece at its base, rotates through nearly or quite 180 , so that the pointed end is directed outward and the middlepiece is turned inward (Fig. 94, A-F)} During or shortly after the rotation appears a minute aster centring in or very near the middlepiece. As it enlarges, the middle-piece itself is thrown to one side (Fig. 12), where it soon degenerates, while in the centre of the aster a minute intensely staining centrosome may be seen. Both spermnucleus and aster now rapidly advance toward the centre of the egg f the aster leading the way and its rays extending far out into the cytoplasm and finally traversing nearly an entire hemisphere. The central mass of the aster comes in contact with the egg-nucleus, divides into two, and the daughter-asters pass to opposite poles of the egg-nucleus, while the sperm-nucleus flattens against the latter and assumes the form of a biconvex lens (Fig. 95). The nuclei now fuse to form the cleavage-nucleus. Shortly afterward the nuclear membrane fades away, a spindle is developed between the asters, and a group of chromosomes arises from the cleavage-nucleus. These are 36 or 38 in number ; and although their relation to the paternal and maternal chromatin cannot in this case be accurately traced, owing to the apparent fusion of the nuclei, there can be no doubt on general grounds that one-half have been derived from each germnucleus. The egg then divides into two, four, etc., by ordinary mitosis (Figs. 4, 52).
In the type of fertilization just described, the polar bodies are formed long before the entrance of the spermatozoon and the germnuclei conjugate immediately upon entrance of the spermatozoon, fusing to form. a true cleavage-nucleus. In a second and more frequent type (Ascaris, Fig. 90; Physa, Fig. 89; Nereis, Fig. 97; Cyclops, Fig. 98) the sperm-nucleus penetrates for a certain distance, often to the centre of the egg, and then pauses while the polar bodies are formed. It then conjugates with the re-formed eggnucleus. In this case the sperm-aster always divides to form an amphiaster before conjugation of the nuclei, while in the first case the aster may be still undivided at the time of union. This difference is doubtless due merely to a difference in the time elapsing between entrance of the spermatozoon and conjugation of the nuclei, the amphiaster having, in the second case, time to
1 The first, as far as I know, to observe the rotation of the sperm-head was Flemming in the echinoderm-egg ('8i, pp. 17-19). It has since been clearly observed in several other cases, and is probably a phenomenon of very general occurrence.
form during extrusion of the polar bodies. The two types just described (Fig. 96) are connected by various gradations. Thus, in the lamprey, the frog, the rabbit, and in Ampkioxus, one polar body is expelled before, and one after, the entrance of the spermatozoon ; in the annelid Ophryotrocha, entrance takes place when the first polar spindle is in the stage of the equatorial plate ;
Fig. 95. — Conjugation of ihe germ-nuclei and division or the sperm-aster in the sea-urchin ToxopntusUs, x 1000. (For later stages see Fig. 53.)
A. Union ofthenuclei; extension of the aster. B. Flattening of the sperm-nucleus against the egg-nucleus ; division of the aster.
while in Chcetopterus and Pieris the first polar spindle has advanced into the anaphase. 1
It is an interesting and significant fact that the aster or amphiaster always leads the way in the march toward the egg-nucleus ; and in many cases it may be far in advance of the sperm-nucleus. 2 Boveri ('87, 1) has observed in sea-urchins that the sperm-nucleus may indeed be left entirely behind, the aster alone conjugating with the egg
Fig. 96. — Diagrams of two ptincipal types of fertilization. /. Polar bodies formed after the entrance of the spermatozoa (annelids, mollusks, flat-worms). //. Polar bodies formed before entrance (echinoderms).
A. Sperm-nucleus and centrosome at <J ; first polar body forming at 9. B. Polar bodies formed ; approach of the nuclei. C. Union of the nuclei. D. Approach of the nuclei. E. Union of the nuclei. /*'. Cleavage-nucleus.
nucleus and causing division of the egg without union of the gennnuclei, though the sperm-nucleus afterward conjugates with one of the nuclei of the two-cell stage. This process, known as " partial fertilization, " is undoubtedly to be regarded as abnormal. It affords, however, a beautiful illustration of the view that it is the centrosome alone that incites division of the egg, and is therefore the fertilizing element proper (Boveri, '87, 2).
The foregoing facts lead us to a consideration of Boveri's theory of fertilization, which has for several years formed a central point of discussion. The ground for this theory had been prepared by Oscar
1 Cf.p. 181.
8 Cf. Kostanecki and Wicrzejski, '96.
Hertwig and Fol. The latter ('73) early reached the conclusion that the asters represented " centres of attraction " lying outside and independent of the nucleus. Oscar Hertwig showed, in 1875, that
sappearing. an sent deutoplasm-spfieres (slightly
disappeared, leaving only I h
ctions. (X400.)
ninule sperm -nucleus at d\ the
mitotic figure forming. The empty spaces repre 1 by the reagents), the firm circles oil-drops, B. Sperm ister in from of it; first polar mitotic figure established;
C. Later stage; second polar body forming. D. The
■r {cf. Fig. 60).
Cgg-Cf
in the sea-urchin egg, the amphiaster arises by the division of a single aster that first appears near the spcrrn-nucleus and accompanies it in its progress toward the egg-nucleus. A similar observation was soon afterward made by Fol ('79) in the eggs of Asterias and Sagitta, and in the latter case he determined the fact that the astral rays do not centre in the nucleus, as Hertwig described, but at a point in advance of it — a fact afterward confirmed by Hertwig himself and by Boveri ('88, i). Hertwig and Fol afterward found that in cases of polyspermy, when several spermatozoa enter the egg, each sperm-nucleus is accompanied by an aster, and Hertwig proved that each of these might give rise to an amphiaster (Fig. 101). In 1886-87 Vejdovsky brought forward strong evidence to show that in the fresh-water annelid Rhynchelmis the cleavage-amphiaster arises directly from the sperm-amphiaster, itself derived by the division of a " periplast " (attraction-sphere) imported into the egg by the spermatozoon, while the polar amphiaster entirely disappears. It was Boveri ('87, 2) who first carefully studied the facts with reference to the centrosome, reaching the conclusion (in the case of Ascaris and the sea-urchin) that a single centrosome is brought in by the spermatozoon, and that it divides to form two centres about which are developed the two asters of the cleavage-figure. He was thus led to the following conclusion, which has received the support of many later investigators : The ripe egg possesses all of the organs and qualities necessary for division excepting the centrosome, by which division is initiated. The spermatozoon, on the other hand, is provided ivith a centrosome, but lacks the substance in which this organ of division may exert its activity. Through the union of the two cells in fertilization, all of the essential organs necessary for division are brought together ; the egg now contains a centrosome which by its oivn division leads the way in the embryonic development} Very numerous observations, supporting this conclusion, have been made by later observers. Bohm could find in Petrotnyzon ('88) and the trout ('91) no radiations near the egg-nucleus after the formation of the polar-bodies, while a beautiful sperm-aster is developed near the sperm-nucleus and divides to form the amphiaster. Platner ('86) had already made similar observations in the snail Arion, and the same result was soon afterward reached by Brauer {'92) in the case of Bratichipus, and by Julin ('93) in Styleopsis. Fick's careful study of fertilization of the axolotl ('93) proved in a very convincing manner not only that the amphiaster is a product of the sperm-aster, but also that the latter is developed about the middle-piece as a centre. The same result was indicated by Foot's observations on the earthworm C94), and it was soon afterward conclusively demonstrated in echinoderms through the independent and nearly simultaneous researches of myself on the egg of Toxopneustes, of Mathews on Arbacia, and of Boveri on Echinus. Nearly at the same, time a careful study was made by Mead ('95, '98, 1) of the annelid Chcetopterus, and of the starfish Asterias by Mathews,
1 '87, 2, p. 155.
both observers independently showing that the polar spindle contains distinct centrosomes, which, however, degenerate after the formation of the polar bodies, their place being taken by the sperm-centrosome, which divides to form an amphiaster before union of the nuclei, as in Rhynchehnis. Exactly the same result has since been reached by Hill C95) and Reinke ('95) in Sphmrechimts, by Hill in the tunicate Phalltisia, by Kostanecki and Wierzejski ('96) in Physa (Fig. 89), and by Van der Stricht ('98) in Thysan ozodn ; and in all of these the centrosome is likewise shown to arise from the middle-piece or in its immediate neighbourhood. Among others who have produced
Fig. 98. — Fertiliiationofthe egg in the copepod, Cyclops stratum. [RlicKERT.] A. Sperm-nucleus soon after entrance, the sperm-aster dividing. B. The germ-nuclei ap. preaching; d\ the enlarged sperm -nucleus with a large aster at each pole; 9, the egg-nucleus re-formed after formation of the second polar body, shown at the right. C. The apposed reticular germ-nuclei, now of equal size; the spindle is immediately afterward developed between (he two enormous sperm-osiers : polar body at Ihe left.
evidence that the cleavage-centrosome stands in definite relation to the spermatozoon, may be mentioned Oppel ('92) in reptiles, Brauer ('92) in Branehipus, Henking ('92) in insects, Riickert ('95,2) in Cyclops, Sobotta ('95) in the mouse and ('98) Amfi/tioxiis, Ziegler ('95) in Diplogaster and Rhabditis, Castle ('96) in dona, Korschelt ('95) in Ophryotrocha, Meyer ('95) in Strongylus, Griffin ('96, '99) in Thalassema, and Coe ('98) in Cerebratulus.
Beside the foregoing evidence may be placed the following additional data based on experiment and the study of pathological fertilization. (1) In the case of sea-urchin eggs, Hertwig, Boveri, and several later observers have shown that egg-fragments, obtained by shaking eggs to pieces, are readily penetrated by the spermatozoa, and that such fragments, though containing no nuclear matter from the egg. may segment and give rise to perfect larvae. 1 (2) Boveri (*88) has observed that in ordinary fertilization the sperm-aster may separate from the sperm-nucleus, travel through the cytoplasm to the egg-nucleus and cause cleavage, the sperm-nucleus afterward fusing with one of the nuclei of the two-cell stage (" partial fertilization "). (3) Most remarkable of all, Boveri, confirmed by Ziegler ('98), has recently observed that during the first cleavage the whole of the chromatin may pass to one pole, so that upon division one of the halves of the egg receives only a centrosome without a nucleus. In the nucleated half cleavage proceeds as usual. In the enucleated half the centrosomes and asters continue for a considerable period to multiply at the same rate as the cleavage of the nucleated half, though the cell-body does not itself divide. 2 Putting these facts together we must conclude (1) that something is introduced into the egg by the middle-piece of each spermatozoon entering it that is either a centrosome or has the power to incite the formation of one ; (2) that the centrosome thus arising is structurally independent of both nuclei and may divide independently of them; (3) that independently of the division of the nucleus or cell-body there is some kind of historical continuity between the centrosomes of successive generations.
