The Cell in Development and Inheritance

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 unite, but in many cases the plastids also (chromatophores). In Spirogyra some interesting variations in this regard have been ob- served. In some species De Bary has observed that the long band- shaped chromatophores unite end to end so that in the zvgote 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 four- celled band receiving paternal, the two end-cells maternal, chro- matophores. In the case of a Spirogyra filament having a single chromatophore it is therefore "wholly immaterial whether the indi- vidual cells receive the chlorophyll-band from the father or the mother" (De Vriesy

All forms of fertilization involve a conjugation of cells bv 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 (^r physiologically. Both in animals and in plants the paternal gerni-

1 De Vries's conclusion is, however, not entirely certain; for it is impossible to deter- mine, save by analogy, whether the chromatophores maintain their indivitluality in the zygote. cell loses most of its cytoplasm, the main bulk of which, and hence the main body of the embryo, is now supi)lied 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 (t^z to develoi)ment, and thus constitutes the fertilizing element in the narrower sense. There is strong ground for the conclusion that in the animal spermatozocin this element is, if not an actual centro- some, 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 replace- ment of a missing or degenerating egg-centrosomc 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 sjiermatid-centrosome is not simply handed on unmodified by the spermatozoon to the Qgg. and the theory wholly breaks down in the case of the higher ])lants. l^ut 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-centrosomes, the formation of the middle-piece, and the origin of the sperm-centro- somcs and sperm-amphiaster in the (tgg, 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 ^^^^ in an unformed state, may still be genetically related to the mitotic apparatus of the preceding division.^ Through the differentiation between the paternal and germ-cells in the higher forms indicated above, their original morphological ecjuivalence 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. Wc tJius find the essential fact of fertilization and sexual reproduction to be a union of equivalent nuclei ; and to tJiis all ot/ier processes are tributary.

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 pro- cesses are controlled and from which they receive the specific stamp of the race. From the father comes the stimulus inducing the organiza- tion of the machinery of mitotic division by which the ^^^^ splits up into the elements of the tissues, and by which each of the.se 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 chap- ter 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.

Van Beneden, E. — Recherches sur la maturation de roeuf, la fecondation et la divi- sion cellulaire : Arch. Biol., IV. 1883. Van Beneden and Neyt. — Nouvelles recherches sur la fecondation et la division mitosique chez TAscaride megalocephale : Bull. Acad. roy. de Belgique. III. 14. No. 8. 1887. Boveri, Th. — Uber den Anteil des Spermatozoon an der Teilung des Eies : Sitz.- Id. — Befruchtung: Merkel intd Bonnefs Ergeb7tisse,l. 1891. Id. — Uber das Verhalten der Centrosomen bei der Befruchtung des Seeigeleies. etc. :

Verhandl. Phys. Med. Ges. Wnrsburg, XXIX. 1895. Biitschli, 0. — Studien Uber die ersten Entwicklungsvorgange der Eizelle, it. s. zc. : Jahrb., XII. Fick, R. — tjber die Reifung and Befruchtuns; des Axolotleies : Zcitsclir. W'iss.Zodl., Griffin, B. B. — Studies on the Maturation, Fertilization, and Cleavage ot Thalassema and Zirphaea: Journ. Morph., XV. 1899. Guignard, L. — Nouvelles etudes sur la fecondation: Ann. d. Sciences nat. Bot., Hertwig, 0. — Beitrage zur Kenntniss der Bildung, Befruchtuiii; unci Teilunij iles

Kostanecki, K. v., and Wierzejski, A. — Uber das \'erhalten der sogen. achromati- schen Substanzcn iiu bctruchteten Ei : Arch. mik. A//a/.,XL\'\l. 2. 1896. Mark, E. L. — Maturation. Fecundation, and Segmentation of Umax ca/Jipcstris : sorio. \11. 18S9. Mead, A. D. The Origin and Heliaviour of the Centrosomes of tlie Annelid Egg: Journ. Morph., X I \'. 2 . 1 898. Ruckert, J. — Uber das Selbstiindigbleiben der vaterlichen und miittcrlichen Kern- substanz wiihrend der ersten Entwickiung des befruchteten Cyclops-Eies : Arch.