In the case of echinoderm-eggs this continuity is not yet known to be effected by actual persistence of the centrosomes. 3 There are, however, a number of cases in which the division of the primary cleavage-centrosomes and the persistence of their descendants as those of the daughter-cells seem to have been conclusively shown — for example on Ascaris (Van Beneden, Boveri, Kostanecki, and Siedlecki), in the trout (Henneguy, '96), in Thalassemia (Griffin, '96, '99), in Chcetoptcrns (Mead, '95, '98), in Physa (Kostanecki and Wierzejski, '96), in Ccrcbratuhis (Coe, '98), and in Rhynchclmis (Vejdovsky and Mrazek, '98). In Thalassemia and Ccrebratulns (Figs. 99, 155) the centrosome is a minute granule at the focus of the sperm-aster, which divides to form an amphiaster soon after the entrance of the spermatozoon. During the early anaphase of the first cleavage, each centrosome divides into two, passes to the outer periphery of the centrosphere, and there forms a minute amphiaster for the second cleavage before the first cleavage takes place. The minute centrosomes of the second cleavage are therefore the direct descendants of the sperm-centrosome ; and there is good reason to believe that the continuity is not broken in later stages. The facts are nearly similar
1 Cf. P . 353. a Cf. p. 108.
8 Erlanger's statement ('98) that the centrosomes persist through the first cleavage in echinoderm-eggs is not supported by his figures ; and I am convinced from my own longcontinued studies of these eggs, as well as by an examination of Erlanger's preparations, kindly placed in my hands by Professor Butschli, that these difficult objects are very unfavourable for a decision of the question.
A. Second polar body fox egg-nucleus and sperm-nucleus, the nuclei. D. Later stage of lasi centrosome divided. G. H. I. the daughter-amphiasters lor the
1 an annelid (armed Gephyrean), Thaiaatma. [Griffin.) .ng ; sperm-nucleus and centrosome below. B, Approach of (he ., the latler accompanied by the spcrm-amphiaster. £'. Union of st. £. Prophase of cleavage-spindle. F, Anaphase of Ihe same; Successive stages in the nuclear reconslitution and formation o( cleavage. J. Two-cell stage.
in the trout, in Chcetoptcrtis, and in Physa. In Ascaris division of the centrosome first occurs at a somewhat later period (Figs. 90, 176). If now the centrosomes were indeed permanent cell-organs, we should thus reach the following result: During cleavage the cytoplasm of the blastomeres is derived from that of the egg, the centrosomes from the spermatozoon, while the nuclei (chromatin) are equally derived from both germ-cells.
There is very strong reason to accept the first part of this conclusion (applying to nucleus and cytoplasm), but the question of the centrosomes remains an open one. The array of evidence given above, derived from the study of so many diverse groups, seems to place Boveri's lucid and enticing hypothesis upon a strong foundation. Two essential points still remain, however, to be determined: first, whether the facts observed in Ascaris, Echinoderms, Physa, T/ialassema f and the like, are typical of all forms of fertilization ; and, second, whether, if so, the primary cleavage-centrosome is actually imported into the egg by the spermatozoon or is only formed under its influence out of the egg-substance. Both these questions have been raised by recent investigators, apparently on good evidence, and some of this evidence is directly opposed to both of the principal assumptions of Boveri's theory. Thus, Wheeler ('97) has found that in Mysostoma both centrosomes are derived from the Ggg) Carnoy and Le Brun ('97) maintain that in Ascaris one centrosome is derived from each of the germ-nuclei; in some mollusks, according to MacFarland ('97) and Lillie ('97), both egg-centrosomes and sperm-centrosomes disappear, to be replaced by two centrosomes of unknown origin ; while recent botanical workers are unable to find any centrosomes in fertilization. These and other divergent results will be critically considered beyond (p. 208) in connection with a more detailed examination of the general subject. It may be pointed out here, however, that recent researches on spermatogenesis (p. 170) render it nearly certain that the centrosome of the sperm-aster cannot be the unmodified centrosome of the spermatid, since the latter, in some cases, enlarges to form a " middle-piece " or analogous structure that is far larger than the sperm-centrosome.
B. Union of the Germ-cells
It does not lie within the scope of this work to consider the innumerable modes by which the germ-cells are brought together, further than to recall the fact that their union may take place inside the body of the mother or outside, and that in the latter case both eggs and spermatozoa are as a rule discharged into the water, where fertilization and development take place. The spermatozoa may live for a long period, either before or after their discharge, without losing their fertilizing power, and their movements may continue throughout this period. In many cases they are motionless when first discharged, and only begin their characteristic swimming movements after coming in contact with the water. There is clear evi
UNION OF THE GERM-CELLS
197
dence of a definite attraction between the germ-cells, which is in some cases so marked (for example in the polyp Renilla) that when spermatozoa and ova are mixed in a small vessel, each ovum becomes in a few moments surrounded by a dense fringe of spermatozoa attached to its periphery by their heads and by their movements actually causing the ovum to move about. The nature of the attraction is not positively known, but Pfeffer's researches on the spermatozoids of plants leave little doubt that it is of a chemical nature, since he found the spermatozoids of ferns and of Selaginella to be as actively attracted by solutions of malic acid or malates (contained in capillary tubes) as by the substance extruded from the
/—
Fig. 100.
— Enlranceof the spermatoiofln into theegg. A~G. In the sea-urchin. Toxefmemttt.
H. In rhe m
-dusa. Mitrocom*. (METSCHNIKOPF.] /. In Ibr slar-fch Aittriai, [FOL.]
A. Spern
aloiodn of Toxafnturlrl. X aoop; 0. ihe apical body. ». nucleus, m. middle-piece.
/ flagellum.
B. Contacl wilh the egg-periphery. C. 1). Entrance uf Ihe head, formation of the
e and of ihe vitelline membrane (t). leaving the lail outside. M. F. Laler stages.
G. Appears
ce of Ihe iperm-asier (j) about 3-5 minutes after first contacl ; entrance-cone break
ing up. H.
Entrance of the spermatozoon into a preformed depression. /. Approach of the
spermatozoon, showing the prefor
neck of the archegonium. Those of mosses, on the other hand, are indifferent to malic acid, but are attracted by cane-sugar. These experiments indicate that the specific attraction between the germcells of the same species is owing to the presence of specific chemical substances in each case. There is clear evidence, furthermore, that the attractive force is not exerted by the egg-nucleus alone, but by the egg-cytoplasm ; for, as the Hertwigs and others have shown, spermatozoa will readily enter egg-fragments entirely devoid of a nucleus.
In naked eggs, such as those of some echinoderms, and ccelenterates, the spermatozoon may enter at any point ; but there are some cases in which the point of entrance is predetermined by the
igS FERTILIZATION OF THE OVUM
presence of special structures through which the spermatozoon enters (Fig. ioo). Thus, the starfish-egg, according to Fol, possesses before fertilization a peculiar protoplasmic "attraction-cone " to which the head of the spermatozoon becomes attached, and through which it enters the egg. In some of the hydromcdusae, on the other hand, the entrance point is marked by a funnel-shaped depression at the egg-periphery ( Metschnikoff ). When no preformed attractioncone is present, an " entrance-cone " is sometimes formed by a rush of protoplasm toward the point at which the spermatozoon strikes the egg and there forming a conical elevation into which the spermhead passes. In the sea-urchin (Fig. ioo) this structure persists only a short time after the spermatozoon enters, soon assuming a ragged flame-shape and breaking up into slender rays. In some cases the egg remains naked, even after fertilization, as appears to be the case in many coelenterates. More commonly a vitelline membrane is quickly formed after contact of the spermatozoon, — e.g. in Atnphioxus, in the echinoderms, and in many plants, — and by means of this the entrance of other spermatozoa is prevented. In eggs surrounded by a membrane before fertilization, the spermatozoon either bores its way through the membrane at any point, as is probably the case with mammals and Amphibia, or may make its entrance through a micropyle.
In some forms only one spermatozoon normally enters the ovum, as in echinoderms, mammals, many annelids, etc., while in others several may enter (insects, elasmobranchs, reptiles, the earthworm, Petromyzotiy etc.). In the former case more than one spermatozoon may accidentally enter (pathological polyspermy), but development is then always abnormal. In such cases each sperm-centrosome gives rise to an amphiaster, and the asters may then unite to form the most complex polyasters, the* nodes of which are formed by the ccntrosomes (Fig. 101). Such eggs either do not divide at all or undergo an irregular multiple cleavage and soon perish. If, however, only two spermatozoa enter, the egg may develop for a time. Thus Driesch has determined the interesting fact, which I have confirmed, that sea-urchin eggs into which two spermatozoa have accidentally entered undergo a double cleavage, dividing into four at the first cleavage, and forming eight instead of four micromeres at the fourth cleavage. Such embryos develop as far as the blastula stage, but never form a gastrula. 1 In cases where several spermatozoa normally enter the egg (physiological polyspermy), only one of the sperm-nuclei normally unites with the egg-nucleus, the supernumerary sperm-nuclei either degenerating, or in rare cases — e.g. in elasmobranchs and reptiles — living for a time and even dividing to form
1 For an account of the internal changes, see p. 355.
UNION OF THE GERM-CELLS
199
"merocytes" or accessory nuclei. The fate of the latter is still in doubt ; but they certainly take no part in fertilization.
It is an interesting question how the entrance of supernumerary spermatozoa is prevented in normal monospermic fertilization. In the case of echinoderm-eggs Fol advanced the view that this is mechanically effected by means of the vitelline membrane formed instantly after the first spermatozoon touches the egg. This is indicated by the following facts. Immature eggs, before the formation
Fig. wt. — Pathological polyspermy.
A. Polyspermy in the egg of Aicaris; below.the egg-nucleus; above, ihree entire spermatozoa. within the egg. (Sala.1
B. Polyspermy in sea-urchin egg treated *ith 0.005% nicotine solution-, tt-n sperm-nuclei Shown. Ihree of which have conjugated with the egg- nucleus. C. Later stage of an vfg similarly treated, showing polyasters formed by union of the sperm -amphiasters. [O. and R. Hektwic]
of the polar bodies, have no power to form a vitelline membrane, and the spermatozoa always enter them in considerable numbers. Polyspermy also takes place, as O. and R. Hcrtwig's beautiful experiments showed ('87), in ripe eggs whose vitality has been diminished by the action of dilute poisons, such as nicotine, strychnine, and morphine, or by subjection to an abnormally high temperature
200 FERTILIZATION OF THE OVUM
(31 C); and in these cases the vitelline membrane is only slowly formed, so that several spermatozoa have time to enter. 1 Similar mechanical explanations have been given in various other cases. Thus Hoffman believes that in teleosts the micropyle is blocked by the polar bodies after the entrance of the first spermatozoon ; and Calberla suggested (Petromyzon) that the same result might be caused by the tail of the entering spermatozoon. It is, however, far from certain whether such rude mechanical explanations are adequate ; and there is considerable reason to believe that the egg may possess a physiological power of exclusion called forth by the first spermatozoon. Thus Driesch found that spermatozoa did not enter fertilized sea-urchin eggs from which the membranes had been removed by shaking. 2 In some cases no membrane is formed (some ccelenterates), in others several spermatozoa are found inside the membrane (ncmertines), in others the spermatozoon may penetrate the membrane at any point (mammals), yet monospermy is the rule.