I'h.inerogamen, als Crundlage t'iir eine Theorie der Zeugung. Jena, 1884. Id. — L'ber Kern- und Zellteilung im Tflanzenreich, nebst einem Anhang liber Bcfnichtung. Jena, 18S8. (See Literature II.) Vejdovsky, F. — EntwickeUmgsgeschichtliche Untersuchungen, Heft i. Reifung, liefruchtung und Furchung des Rhynchehnis-Eies. Prag, 1888. Waldeyer, W. — Befruchlung und \'ererbung : / 'crh. Ges. deutsch. iVaturf. u. Aerzte^ \k\\. 1897. Wilson, Edm. B. — Atlas of Fertilization and Karyokinesis. A\^d' York, 1895. Zoja, R. — Stato Attuale degli Studi sulla Fecondazione : Boll. Scientif. di Pavia-,

" Es konimt also in der Generationenreihe der Keimzelle irgendwo zu einer Reduktion der ursprunglich vorhandenen Chromosomenzahl auf die Halfte, und diese Zr?///^';;. reduk- tion ist demnach nicht etwa nur ein theoretisches Postulat, sondern eine Thatsache." Van Beneden's epoch-making discovery that the nuclei of the con- jugating germ-cells contain each one-half the number of chromosomes characteristic of the body-cells has now been extended to so many plants and animals that it may probably be regarded as a universal law of development. The process by which the reduction in number is effected, forms the most essential part of the phenomena of matura- tion by which the germ-cells are prepared for their union. No phe- nomena of cell-life possess a higher theoretical interest than these. For, on the one hand, nowhere in the history of the cell do we find so unmistakable and striking an adaptation of means to ends or one of so marked a prophetic character, since maturation looks not to the present but to the future of the germ-cells. On the other hand, the chromatin-reduction suggests questions relating to the morphological constitution of nucleus and chromatin, which have an important bearing on all theories of the ultimate structure of living matter and now stand in the foreground of scientific discussion among the most debatable and interesting of biological problems.

Two fundamentally different views have been held of the manner in which the reduction is effected. The earlier and simpler view, which was suggested by Van Beneden and adopted in the earlier works of Weismann, Boveri, and others, assumed an actual degenera- tion or casting out of half of the chromosomes during the growth of the germ-cells — a simple and easily intelligible process. Later researches conclusively showed, however, that this view cannot be sustained, and that reduction is effected by a rearrauiienient and redis- tribution of the 7iuciear siibstance without loss of an\' of its essential constituents. It is true that a lars^e amount of chromatin is lost dur- ing the growth of the Qggr- It is nevertheless certain that this loss is not directly connected with the process of reduction ; for, as Hertwig

and others have shown, no such loss occurs during spermatogenesis, and even in the oogenesis the evid^ence is clear that an explanation must be sought in another direction. The attempts to find such an explanation have led to some of the most interesting researches of modern cytology ; and though only partially successful, they have raised manv new questions which ])romise to give in the end a deeper insight into some of the fundamental questions of cell-morphology. Kor this reason they deserve careful consideration, despite the fact that taken as a whole the subject still remains an unsolved riddle in the face of which we can only return again and again to Boveri's remark that whatever be its theoretical interpretation the numerical reduction of the chromosomes is itself not a theory but a fact.

Fig. 114. — Kormation of the polar bodies before entrance of the spermatozoon, as seen in the living ovarian egg of the sea-urchin, Toxopneustes (X 365). A. Preliminary change of form in the germinal vesicle. B. The first polar body formed, the second forming, C. The ripe egg, ready for fertilization, after formation of the two polar bodies (/. ^. 1. 2) ; c. the egg-nucleus. In this animal the first polar body fails to divide. For its division see Fig. 89.