1. Immediate Results of Union
The union of the germ-cells calls forth profound changes in both.
(a) The Spermatozoon. — Almost immediately after contact the tail ceases its movements. In some cases the tail is left outside, being carried away on the outer side of the vitelline membrane, and only the head and middle-piece enter the egg (echinoderms, Fig. 100). In other cases the entire spermatozoon enters (amphibia, earthworm, insects, etc., Fig. 89), but the tail always degenerates within the ovum and takes no part in fertilization. Within the ovum the sperm-nucleus rapidly grows, and both its structure and stainingcapacity rapidly change (cf. p. 182). The most important and significant result, however, is an immediate resumption by the sperm-nucleus and sperm-centrosome of the power of division^ which has hitherto been suspended. This is not due to the union of the germ-nuclei ; for, as the Hertwigs and others have shown, the supernumerary sperm-nuclei in polyspermic eggs may divide freely without copulation with the egg-nucleus, and they divide as freely after entering enucleated egg-fragments. The stimulus to division must therefore be given by the egg-cytoplasm. It is a very interesting fact that in some cases the cytoplasm has this effect on the sperm-nucleus
1 The Hertwigs attribute this to a diminished irritability on the part of the egg-substance. Normally requiring the stimulus of only a single spermatozoon for the formation of the vitelline membrane, it here demands the more intense stimulus of two, three, or more before the membrane is formed. That the membrane is not present before fertilization is admitted by Hertwig on the ground stated at page 132.
1 On the other hand, Morgan states ('95, 5, p. 270) that one or more spermatozoa will enter nucleated or enucleated egg-fragments whether obtained before or after fertilization.
UNION OF THE GERM-CELLS 201
only after formation of the polar bodies ; for when in sea-urchins the spermatozoa enter immature eggs, as they freely do, they penetrate but a short distance, and no further change occurs.
(6) The Ovum. — The entrance of the spermatozoon produces an extraordinary effect on the egg, which extends to every part of its organization. The rapid formation of the vitelline membrane, already described, proves that the stimulus extends almost instantly throughout the whole ovum. 1 At the same time the physical consistency of the cytoplasm may greatly alter, as for instance in echinoderm eggs, where, as Morgan has observed, the cytoplasm assumes immediately after fertilization a peculiar viscid character which it afterward loses. In many cases the egg contracts, performs amoeboid movements, or shows wave-like changes of form. Again, the egg-cytoplasm may show active streaming movements, as in the formation of the entrance-cone in echinoderms, or in the flow of peripheral protoplasm toward the region of entrance to form the germinal disc, as in many pelagic fish-eggs. An interesting phenomenon is the formation, behind the advancing sperm-nucleus, of a peculiar funnelshaped mass of deeply staining Pj I01 _ material extending outward to the during fenitimtu periphery. This has been carefully M p°i" bodies; /... polar rmg 5;
j i_ j i_ r> . ,1 \ • lL _j.iT cleavage-nucleus near Ihe centre.
described by Foot ( 94) in the earthworm, where it is very large and conspicuous, and I have since observed it also in the sea-urchin (Fig. 94).
The most profound change in the ovum is, however, the migration of the germinal vesicle to the periphery and the formation of the polar bodies. In many cases either or both these processes may occur before contact with the spermatozoon (echinoderms, some vertebrates). In others, however, the egg awaits the entrance of the spermatozoon (annelids, gasteropods, etc.), which gives' it the necessary stimulus. This is well illustrated by the egg of Nereis. In the newly discharged egg the germinal vesicle occupies a central position, the yolk, consisting of deutoplasm-spheres and oil-globules, is uniformly distributed, and at the periphery of the egg is a zone of clear perivitelline protoplasm (Fig. 60). Soon after entrance of the sperma
202 FERTILIZATION OF THE OVUM
tozoon the germinal vesicle moves toward the periphery, its membrane fades away, and a radially directed mitotic figure appears, by means of which the first polar body is formed (Fig. 97). Meanwhile the protoplasm flows toward the upper pole, the peri-vitelline zone disappears, and the egg now shows a sharply marked polar differentiation. A remarkable phenomenon, described by Whitman in the leech ('78), and later by Foot in the earthworm ('94), is the formation of " polar rings," a process which follows the entrance of the spermatozoon and accompanies the formation of the polar bodies. These are two ring-shaped cytoplasmic masses which form at the periphery of the egg near either pole and advance thence toward the poles, the upper one surrounding the point at which the polar bodies are formed (Fig. 102). Their meaning is unknown, but Foot ('96) has made the interesting- discovery that they are probably of the same nature as the yolk-nuclei (p. 1 56).
2. Paths of the Germ-nuclei (Pro-nuclei ) x
After the entrance of the spermatozoon, both germ-nuclei move through the egg-cytoplasm and finally meet one another. The paths traversed by them vary widely in different forms. In general two classes are to be distinguished, according as the polar bodies are formed before or after entrance of the spermatozoon. In the former case (echinoderms) the germ-nuclei unite at once. In the latter case the sperm-nucleus advances a certain distance into the egg and then pauses while the germinal vesicle moves toward the periphery, and gives rise to the polar bodies (Ascaris, annelids, etc.). This significant fact proves that the attractive force between the two nuclei is only exerted after the formation of the polar bodies, and hence that the entrance-path of the sperm-nucleus is not determined by such attraction. A second important point, first pointed out by Roux, is that the path of the sperm-nucleus is curved, its " entrance-path " into the egg forming a considerable angle, with its " copulation-path " toward the egg-nucleus.
These facts are well illustrated in the sea-urchin egg (Fig. 103), where the egg-nucleus occupies an eccentric position near the point at which the polar bodies are formed (before fertilization). Entering
1 The terms female pro-nucleus, male pro-nucleus (Van Beneden), are often applied to the germ-nuclei before their union. These should, I think, be rejected in favour of Hertwig's terms egg-nucleus and sperm-nucleus y on two grounds: (i) The germ-nuclei are true nuclei in every sense, differing from the somatic nuclei only in the reduced number of chromosomes. As the latter character has recently been shown to be true also of the somatic nuclei in the sexual generation of plants (p. 275), it cannot be made the ground for a special designation of the germ-nuclei. (2) The germ-nuclei are not male and female in any proper sense (p. 243).
UNION OF THE GERM-CELLS
203
the egg at any point, the sperm-nucleus first moves rapidly inward along an entrance-path that shows no constant relation to the position of the egg-nucleus and is approximately but never exactly radial, i.e. toward a point near the centre of the egg. After penetrating a
Fig. 103. — Diagrams showing the paths of the germ-nuclei in four different eggs of the seaurchin, ToxopneusUs. From camera drawings of the transparent living eggs.
In all the figures the original position of the egg-nucleus (reticulated) is shown at 9 ; the point at which the spermatozoon enters at E (entrance-cone). Arrows indicate the paths traversed by the nuclei. At the meeting-point (Af) the egg-nucleus is dotted. The cleavage-nucleus in its final position is ruled in parallel lines, and through it is drawn the axis of the resulting cleavagefigure. The axis of the egg is indicated by an arrow, the point of which is turned away from the micromere-pole. Plane of first cleavage, passing near the entrance-point, shown by the curved dotted line.
certain distance its direction changes slightly to that of the copulation-path, which, again, is directed not precisely toward the eggnucleus, but toward a meeting-point where it comes in contact with the egg-nucleus. The latter does not begin to move until the
204 FERTILIZATION OF THE OVUM
entrance-path of the sperm-nucleus changes to the copulation-path. It then begins to move slowly in a somewhat curved path toward the meeting-point, often showing slight amoeboid changes of form as it forces its way through the cytoplasm. From the meeting-point the apposed nuclei move slowly toward the point of final fusion, which in this case is near, but never precisely at, the centre of the egg.
These facts indicate that the paths of the germ-nuclei are determined by at least two different factors, one of which is an attraction or other dynamical relation between the nuclei and the cytoplasm, the other an attraction between the nuclei. The former determines the entrance-path of the sperm-nucleus, while both factors probably operate in the determination of the copulation-path along which it travels to meet the egg-nucleus. The real nature of neither factor is known.
Hertwig first called attention to the fact — which is easy to observe in the living sea-urchin egg — that the egg-nucleus does not begin to move until the spermnucleus has penetrated some distance into the egg and the sperm-aster has attained a considerable size ; and Conklin ('94) has suggested that the nuclei are passively drawn together by the formation, attachment, and contraction of the astral rays. While this view has some facts in its favour, it is, I believe, untenable, for many reasons, among which may be mentioned the fact that neither the actual paths of the pro-nuclei nor the arrangement of the rays support the hypothesis ; nor does it account for the conjugation of nuclei when no astral rays are developed (as in Protozoa or in plants). I have often observed in cases of dispermy in the sea-urchin, that both sperm-nuclei move at an equal pace toward the egg-nucleus ; but if one of them meets the egg-nucleus first, the movement of the other is immediately retarded, and only conjugates with the egg-nucleus, if at all, after a considerable interval ; and in polyspermy the egg-nucleus rarely conjugates with more than two sperm-nuclei. Probably, therefore, the nuclei are drawn together by an actual attraction which is neutralized by union, and their movements are not improbably of a chemotactic character. Conklin ('99) has recently suggested that the nuclei are drawn together by the agency of protoplasmic currents in the egg-substance.
3. Uniofi of the Germ-nuclei. The Chromosomes
The earlier observers of fertilization, such as Auerbach, Strasburger, and Hertwig, described the germ-nuclei as undergoing a complete fusion to form the first embryonic nucleus, termed by Hertwig the cleavage- or segmentation-nucleus. As early as 1881, however, Mark clearly showed that in the slug Limax this is not the case, the two nuclei merely becoming apposed without actual fusion. Two years later appeared Van Beneden's epoch-making work on Ascaris, in which it was shown not only that the nuclei do not fuse, but that they give rise to two independent groups of chromosomes which separately enter the equatorial plate and whose descendants pass separately into the daughter-nuclei. Later observations have given the strongest reason to believe that, as far as the chromatin is con
UNION OF THE GERM-CELLS 205
cerned, a true fusion of the nuclei never takes place during fertilization, and that the paternal and maternal chromatin may remain separate and distinct in the later stages of development — possibly throughout life (p. 299). In this regard two general classes may be distinguished. In one, exemplified by some echinoderms, by Amphioxtts, Phallusia, and some other animals, the two nuclei meet each other when in the reticular form, and apparently fuse in such a manner that the chromatin of the resulting nucleus shows no visible distinction between the paternal and maternal moieties. In the other class, which includes most accurately known cases, and is typically represented by Ascaris (Fig. 90) and other nematodes, by Cyclops (Fig. 98), and by Pterotrachea (Fig. 93), the two nuclei do not fuse, but only place themselves side by side, and in this position give rise each to its own group of chromosomes. On general grounds we may confidently maintain that the distinction between the two classes is only apparent, and probably is due to corresponding differences in the rate of development of the nuclei, or in the time that elapses before their union. 1 If this time be very short, as in echinoderms, the nuclei unite before the chromosomes are formed. If it be more prolonged, as in Ascaris, the chromosome-formation takes place before union.