The general phenomena of maturation fall under two heads : viz. Oflgcficsis, which includes the formation and maturation of the ovum, and spcrmatoi:;cn€sis, comprising the corresponding phenomena in case of the spermatozoon. Recent research has shown that maturation conforms to the same type in both sexes, which show as close a paral- lel in this regard as in the later history of the germ-nuclei. Stated in the most general terms, this parallel is as follows : ^ In both sexes the final reduction in the number of chromosomes is effected in the course of the last two cell-divisions, or Diatiiration-divisiojis, by which the definitive germ-cells arise, each of the four cells thus formed having but half the usual number of chromosomes. In the female but one

^ The parallel was first clearly pointed out by Plainer in 1 889, and was brilliantly demon- strated by Oscar Hertwig in the following year. of the four cells forms the " ovum " proper, while the other three, known as the polar bodies, are minute, rudimentary, and incapable of development (Figs. 89, 97, 114). In the male, on the other hand, all four of the cells become functional spermatozoa. This difference between the two sexes is probably due to the physiological division of labour between the germ-cells, the spermatozoa being motile and very small, while the ^gg contains a large amount of protoplasm and yolk, out of which the main mass of the embryonic body is formed. In the male, therefore, all of the four cells may become functional ; in the female the functions of development have become restricted to but one

■ Division-period (the number of divi- sions is much greater). Fig. 115. — Diagram showing the genesis of the egg. [After BOVERI.] of the four, while the others have become rudimentary (r/. p. 124). The polar bodies are therefore not only rudimentary cells (Giard, '76), but may further be regarded as abortive eggs — a view first put forward by Mark in 1881, and ultimately adopted by nearly all investigators.^ The evidence is steadily accumulating that reduction is accomplished by two maturation-divisions throughout the animal kingdom, even in the unicellular forms ; though in certain Infusoria an additional divi- sion occurs, while in some other Protozoa only one maturation-division has thus far been made out. Among plants, also, two maturation-

1 A beautiful confirmation of this view is given by Francottes's ('97) observations on a turbellarian, Prosthecerceus. The first polar body is here often abnormally larj^c, all grada- tions having been observed from the normal size up to cells nearly as large as the egg itself. Such polar bodies are occasionally fertilized ^nA develop into small gastrulas, first forming a single polar body like the second polar body of the egg. Here, therefore, two of the four cells are exceptionally capable of development. It may be added that Fol long ago observed the penetration of the small polar bodies by spermatozoa in the echinoderms; and this has been more recently observed by Kostanecki in mollusks.

divisions occur in all the hi^i^her forms (Muscineoe, pteridophytes, and phanerogams), and in some, at least, of the lower ones. Here, how- ever, the phenomena are complicated by the fact that the two divi- sions do not as a rule give rise directly to the four sexual germ-cells, but to four asexual spores which undergo additional divisions before the detinitive germ-cells are produced. In the flowering plants there are onlv a few such divisions, which give rise to structures within the pollen-tube or embryo-sac. In the archegoniate cryptogams, on the other hand, each spore gives rise, by repeated divisions, to a " sexual generation" (prothallium, etc.) that intervenes between the process of reduction and that of fertilization. The following account deals primarily with reduction in animals, the plants being afterward con- sidered.

I . Reduction ill tlic Female. Fonnation of the Polar Bodies As described in Chapter III., the Q.gg arises by the division of cells descended from the primordial egg-cells of the maternal organism, and these may be differentiated from the somatic cells at a very early period, sometimes even in the cleavage-stages. As development pro- ceeds, each primordial cell gives rise, by division of the usual mitotic type, to a number of descendants known as oogonia (Fig. 115), which are the immediate predecessors of the ovarian (tgg. At a certain period these cease to divide. Each of them then grows to form an ovarian egg, its nucleus enlarging to form the germinal vesicle, its cytoplasm becoming more or less laden with food-matters (yolk or deutoj)lasm), while egg-membranes may be formed around it. The ovum may now be termed the oocyte (Boveri) or ovarian (igg.

In this condition the egg-cell remains until near the time of fertili- zation, when the process of maturation proper — i.e. the formation of the polar bodies — takes place. In some cases, e.o;. in the sea-urchin, the polar bodies are formed before fertilization, while the (igg is still in the ovary. More commonly, as in annelids, gasteropods, nema- todes, they are not formed until after the spermatozoon has made its entrance ; while in a few cases one polar body may be formed before fertilization and one afterward, as in the lamprey-eel, the frog, and Ainphioxiis.^ In all these cases the essential phenomena are the same. Two minute cells are formed, one after the other, near the upper or animal pole of the ovum (Figs. 97, 116); and in many cases the first of these divides into two as the second is formed (Fig. 89).