With a few exceptions, which are of such a character as not to militate against the rule, the number of chromosomes arising from the germ-nuclei is always the same in both, and is one-half the number characteristic of the tissue-cells of the species. By their union, therefore, the germ-nuclei give rise to an equatorial plate containing the typical number of chromosomes. This remarkable discovery was first made by Van Beneden in the case of Ascaris, where the number of chromosomes derived from each sex is either one or two. It has since been extended to a very large number of animals and plants, a partial list of which follows.
1 Indeed, Boveri has found that in Ascaris both modes occur, though the fusion of the germ-nuclei is exceptional. (Cf. p. 296.)
206
FERTILIZATION OF THE OVUM
A Partial List showing the Number of Chromosomes Characteristic of the Germ-nuclei and Somatic Nuclei in Various Plants and Animals 1
1
Germ- , Nuclei.
Somatic Nuclei.
Name.
Group.
Authority.
I
2
Ascaris megalocephala, var. univalens.
Nematodes.
Van Beneden, Boveri.
2
4
Id., var. bivalens.
»
»
n
M
Ophryotrocha.
Annelids.
Korschelt.
n
w
Styleopsis.
Tunicates.
Julin.
4
8
Coronilla.
Nematodes.
Carnoy.
»
»
Pallavicinia.
Hepaticae.
Farmer.
99
99
Anthoceras.
»»
Davis.
6
12
Spiroptera.
Nematodes.
Carnoy.
»
»
Prostheceraeus.
Polyclades.
Klinckostrom, Francotte.
rt
»
Nais.
Phanerogams.
Guignard.
W
»»
Spirogyra.
Conjugatae.
Strasburger.
»
W
Gryllotalpa.
Insects.
Vom Rath.
»
?>
Caloptenus.
99
Wilcox.
w
»>
vEquorea.
Hydromedusse.
Hacker.
7
14
Pentatoma.
Insects.
Montgomery.
8
16
Filaroides.
Nematodes.
Carnoy.
V
w
Prosthiostomum.
Polyclades.
Francotte.
»
w
Leptoplana.
»
?>
»
w
Cycloporus.
•«
»»
»>
»»
Hydrophilus.
Insects.
Vom Rath.
»»
«7
Phallusia.
Tunicates.
Hill.
»
«•
Limax.
Gasteropods.
Vom Rath.
»
[••]
Rat.
Mammals.
Moore.
»
Ox, guinea-pig, man.
»
Bardeleben.
»»
J?
Ceratozamia.
Cyads.
Overton, Guignard.
M
»y
Pinus.
Coniferae.
Dixon.
»*
??
Scilla, Triticum.
Angiosperms.
Overton.
V
V
Allium.
>?
Strasburger, Guignard.
V
»»
Podophyllum.
•»
Mottier.
9
! 18
, Echinus.
Echinoderms.
Boveri.
i»
1
Thysanozoon.
Polyclades
Van der Stricht.
•>
1
1
Sagitta.
Chai'tognaths.
Boveri.
»»
1
! *'
Chactopterus.
Annelids.
Mead.
»
, ••
1 Ascidia.
Tunicates.
Boveri.
IO
1
i 20
1
Lasius.
Insects.
Henking.
ii
! [33]
Allolobophora.
Annelids.
Foot.
12
1 *>A
1 " 4
1
Myzostoma.
»
Wheeler.
1 This table is compiled from papers both on brackets are inferred.
fertilization and maturation. Numbers in
UNION OF THE GERM- CELLS
207
GermNuclei.
Somatic Nuclei.
Name.
•
Group.
Authority.
12
24
Thalassema.
Annelids.
Griffin.
II (12)
22 (24)
Cyclops strenuus.
Copepods.
Ruckert.
12
24
„ brevicornis.
•?
Hacker.
»>
j
Helix.
Gasteropods.
Platner,VomRath.
»
?>
Branchipus.
Crustacea.
Brauer.
Yt
»
Pyrrhocoris.
Insects.
Henking.
»
»>
Salmo.
Teleosts.
Bohm.
»
»
Salamandra.
Amphibia.
Flemming.
»
»
Rana.
««
Vom Rath.
»
»>
Mouse.
Mammals.
Sobotta.
»
»
Osmunda.
Ferns.
Strasburger.
»
•»
Lilium.
Angiosperms.
Strasburger, Guignard.
>♦
«>
• Helleborus.
»
Strasburger.
»
??
Leucojum, Pseonia, Aconitum.
yy
Overton.
14
28
Tiara.
Hydromedusae.
Boveri*
>»
44
Pieris.
1 nsects.
Henking.
16
3 2
Cerebratulus, Micrura.
Nemertines.
Coe.
>»
»
Pterotrachea, Carinaria,
Phyllirhoc.
Gastropods.
Boveri.
»
w
Diaptomus, Heterocope.
Copepods.
Ruckert.
»*
-J
Anomalocera, Euchaeta.
•«
Vom Rath.
»»
[„]
Lumbricus.
Annelids.
Calkins.
18
36
Torpedo, Pristiurus.
Elasmobranchs.
RUckert.
['8(19)] 36(38)
Toxopneustes.
Echinoderms.
Wilson.
3°
[60]
Crepidula.
Gasteropods.
Conklin.
84
168
Artemia.
Crustacea.
Brauer.
The above data are drawn from sources so diverse and show so remarkable a uniformity as to establish the general law with a very high degree of probability. The few known exceptions are almost certainly apparent only and are due to the occurrence of plurivalent chromosomes. This is certainly the case with Ascaris (cf. p. 87). It is probably the case with the gasteropod Avion, where, as described by Platner, the egg-nucleus gives rise to numerous chromosomes, the sperm-nucleus to two only ; the latter are, however, plurivalent, for Garnault showed that they break up into smaller chromatin-bodies, and that the germ-nuclei are exactly alike at the time of union. We may here briefly refer to remarkable recent observations by Ruckert and others, which seem to show that not only the paternal and maternal chromatin, but also the chromosomes, may retain their individuality throughout development. 1 Van Beneden, the pioneer observer
1 '89, pp. 10, 33.
208 FERTILIZATION OF THE OVUM
in this direction, was unable to follow the paternal and maternal chromatin beyond the first cleavage-nucleus, though he surmised that they remained distinct in later stages as well ; but Rabl and Boveri brought forward evidence that the chromosomes did not lose their identity, even in the resting nucleus. Ruckert ('95, 3) and Hacker C95, 1 ) have recently shown that in Cyclops the paternal and maternal chromatin-groups not only remain distinctly separated during the anaphase, but give rise to double nuclei in the two-cell stage (Fig. 146). Each half again gives rise to a separate group of chromosomes at the second cleavage, and this is repeated at least as far as the blastula stage. Herla and Zoja have shown furthermore that if in Ascaris the egg of variety bivalens , having two chromosomes, be fertilized with the spermatozoon of variety univalens having one chromosome, the three chromosomes reappear at each cleavage, at least as far as the twelve-cell stage (Fig. 145); and according to Zoja, the paternal chromosome is distinguishable from the two maternal at each step by its smaller size. We have thus what must be reckoned as more than a possibility, that every cell in the body of the child may receive from each parent not only half of its chromatin-substance, but one-half of its chromosomes, as distinct and individual descendants of those of the parents.
C. The Centrosome in Fertilization
In examining more critically the history of the centrosomes we may conveniently take Boveri's hypothesis of fertilization as a point of departure, since it has long formed the focus of discussion of the entire subject. Before the hypothesis is more closely scrutinized we may first eliminate two other views, both of which are irreconcilable with it, though neither has stood the test of later research. The first of these, doubtfully suggested by Van Beneden ('87) and definitely maintained by Wheeler ('97) in the case of Myzostoma, is that the cleavage-centrosomes have no definite relation to the spermatozoon, but are derived from the egg — a conclusion that has the a priori support of the fact that in parthenogenesis the centrosomes are certainly of maternal origin.
Van Beneden's early statement may be passed by, since it was no more than a surmise. Wheeler, after a careful research, found that no sperm-aster accompanied the sperm-nucleus — a fact correlated with the absence of a middle-piece in the spermatozoon, — and reached the conclusion that after formation of the polar bodies, the egg-centrosomes persisted to become directly converted into the cleavage-centrosomes (Fig. 104). That the absence of a distinct middle-piece is not a valid argument is shown by the insect-spermatozoon, where the region
THE CENTROSOME IN FERTILIZATION
of the middle-piece is likewise not marked off from the tail, yet as we have seen (p. 165) the centrosome passes into this part of the spermaKostanecki's later examination of the fertilization of the
Pig. 104. — Fertilisation of the egg of the parasitic annelid. Mytosloma.
A. Soon after enlrance of the spermaiotoon ; the sperm-nucleus at rf ;
esiele; at c (he double centrosome. B. First polar body forming si i ; «.
lus or germinal spot. C. The polar bodies formed (fi.t.) ; germ-nuclei of
D. Approach of the germ-nuclei ; [he amphiaster formed.
[Wheelek.J at 9 the germinal
same animal ('98), while inconclusive on the main point, leaves little doubt that Wheeler's evidence was equally so ; for he has on the one hand shown that the sperm-nucleus is often accompanied by a sperm
2IO FERTILIZATION OF THE OVUM
aster containing a pair of centrosomes, on the other hand that these, like the egg-centrosomes, wholly disappear from view at a later period, the cleavage-centrosomes having only a conjectural origin.
The second of the views in question is that the cleavage-centrosomes are derived from both germ-cells ; and this in turn has in its favour the a priori evidence that in the Infusoria conjugation takes place between two mitotic figures (p. 224). It appears in two forms, of which the first, though undoubtedly erroneous, has had so interesting a history as to deserve a brief review. It was predicted by Rabl in 1889 that if the centrosome be a permanent cell-organ, the conjugation of germ-cells and germ-nuclei would be found to involve also a conjugation of centrosomes. Unusual interest was therefore aroused when Fol, in 1891, under the somewhat dramatic title of the "Quadrille of Centres," described precisely such a conjugation of centrosomes as Rabl had predicted. The results of this veteran observer were very positively and specifically set forth, and were of so logical and consistent a character as to command instant acceptance on the part of many authorities. In the eggs of the sea-urchin the sperm-centrosome and egg-centrosome were asserted to divide each into two, the daughter-centrosomes then conjugating two and two, paternal with maternal, to form the cleavage-centrosomes. The same result was announced by Guignard ('91 ) in the lily, by Conklin ('93) in the gasteropod Crcpidula, less definitely by Blanc O93) in the trout, and still later by Van der Stricht ('95) in Amphioxus. None of these results have stood the test of later work. Fol's result was opposed to the earlier conclusions of Boveri and Hertwig, and a careful reexamination of the fertilization of the echinoderm egg, independently made in 1894-95 by Boveri (Echinus), by myself (Toxopneustes), and Mathews (Arbacia, Asterias), and slightly later by Hill ('95) and Reinke (*95) in Sphcerechituts, demonstrated its erroneous character. Various attempts have been made to explain Fol's results as based on double-fertilized eggs, on imperfect method, on a misinterpretation of the double centrosomes of the cleavage-spindle, yet they still remain an inexplicable anomaly of scientific literature.
Serious doubt has also been thrown on Conklin's conclusions by subsequent research. Kostanecki and Wierzejski ('96) made a very thorough study, by means of serial sections, of the fertilization of the gasteropod Physa> and reached exactly the same result as that obtained in the echinoderms. Here, also, the egg-centres degenerate, their place being taken by a new pair, arising in intimate relation with the middle-piece of the spermatozoon, about which forms a spermamphiaster (Fig. 89). Conklin, after renewed research, himself admitted that no quadrille occurs in Crepidula, though he still believes that a union of paternal and maternal attraction-spheres takes place.
THE CENTROSOME IN FERTILIZATION 211
Guignard's results, too, have entirely failed of confirmation by later observers (p. 221), and in his own latest contribution to the subject ('99) the centrosomes are conspicuous by their absence in both the text and the figures. In like manner Van der Stricht's conclusions have been shown by Sobotta('97) to be without substantial foundation, while Blanc's account, opposed to the earlier work of Bohm, is too incomplete to carry any weight. The entire case for the " quadrille " has thus fallen to the ground. In its second form the supposed double origin of the centrosomes rests upon a single research upon Ascaris by Carnoy and Le Brun ('97, 2), who assert that the cleavagecentrosomes arise de novo and separately, one inside of each of the germ-nuclei, to migrate thence out into the cytoplasm. At the close of mitosis they wholly disappear, to be replaced by a new pair, likewise of intranuclear origin. Since this result is totally opposed to those of Van Beneden, Boveri, Erlanger, and Kostanecki and Siedlecki on the same object, and is contradicted in the most positive manner by Fiirst, 1 it may be received with some scepticism. The work of Kostanecki and Siedlecki ('96) demonstrates the division of the spermcentrosome in Ascaris as described by Boveri; and while it still remains possible that the daughter-centrosomes may for a very brief period disappear (as in some of the mollusks described beyond), no ground is given for such a conclusion as Carnoy has drawn. No one familiar with the object can repress the suspicion that Carnoy and Le Brun have confused the centrosomes with the nucleoli ; but only renewed research can determine the point.
The ground is now clear for a closer study of Boveri's hypothesis in the light of more recent research. It should first be pointed out that that hypothesis is based upon and forms a part of the more general theory of the autonomy of the centrosome ; and if the latter theory cannot be sustained, the a priori side of Boveri's hypothesis assumes a different aspect. In point of fact the general outcome of recent research on fertilization has been on the whole unfavourable to the view that the cleavage-centrosomes must necessarily be individually identical with permanent preexisting centrosomes — indeed, it is in this very field that some of the most convincing evidence against the persistence of the centrosome has been produced. The mode of origin of the cleavage-centrosomes is nevertheless a question of high interest on account of the unmistakable genetic relations existing between the centrosome of the spermatid and spermatozoon and those of the sperm-amphiaster within the egg.
There are two points of capital importance to be determined before a definite decision regarding the origin of the cleavage-centrosomes can be reached. First, are the centrosomes of the sperm-aster within
1 '98, p. 105.
212 FERTILIZATION OF THE OVUM
the egg identical with, or the descendants of, a centrosome or pair of centrosomes in the middle-piece of the spermatozoon ? Second, do they actually persist to form those of the cleavage-amphiaster ? In the present state of knowledge we are not in a position to give an affirmative answer to the first of these questions. As has been shown in Chapter III., it is no longer possible to doubt that the middlepiece either contains or is itself a metamorphosed centrosome ; but, as pointed out at page 196, it does not seem possible that the extremely minute centrosome of the sperm-aster can represent the entire centrosome of the middle-piece (however we conceive the origin of the latter). At most we can only assume that a part of the latter persists as the sperm-centrosome within the egg. The exact origin of the latter still remains problematical. A large number of observers are now agreed that the sperm-aster is formed about a focus that is either in or very near the middle-piece ; * but no one, I believe, has yet succeeded in showing that the centrosome actually is the metamorphosed middle-piece, or escapes from it. 2 The possibility therefore remains that the centrosome of the sperm-aster is not actually imported as such into the egg, but is either only a portion of the original spermatid-centrosome, or, as was first suggested by Miss Foot C97) and further discussed by Mead ('98, 2), is, like the aster, formed anew in the egg-cytoplasm. If the latter alternative be the case, the original form of Boveri's hypothesis would have to be abandoned;
1 For example, in echinoderms (Flemming, *8i, O. and R. Hertwig, '86, Boveri, '95, Wilson and Mathews, '95, Hill, '95, Reinke, '95, R. Hertwig, '96, Doflein, '97, 2, Erlanger, '98), in Pterotrachea and Pieris (Henking, '91, '92), in the axolotl (Fick, '93), and Triton (Michaelis, '97), in Phallusia (Hill, '95), in Ophryotrocha (Korschelt, '95), in Physa (Kostanecki and Wierzejski, '96), in Strongylus (Meyer, '95), in Thysanozo'dn (Van der Stricht, '98), and Prosthiostomum (Francotte, '98). In a large number of other cases the sperm -aster is found near the sperm-nucleus, but its relation to the middle-piece has not been demonstrated.
2 I myself formerly concluded ('95, 2) that the entire middle-piece of echinoderms is the centrosome — a result apparently confirmed in a most positive manner by Erlanger ('98), as well as by R. Hertwig ('96) and Doflein ('97, 2). I have, however, demonstrated this to be an error, showing that the extremely minute centrosome is quite distinct from the middle-piece, the latter being thrown aside and degenerating in the egg-cytoplasm outside of the newly formed sperm-aster (Figs. 12, 94). This fact, of which the phenomena in
Toxopnemtes leave no doubt (see Wilson, '97, '99), is, I think, fatal to Kostanecki's and Wierzejski's theory of fertilization ('96, pp. 374-375), according to which the archoplasm of the middle-piece gives rise to the new astral system and is thus the essential fertilizing substance (the centrosome being merely a mechanical centre for the attachment of the rays) ; but the most careful examination has still failed to show whether the centrosome actually escapes from the middle-piece, nor have other observers had better success with any animal. Erlanger ('96, 2, '97, 4) believes he has seen the centrosome in the Ascaris spermatozoon as a distinct body lying behind the nucleus, and that it can be traced continuously into the egg and after its division into the two poles of the cleavage-figure. Neither the schematic figures of his preliminary nor the photographic ones of his final paper seem sufficient to establish either the identity or the subsequent history of the granule in question.
THE CENTROSOME IN FERTILIZATION 21 3
though in substance it would still retain an element of truth, as pointed out beyond.
We may now examine the question whether the sperm-centrosomes are actually identical with the cleavage-centrosomes. That such is the case is positively maintained in the case of Ascaris by Boveri, Kostanecki, and Erlanger, in Physa by Kostanecki and Wierzejski C96), in Thalassema by Griffin ('96, '99), and in Chatopterus by Mead (95i '98)- The two last-mentioned observers, who have followed the phenomena with especial care, produce very strong evidence that at no time do the sperm-centrosomes and asters disappear, and that the former may be traced in unbroken continuity from the time of their first appearance to the daughter-cells resulting from the first cleavage (Figs. 99, 155). On the other hand, a considerable number of observers, beginning with Hertwig (Phyllirrhoe, Pterotrachea, '75), have found that as the sperm-nucleus enlarges the sperm-asters diminish in size, until, in many cases, they nearly or quite disappear ; for example, in Prosthecerams (Klinckowstrom, '97), in the mouse (Sobotta^ '95), in Pleurophyllidia (MacFarland, '97), Physa (Kostanecki and Wierzejski, '96), Arenicola (Child, '97), Unto (Lillie, '97), Myzos4ema (Kostanecki, '98), and Ccrebratnlus (Coe, '98). * Several of these observers (Klinckowstrom, MacFarland, Lillie, Child) have found that not only the asters but also the centrosomes totally disappear about the time the germ-nuclei come together, a new pair of cleavagecentrosomes and asters being afterward developed at the poles of the united nuclei. These conclusions, if correct, place in a new light the disappearance of the egg-centrosomes ; for this process
1 Coe has pointed out that the eggs of various animals may be arranged in a series showing successive graduations in the disappearance of the sperm-asters. " At the head of the series we must place the eggs of Ascaris and Myzostoma (according to Kostanecki) and similar ones in which the sperm-asters make their appearance only a short time before the formation of the cleavage-spindle, and which, consequently, suffer no diminution in size. Following these are the eggs of Ch<etopterus (Mead) and Ophryotrocha (Korschelt) and of some echinoderms in which the sperm-asters develop very early, but are not described as decreasing in size before the formation of the cleavage-spindle. Then come the eggs of Toxopneustes (Wilson) and Thalassema (Griffin), where the sperm-asters appear early and develop to a very considerable size, but nevertheless become very much smaller and less conspicuous after the germ-nuclei have come together. After these we must place the eggs of Physa (Kostanecki and Wierzejski), for here the sperm -asters, after becoming very large and conspicuous, degenerate to such an extent that only a very few exceedingly delicate fibres remain. Those of Cerebratulus follow next.
" Here the sperm-asters increase in size until they extend throughout the whole body of the cell, but at the time of fusion of the germ-nuclei they degenerate completely. The peripheral portions of their fibres, however, may be followed, as stated above of Pleurophyllidia, Prostheceraus, etc., where the sperm-asters degenerate soon after their formation, so that for a considerable period the egg is without trace of aster-fibres. Yet in all of those cases where the sperm -asters disappear and their centrosomes become lost among the other granules of the cell, we are justified in believing that the sperm-centrosomes nevertheless retain their identity, and later reappear in the cleavage-asters " ('98, p. 455).
214 FERTILIZATION OF THE OVUM
would thus seem to be of the same nature as the disappearance of the sperm-centrosomes, and both Boveri's theory of fertilization and the general hypothesis of the permanence of the centrosomes would receive a serious blow.
The investigators to whom these observations are due have ranged themselves in two groups in the interpretation of the phenomena. On the one hand, Lillie and Child do not hesitate to maintain that the centrosomes actually go out of existence as such, to be re-formed like the asters out of the egg-substance ; and that such a new formation of centrosomes is. possible seems to be conclusively shown by the experiments of Morgan and Loeb described at pages 2 1 5 and 307. On the other hand, Sobotta, MacFarland, Kostanecki, and Coe, relying partly on the analogy of other forms, partly on the occasional presence of the centrosomes during the critical stage, urge that the disappearance of the sperm-centrosomes is only apparent, and is due to the disappearance of the asters, which renders difficult or impossible the identification of the centrosomes among the other protoplasmic granules of the egg. These authors accordingly still uphold Boveri's theory.
It is difficult to sift the evidence at present, for it has now become very important to reexamine, in the light of these facts, those cases in which the absolute continuity of the centrosome has been maintained — for example, in Ascaris, Ckatopterus, and Thalassema — in order to determine whether there may not be here also a brief critical period in which the centrosomes disappear. There are, however, some facts which tend to sustain the conclusion that even though the sperm-centrosomes disappear from view, there is some kind of genetic continuity between them and the cleavage-centrosomes. First, both Kostanecki and Wierzejski ('96) and Coe ('98) have found that there is sojne variation in eggs apparently equally well preserved, a few individuals showing the sperm-centrosomes at the poles of the united nuclei at the same period when they are invisible in other individuals. Second, both these observers, Coe most clearly, have shown that the egg-centrosomes disappear considerably earlier than the sperm-centrosomes, and Coe has traced the sperm-centrosomes continuously to the exact points (the poles of the united nuclei} at which the cleavagecentrosomes afterward appear (Y\g. 155). This important observation leads to the suspicion that the apparent disappearance of the centrosomes may be due to a loss of staining-capacity at the critical period, or that even though the formed centrosome disappears its substance reappears in its successor. Here again we come to the view suggested at page 1 1 1, that the centrosome may be regarded as the vehicle of a specific chemical substance which is transported to the nuclear poles by its division, and may there persist even though the body of the
FER T I LIZ A TION IN PLANTS 2 1 5
centrosome be no longer visible. On such a basis we may perhaps find a reconciliation between these observations and Boveri's theory, and may even bring the fertilization of plants into relation with it (p. 221). Even in case of the nucleus, universally recognized as a permanent cell-organ, it is not the whole structure that persists as such during division, but only the chromatin-substance — in some cases only a small fraction of that substance. The law of genetic continuity therefore would not fail in case of the centrosome, though only a portion of its substance were handed on by division ; and even if we take the most extreme negative position, assuming that the sperm-centrosome is wholly formed anew under the stimulus of the spermatozoon, we should still not escape the causal nexus between it and the centrosome of the spermatid.
Boveri himself has suggested 1 that the egg may be incited to development by a specific chemical substance carried by the spermatozoon, and the same view has been more recently urged by Mead, 2 while Loeb's recent remarkable experiments on sea-urchins ('99) show that the egg may in this case (Arbacia) undergo complete parthenogenetic development as the result of artificial chemical stimulus. 8 Assuming such a substance to exist, by what part of the spermatozoon is it carried ? It is possible that the vehicle may be the nucleus, which forms the main bulk of that which enters the egg ; and this view seems to be supported by what is at present known of fertilization in the plants (p. 221). Yet when we regard the facts of fertilization in animals, taken in connection with the mode of formation of the spermatozoon, we find it difficult to avoid the conclusion that the substance by which the stimulus to development is normally given is originally derived from the spermatid-centrosome, is conveyed into the egg by the middle-piece, and is localized in the sperm-centrosomes which are conveyed to the nuclear poles during the amphiaster-formation. Accepting such a view, we could gain an intelligible view of the genetic relation between spermatid-centrosome, middlepiece, sperm-centrosome, and cleavage-centrosomes, without committing ourselves to the morphological hypothesis of the persistence of the centrosome as an individualized cell-organ. Such a conclusion, I believe, would retain the substance of Boverfs theory while leaving room for the abandonment of the too simple morphological form in which it was originally cast.
D. Fertilization in Plants
The investigation of fertilization in the plants has always lagged somewhat behind that of the animals, and even at the present time
1 '91, p. 431. a '98, 2, p. 217. 8 QCp. in.
2l6
FERTILIZATION OF THE OVUM
our knowledge of it is rather incomplete. It is, however, sufficient to show that the essential fact is everywhere a union of two germnuclei — a process agreeing fundamentally with that observed in animals. On the other hand, almost nothing is known regarding the centrosome and the archoplasmic or kinoplasmic structures; and most recent observations point to the conclusion that in the lowering plants and pteridophytes no centrosomes are concerned in fertilization. Many early observers from the time of Pringsheim ('55) onward described a conjugation of cells in the lower plants, but the union of germ-nuclei, as far as I can find, was first clearly made out in the flowering plants by Strasburger in 1877-78, and carefully described by him in 1884. Schmitz observed a union of the nuclei of the
B
Pig. io$. —Fertilization in Pilularia. [Campbkll.]
A, B. Early stages in the formation of the spermatozoid. C. The mature spermatozoid ; the
nucleus lies above in the spiral turns; below is a cytoplasmic mass containing starch-grains (cf.
the spermatozoids of ferns and of Mars ilia, Fig. 71). D. Archegonium during fertilization. In the centre the ovum containing the apposed germ-nuclei (0*1 9).
conjugating cells of Spirogyra in 1879, and made similar observations on other algae in 1884. Among other forms in which the same phenomenon has been described may be mentioned Gidigonium (Klebahn, '92), Vauchcria (Oltmanns, '95), Cystopus (Wager, '96), Sphcerotheca and Etysiphc{ Harper, '96), Fucus ( Farmer and Williams, '96, Strasburger, '97), Basidiobolus (Fairchild, '97), Pilularia (Fig. 105, Campbell, '88), Onoclca (Shaw, '98, 2), Zamia (Webber, '97, 2), and Lilium (Guignard, '91, Mottier, '97), Ginkgo (Hirase, '97). 1 In all of these forms and many others fertilization is effected by the union of a single paternal and a single maternal uninucleated cell, such as occurs throughout the animal kingdom. There are, however, some apparently well-determined exceptions to this rule occurring in the "compound" multinucleate oospheres of some of the lower
1 For unicellular forms see pp. 228, 280.
FERTILIZATION IN PLANTS
217
plants. In Albugo btiti (one of the Peronosporeae), for example, as shown by the recent work of Stevens ('99), the mature ovum contains about a hundred nuclei, and is fertilized by a multinucleate protoplasmic mass derived from the antheridium, each nucleus of the latter conjugating with one of the egg-nuclei. But although the conjugating bodies are here multinucleate, the germ-nuclei conjugate two and two (as is also the case in the multinucleate cysts of Actinospharium, p. 279); and the case therefore forms no real exception to the general rule that one paternal nucleus unites with one maternal.
Fij. 1°"- — Formation of the ovum and penetration of [lie pollen-lube in flowering plants. [Strasburc.er.]
A. Embryo-sac of Mtmotropa, showing ihc division that follows the two mat urat ion-divisions and produces the upper and lower "tetrads." B. The same, ready for fertilization, showing ovum (b). synergidce (j), upper and lower polar cells (/). and antipodal tells (a), C. Penetration of the pollen-tube (/./.) in Orchis ; e. ovum, with synergidie at either side. g.n. generative nuclei in the pollen-tube. D. Slightly later stage with generative nuclei entering the micropyle.
Whether a union of more than two germ-nuclei occurs in any of the lower plants is a question still disputed by botanists. 1 Such plural fusion is rendered a priori improbable by the observations thus far made upon the one-celled forms both in plants and in animals; and the known facts are sufficient to show that it must be, to say the least, an exceptional process.
In cases where the paternal germ-cell is a ciliated spermatozoid, as in Fucus, Pilnlaria, and the ferns and cycads, the germ-nuclei differ
1 Cf. Hattog, '91, '96, Trow, '95, Stevens, '99, Zimmerman, '96, and literature there cited.
2l8 FERTILIZATION OF THE OVUM
more or less widely at the time of union, the sperm-nucleus being smaller, more compact, and deeply staining (Figs. 105, 108), as is the case in such forms of fertilization as the echinoderm-egg. In the case of angiosperms all earlier observers, including Strasburger ('78, '84), Guignard ('91, 1), and Mottier ('97, 1), found the conjugating nuclei to be closely similar at the time of union. The recent observations of Guignard C99) and Nawaschin ('99) show, however, that even here the sperm-nucleus is smaller, more compact, and of different form (spindle-shaped) from the egg-nucleus (Fig. 107).
The ovum or oosphere of the flowering plant is a large, rounded cell containing a large nucleus and numerous minute colourless plastids from which arise, by division, the plastids of the embryo (chromatophores, amyloplasts). In the angiosperms the ovum forms one of the eight cells constituting the embryo-sac which morphologically represents the female prothallium or sexual generation of the pteridophyte and is itself embedded in the ovule within the ovary. 1 The male germ-cells are represented in the cycads by two ciliated spermatozoids (p. 175), in the angiosperms by two spindle-shaped "generative nuclei" which are suspected by Guignard and Nawaschin to be motile bodies, though no cilia were seen. These lie near the tip of the pollen-tube (Fig. 107), which is developed as an outgrowth from the pollen-grain and represents a rudimentary male prothallium or sexual generation. 2
The formation of the pollen-tube, and its growth down through the tissue of the pistil to the ovule, was observed by Amici ('23), Brongniart ('26), and Robert Brown C31); and in 1833-34 Corda was able to follow its tip through the micropyle into the ovule. 8 Strasburger first demonstrated the fact that the generative nucleus, carried at the tip of the pollen-tube, enters the ovum and unites with the eggnucleus, and the facts have been since carefully studied by himself, by Guignard, Mottier, Webber, Ikeno, Hirase, and a number of others. In the cycads, according to the last-named two observers, a single spermatozoid enters the egg f its nucleus soon fusing with that of the
1 The eight cells arc at first arranged in an upper and a lower " tetrad " of four cells each, the former including the ovum, two synergida.% and an •• upper polar cell," the latter a " lower polar cell " and three antipodal cells (Figs. 106, 107) ; cf. p. 263.
2 Cf. p. 264.
8 It is interesting to note that the botanists of the eighteenth century engaged in the same fantastic controversy regarding the origin of the embryo as that of the zoologists of the time. Moreland (1703), followed by Etienne Francois Geoff roy, Needham, and others, placed himself on the side of I^eeuwenhoek and the spermatists, maintaining that the pollen supplied the embryo which entered the ovule through the micropyle (the latter had been described by drew in 1672); and even Schleiden adopted a similar view. On the other hand, Adanson (1763) and others maintained that the ovule contained the germ which was excited to development by an aura or vapour emanating from the pollen and entering through the tracheae of the pistil.
FERTILIZATION IN PLANTS
egg (Fig. 108); and the earlier observers of the angiosperms, including Strasburger ('84, '88) and Guignard ('91, 1), likewise found that only one of the generative nuclei entered the embryo-sac. Guignard
Pig. 107. — Fertilizal
on in ih
lily.
[a from MOTTIER
Ihe o(he
■sfrom Guignard.]
A. Embryo-sac
ready for ferlil
tition
B.
Both general i
e nuclei
have enicre
the em
ac ; one isapproac
ingth
egg-nu
-r uniting with
C.Un
ion of ill
dge
and the
rrtiliied egg, showi
gfus
an of tin
. S. The fen
liied egg
dividing; b
low, div
f the endo;perm-n
r. entlosperm
' the oospli
». polar nuclei; p.t
poUe
•lube.
and Nawaschin have, however, recently made the remarkable discovery that in Lilium and Fritillaria both generative nuclei enter the embryo-sac. One of these conjugates with the egg-nucleus and
220 FERTILIZATION OF THE OVUM
thus effects fertilization (Fig. 107). The other conjugates with one of the polar nuclei (usually the upper), which then unites with the other polar nucleus (cf. p. 264). By division of the fertilized egg arises the embryo ; while by division of the compound nucleus resulting from the fusion of the polar nuclei .£$& and the second sperm nu cleus are formed the endosperm-cells, which serve for the nourishment of the embryo. This remarkable double copulation within the embryo-sac is without a parallel and is of wholly problematical meaning, but in no way contradicts the general rule regarding the union of two germ -nuclei toproduce the embryo. 1
1 As in the cue of animals (p. 176), Ihe germ-nuclei of phanerogams alio show marked differences in structure and staining- reaction before their union, though they ultimately become exactly equivalent. Thus, according to Rosen (•92, p. 443), on treatment by fuchsin-methyl-hlue the male germnucleus is " cyanophilous," the female " erythrophiloua," as described by Auerhach in animals. Strashurger, while confirming this observation in some cases, finds the reaction to lie inconstant, though the germ-nuclei usually show marked differences in their ■taining-capacity. These arc ascribed by Strasburger ('02, '94) to diftcrences in the conditions of nutrition ; by Zacharias and Schwtci lo corresponding differences in chemical (.omposiiinii, the male nucleus being
Pig. 108
- Fertilize
iio
ina
yea
, Zamia.
A. Sper
11a toxoid.
B.
The
sail
e after r
the cge. si
owing nuc
■) .
C. The ov
m shortly
ate
oflhesp
D. Union
of the germ-n
cilia- bearing
periphery
c)
in gen
ral ri
d the
female nucleus poorer. This distinction disappears during fertilization, and -Strasburger has observed, in the case of gymnosptrms (after treatment with a mixture of fuchsin-iodinc-green), that the paternal nucleus, which is al hrst "cyanophilous," becomes "erythrophilous," like Ihe egg-nucleus before the pollen-tube has reached the egg. Within the egg both stain exactly alike. These facts indicate, as Strasburger insists, that the differences between the germ-nuclei of plants are, as in animals, of a temporary and non-essential character.
FERTILIZATION IN PLANTS 221
The nature and origin of the achromatic elements involved in the fertilization of plants is still almost wholly in the dark. No observer has yet succeeded in observing either centrosomes or asters in the fertilization of the thallophytes, despite the fact that in some of these forms mitosis takes place with both these structures in a manner nearly analogous to that observed in animals. 1 In the cycads Zatnia and Cycas, Webber and Ikeno ('98) agree that the entire spermatozoid enters, but only the nucleus appears to be concerned in fertilization. The cilia-bearing band — a product of the blepharoplast, and, as described at page 175, probably the analogue of the middle-piece of the animal spermatozoon — remains near the egg-periphery, gives rise to no astral or other fibrillar formations, and apparently remains quite passive (Fig. 108).
In angiosperms, too, the evidence seems to show that no centrosomes are concerned in fertilization. Guignard ('91, 1), in a very detailed and clearly illustrated paper, gave an account of the centrosomes in the lily agreeing almost exactly with the "quadrille of centres" as described by Fol, 2 paternal and maternal centrosomes conjugating two by two. The later and very careful studies of Mottier and others have, however, entirely failed to confirm Guignard's results, the germ-nuclei fusing without the participation of centrosomes or astral formations, and after a time dividing, without centrosomes, in the manner characteristic of the higher plants. 8 Neither in the cryptogams has any one thus far succeeded in finding fertilization-centrosomes or asters at the time the germ-nuclei unite. Strasburger contributes, however, the interesting observation that in Fucus the cleavage-centrosomes afterward appear on that side of the cleavage-nucleus derived from the sperm-nucleus, which he believes from analogy may indicate the importation of a "new dynamic centre " into the egg by the spermatozoid. 4 Combining these facts with the phenomena involved in the origin of the spermatozoids, Strasburger suggests that the sperm-nucleus may import into the egg either a formed centrosome (probably thus in Fucus) or a certain quantity of " kinoplasm," which incites the mitotic phenomena in the absence of individualized centrosomes. 6 This view harmonizes with that suggested at pages 11 1 and 214, and we may perhaps here in the end find a reconciliation between the various types, not only of fertilization but also of mitosis, in plants and animals.
On their face the facts of fertilization in plants, especially in the phanerogams, seem to indicate that the stimulus to development is given by the paternal germ-nucleus. Nevertheless, the analogy of animal fertilization would lead us to expect that the fertilizing sub 1 Cf. p. 82. 8 Cf. p. 82. 6 '97, p. 420.
2 Cf. p. 210. 4 '97, p. 418.
222 FERTILIZATION OF THE OVUM
stance is contained not in the nucleus but in the cytoplasm — more specifically, in the case of spermatozoids, in the cilia-bearing body derived from the blepharoplast, which in its development so strongly suggests a centrosome (p. 172). Webber's and Ikeno's observations on the cycads are not necessarily fatal to this view; for, as I have shown (p. 188), the middle-piece in the echinoderm is likewise cast off and degenerates near the periphery of the egg, and the centrosome is a body far more minute. The possibility has been admitted that this centrosome may be formed de novo under the influence of the middle-piece, which itself perishes. In like manner it may also be possible that the primary stimulus in Zamia and like cases is given by the cilia-bearing body, even though this body itself disappears and the mitotic apparatus is not formed until long afterward.
E. Conjugation in Unicellular Forms
The conjugation of unicellular organisms possesses a peculiar interest, since it is undoubtedly a prototype of the union of germ-cells in the multicellular forms. Biitschli and Minot long ago maintained that cell-divisions tend to run in cycles, each of which begins and ends with an act of conjugation. In the higher forms the cells produced in each cycle cohere to form the multicellular body ; in the unicellular forms the cells separate as distinct individuals, but those belonging to one cycle are collectively comparable with the multicellular body. The validity of this comparison, in a morphological sense, is generally admitted. 1 No process of conjugation, it is true, is known to occur in many unicellular and in some multicellular forms, and the cyclical character of cell-division still remains sub judice. 2 It is none the less certain that a key to the fertilization of higher forms must be sought in the conjugation of unicellular organisms.
The difficulties of observation are, however, so great that we are as yet acquainted with only the outlines of the process, and have still no very clear idea of its finer details or its physiological meaning. The phenomena have been most closely followed in the Infusoria by Biitschli, Engelmann, Maupas, and Richard Hertwig, though many valuable observations on the conjugation of unicellular plants have been made by De Bary, Schmitz, Klebahn, and Overton. All these observers have reached the same general result as that attained through study of the fertilization of the egg ; namely, that an essential phenomenon of conjugation is a union of the nuclei of the conjugating- cells. Among the unicellular plants both the cell-bodies and the nuclei completely fuse. Among animals this may occur ; but in
c/.p. 58. 2 Cf. p. 178.
CONJUGATION IN UNICELLULAR FORMS
223
many of the Infusoria union of the cell-bodies is only temporary, and the conjugation consists of a mutual exchange and fusion of nuclei. It is impossible within the limits of this work to attempt more than a sketch of the process in a few forms.
We may first consider the conjugation of Infusoria. Maupas's beautiful observations have shown that in this group the life-history
Pit. 109. — Diagram showing the history of the ir mini, [Modified from Maufas.]
X and Y represent the opposed macro- and micron epresent degenerating nuclei ; black dots, persisiing in
during the conjugation
of the species runs in cycles, a long period of multiplication by celldivision being succeeded by an "epidemic of conjugation," which inaugurates a new cycle, and is obviously comparable in its physiological aspect with the period of sexual maturity in the Metazoa. If conjugation does not occur, the race rapidly degenerates and dies out ; and Maupas believes himself justified in the conclusion that conju
224 FERTILIZATION OF THE OVUM
gation counteracts the tendency to senile degeneration and causes rejuvenescence, as maintained by Biitschli and Minot. 1
In Stylonychia pustulata, which Maupas followed continuously from the end of February until July, the first conjugation occurred on April 29th, after 128 bi-partitions; and the epidemic reached its height three weeks later, after 175 bi-partitions. The descendants of individuals prevented from conjugation died out through " senile degeneracy," after 316 bi-partitions. Similar facts were observed in many other forms. The degeneracy is manifested by a very marked reduction in size, a partial atrophy of the cilia, and especially by a more or less complete degradation of the nuclear apparatus. In Stylonychia pustulata and Onychodromus grandis this process especially affects the micronucleus, which atrophies, and finally disappears, though the animals still actively swim, and for a time divide. Later, the macronucleus becomes irregular, and sometimes breaks up into smaller bodies. In other cases, the degeneration first affects the macronucleus, which may lose its chromatin, undergo fatty degeneration, and may finally disappear altogether {Stylonychia tnytilus), after which the micronucleus soon degenerates more or less completely, and the race dies. It is a very significant fact that toward the end of the cycle, as the nuclei degenerate, the animals become incapable of taking food and of growth ; and it is probable, as Maupas points out, that the degeneration of the cytoplasmic organs is due to disturbances in nutrition caused by the degeneration of the nucleus.
The more essential phenomena occurring during conjugation are as follows. The Infusoria possess two kinds of nuclei, a large macronucleus and one or more small micronuclei. During conjugation the macronucleus degenerates and disappears, and the micronucleus alone is concerned in the essential part of the process. The latter divides several times, one of the products, the germ-nucleus, conjugating with a corresponding germ-nucleus from the other individual, while the others degenerate as "corpuscules de rebut." The dual nucleus thus formed, which corresponds with the cleavagenucleus of the ovum, then gives rise by division to both macronuclei and micronuclei of the offspring of the conjugating animals (Fig. 109).
These facts may be illustrated by the conjugation of Paramecium caudatum, which possesses a single macronucleus and micronucleus, and in which conjugation is temporary and fertilization mutual. The two animals become united by their ventral sides and the macronucleus of each begins to degenerate, while the micronucleus divides twice to form four spindle-shaped bodies (Fig. no, A, B). Three of these degenerate, forming the "corpuscules de rebut," which play no further part. The fourth divides into two, one of which, the "female pronucleus/' remains in the body, while the other, or "male pronucleus," passes into the other animal and fuses with the female pronucleus (Fig. no, C-H). Each animal now contains a cleavagenucleus equally derived from both the conjugating animals, and the latter soon separate. The cleavage-nucleus in each divides three
1 Cf. p. 179.
Fig. no. — Conjugation of Param. Maufas.] (The macronuclei dolled ii
A. Micronuclei preparing for their three polar bodies or"corpusculcsde n Exchange of the germ-nuclei. E. The same, enlarged. H. Cleavage nuclei: nucleus has divided twice. J. After
tcium caudal**. [A-C, after R. HPRTWIC; D-K. after all the figures.)
but,"andone dividing germ-nucleus in each animal. D, same, enlarged. F. Fusion of the germ-nuclei. (7. The , (t) preparing for the first division. /. The cleavagethree divisions of the cleavage-nucleus: macronueleus Urging to form new macronuclei. The first fission soca
226
FERTILIZATION OF THE OVUM
times successively, and of the eight resulting bodies four become macronuclei and four micronuclei (Fig. no, H-K\ By two succeeding fissions the four macronuclei are then distributed, one to each of the four resulting individuals. In some other species the micronuclei are equally distributed in like manner, but in P. caudatutn the process is more complicated, since three of them degenerate, and the fourth divides twice to produce four new micronuclei. In either case at the close of the process each of the conjugating individuals
B
Fig. m. — Conjugation of Vorticellids. [Maupas.]
A. Attachment of the small free-swimming microgamete to the large fixed macrogamete; micronucleus dividing in each (Carchesium). B. Microgamete containing eight micronuclei; macrogamete four ( Vorticella). C. All but one of the micronuclei have degenerated as polar bodies or " corpuscules de rebut." D. Each of the micronuclei of the last stage has divided into two to form the germ-nuclei ; two of these, one from each gamete, have conjugated to form the cleavage-nucleus seen at the left; the other two, at the right, are degenerating.
has given rise to four descendants, each containing a macronucleus and micronucleus derived from the cleavage-nucleus. From this time forward fission follows fission in the usual manner, both nuclei dividing at each fission, until, after many generations, conjugation recurs. Essentially similar facts have been observed by Richard Hertwig and Maupas in a large number of forms. In cases of permanent conjugation, as in Vorticella, where a smaller microgamete unites with a larger macrogamete, the process is essentially the same, though the details are still more complex. Here the germ-nucleus derived from each gamete is in the macrogamete one-fourth and in the microgamete
CONJUGATION IN UNICELLULAR FORMS
22J
one-eighth of the original micronucleus (Fig. in). Each germnucleus divides into two, as usual, but one of the products of each degenerates, and the two remaining pronuclei conjugate to form a cleavage-nucleus.
The facts just described show a very close parallel to those observed in the maturation and fertilization of the egg. In both cases there is a union of two similar nuclei to form a cleavage-nucleus or its equivalent, equally derived from both gametes, and this is the progenitor of all the nuclei of the daughter-cells arising by subsequent divisions. In both cases, moreover (if we confine the comparison to the egg), the original nucleus does not conjugate with its fellow until it has by division produced a number of other nuclei all but one of which degenerate. Maupas does not hesitate to compare
Fit. im. — Conjugation of Ntxtiluca. [Ishikawa.]
A, Union of the gametes, apposition of the nuclei. B. Complete fusion of the gan ibove and below the apposed nuclei are the cenlrosomes. C. Cleavage-spindle, consisli
o separate halves.
these degenerating nuclei or " corpuscules de rebut " with the polar bodies (p. 181), and it is a remarkable coincidence that their number, like that of the polar bodies, is often three, though this is not always the case.
A remarkable peculiarity in the conjugation of the Infusoria is the fact that the gernt-nuclei unite when in the form of spindles or mitotic figures. These spindles consist of achromatic fibres, or "archoplasm," and chromosomes, but no asters or undoubted centrosomes have been thus far seen in them. During union the spindles join side by side (Fig. 1 10, G\ and this gives good reason to believe that the chromatin of the two gametes is equally distributed to the daughter-nuclei as in Metazoa. In the conjugation of some other Protozoa the nuclei unite while in the resting state; but very little is known of the process save in the cystoflagellate Noctiluca, which has been studied with some care by Cienkowsky and Ishikawa (Fig. 1 12). Here the conjugating animals completely fuse, but the nuclei are merely apposed and give rise each to one-half of
228
FERTILIZATION OF THE OVUM
the mitotic figure. At either pole of the spindle is a centrosome, the origin of which remains undetermined.
It is an interesting fact that in Noctiluca, in the gregarines, and probably in some other Protozoa, conjugation is followed by a very rapid multiplication of the nucleus followed, by a corresponding division of the cell-body to form " spores," which remain for a time closely aggregated before their liberation. The resemblance of this
Fig. IIJ — Conjugation of Spirogyta. [OVERTON.] A. Union of the conjugating cells (S. communis). B. The typical, though not invariable, mode of fusion in S. Wtdtri ; the chromatophore of the "female" cell breaks in Ihe middle, while thai of the " male " cell passes into the interval. C. The resulting zygospore filled with pyrenoids. before union of the nuclei. D. Zygospore after fusion of the nuclei and formation of the membrane.
process to the fertilization and subsequent cleavage of the ovum is particularly striking.
The conjugation of unicellular plants shows some interesting features. Here the conjugating cells completely fuse to form a "zygospore" (Figs. 113, 140), which as a rule becomes surrounded by a thick membrane, and, unlike the animal conjugate, may long remain in a quiescent state before division. Not only do the nuclei
SUMMARY AND CONCLUSION 229
unite, but in many cases the plastids also (chromatophores). In Spirogyra some interesting variations in this regard have been observed. In some species De Bary has observed that the long bandshaped chromatophores unite end to end so that in the zygote the paternal and maternal chromatophores lie at opposite ends. In 5. Weberi, on the other hand, Overton has found that the single maternal chromatophore breaks in two in the middle and the paternal chromatophore is interpolated between the two halves, so as to lie in the middle of the zygote (Fig. 113). It follows from this, as De Vries has pointed out, that the origin of the chromatophores in the daughter-cells differs in the two species, for in the former case one receives a maternal, the other a paternal, chromatophore, while in the latter, the chromatophore of each daughter-cell is equally derived from those of the two gametes. The final result is, however, the same; for, in both cases, the chromatophore of the zygote divides in the middle at each ensuing division. In the first case, therefore, the maternal chromatophore passes into one, the paternal into the other, of the daughter-cells. In the second case the same result is effected by two succeeding divisions, the two middle-cells of the fourcelled band receiving paternal, the two end-cells maternal, chromatophores. In the case of a Spirogyra filament having a single chromatophore it is therefore "wholly immaterial whether the individual cells receive the chlorophyll-band from the father or the mother" (De Vries). 1
F. Summary and Conclusion
All forms of fertilization involve a conjugation of cells by a process that is the exact converse of cell-division. In the lowest forms, such as the unicellular algae, the conjugating cells are, in a morphological sense, precisely equivalent, and conjugation takes place between corresponding elements, nucleus uniting with nucleus, cell-body with cell-body, and even, in some cases, plastid with plastid. Whether this is true of the centrosomes is not known, but in the Infusoria there is a conjugation of the achromatic spindles which certainly points to a union of the centrosomes or their equivalents. As we rise in the scale, the conjugating cells diverge more and more, until in the higher plants and animals they differ widely not only in form and size, but also in their internal structure, and to such an extent that they are no longer equivalent either morphologically or physiologically. Both in animals and in plants the paternal germ 1 De Vries's conclusion is, however, not entirely certain; for it is impossible to determine, save by analogy, whether the chromatophores maintain their individuality in the zygote.
cell loses most of its cytoplasm, the main bulk of which, and hence the main body of the embryo, is now supplied by the egg', and in the higher plants, the egg alone retains the plastids which are thus supplied by the mother alone. On the other hand, the paternal germ-cell is the carrier of something which incites the egg to development, and thus constitutes the fertilizing element in the narrower sense. There is strong ground for the conclusion that in the animal spermatozoon this element is, if not an actual centrosome, a body or a substance directly derived from a centrosome of the parent body and contained in the middle-piece. Boveri's theory, according to which fertilization consists essentially of the replacement of a missing or degenerating egg-centrosome by the importation of a sperm-centrosome, was stated in too simple and mechanical a form ; for the facts of spermatogenesis show conclusively that the spermatid-centrosome is not simply handed on unmodified by the spermatozoon to the egg, and the theory wholly breaks down in the case of the higher plants. But although the theory probably cannot be sustained in its morphological form, it may still contain a large element of truth when recast in physiological terms. Like mitosis, fertilization is perhaps at bottom a chemical process, the stimulus to development being given by a specific chemical substance carried in some cases by an individualized centrosome or one of its morphological products, in other cases by less definitely formed material. In the case of animals, we cannot ignore the historical continuity shown in the origin of the spermatid-centrosonies, the formation of the middle-piece, and the origin of the sperm-centrosomes and sperm-amphiaster in the egg f even though we do not yet know whether the sperm-centrosome is as such imported into the egg. And this chain of phenomena suggests that even in the higher plants, where no centrosomes seem to occur, the fertilizing substance, even if brought into the egg in an unformed state, may still be genetically related to the mitotic apparatus of the preceding division. 1
Through the differentiation between the paternal and germ-cells in the higher forms indicated above, their original morphological equivalence is lost and only the nuclei remain of exactly the same value. This is shown by their history in fertilization, each giving rise to the same number of chromosomes exactly similar in form, size, and staining-reactions, equally distributed by cleavage to the daughter-cells, and probably to all the cells of the body. We thus find the essential fact of fertilization and sexual reproduction to be a union of equivalent nuclei ; and to this all other processes are tributary.
1 Cf. Strasburger's view, p. 221.
As regards the most highly differentiated type of fertilization and development we reach therefore the following conception . From the mother comes in the main the cytoplasm of the embryonic body which is the principal substratum of growth and differentiation. From both parents comes the hereditary basis or chromatin by which these processes are controlled and from which they receive the specific stamp of the race. From the father comes the stimulus inducing the organization of the machinery of mitotic division by which the egg splits up into the elements of the tissues, and by which each of these elements receives its quota of the common heritage of chromatin. Huxley hit the mark two score years ago when in the words that head this chapter he compared the organism to a web of which the warp is derived from the female and the woof from the male. Our principal advance upon this view is the knowledge that this web is probably to be sought in the chromatic substance of the nuclei ; and perhaps we shall not push the figure too far if we compare the amphiaster to the loom on which the fabric is woven.
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