A group of four cells thus arises, namely, the mature Q,gg, w^hich gives rise to the embryo, and three small cells or polar bodies which take no part in the further development, are discarded, and soon die without further change. The egg-nucleus is now ready for union with the sperm-nucleus. Fig. ii6. — Diagrams showing the essential facts in the maturation of the egg. The somatic number of chromosomes is supposed to be four. A. Initial phase; two tetrads have been formed in the germinal vesicle. />'. The two tctr.ids have been drawn up about the spindle to form the equatorial plate of the first polar mitotic figure. C. The mitotic figure has rotated into position, leaving the remains of the germinal vesicle at g.v. D. P'ormation of the first polar body ; each tetrad divides into two dyads. E. First polar body formed; two dyads in it and in the egg. F. lYeparation for the second division. G. Second polar body forming and the first dividing; each dyad divides into two single chromosomes. H. Final result; three polar bodies and the egg-nucleus (9). each con- taining two single chromosomes (half the somatic number) ; c. the egg-centrosome which now degenerates and is lost.

A Study of the nucleus during; these changes brings out the follow- ing facts. During the multijilication of the oogonia the number of chromosomes is the same as that occurring in the division of the somatic cells, and the same number enters into the formation of the chromatic reticulum of the germinal vesicle. During the formation of the polar bodies this number becomes reduced to one-half, the nucleus of each polar body and the egg-nucleus receiving the reduced number. In some manner, therefore, the formation of the polar bodies is connected with the process by which the reduction is ef- fected. The precise nature of this process is, however, a matter which has been certainly determined in only a few cases.

We need not here consider the history of opinion on this subject further than to point out that the early observers, such as Purkinje, \'on Haer, Bischoff, had no real understanding of the process and believed the germinal vesicle to disappear at the time of fertilization. To Hiitschli ('76), Hertwig, and Giard ('76, ^TT) we owe the discovery that the formation of the polar bodies is through mitotic division, the chromosomes of the equatorial plate being derived from the chro- matin of the germinal vesicle.^ In the formation of the first polar body the group of chromosomes splits into two daughter-groups, and this process is immediately repeated in the formation of the second icithout ati intcrccniiig reticular rcstiiig stage. The egg-nucleus therefore receives, like each of the polar bodies, one-fourth of the mass of chromatin derived from the germinal vesicle.

But alth(jugh the formation of the polar bodies was thus shown to be a process of true cell-division, the history of the chromosomes was found to differ in some very important particulars from that of the tissue-cells. The essential facts, which were first carefully studied in Ascaris by Van Beneden ('83, '87), and especially by Boveri ('87, i), are in a typical case as follows (Figs. 116, 117): As the &gg prepares for the formation of the first polar body, the chromatin of the ger- minal vesicle groups itself in a number of masses, each of which splits up into a group of four bodies united by linin-threads to form a "quadruple group" or tetrad (Vierergruppe). T/ie number of tetrads is ahuays oie-half the usual nundier of chromosomes. Thus in Ascaris {megalocephalay bivalens) \.\\c.gQrm\v\-a.\ vesicle gives rise to two tetrads, the normal number of chromosomes in the earlier divisions being four ; in the mole-cricket there are six tetrads, the somatic number of chromosomes being twelve ; in Cyclops the respectiv^e numbers are twelve and twenty-four (one of the most frequent cases); while in Artemia there are eighty-four tetrads and one hundred and sixty-

' The early accounts asserting the disappearance of the germinal vesicle were based on the fact that in many cases only a small fraction of the chromatic network gives rise to chromosomes, the remainder disintegrating and being scattered through the yolk. while the other passes into the polar body. Both the (i^g and the first polar body therefore receive each a number of dyads equal to one-half the usual number of chromosomes. The e^*; now proceeds at once to the formation of the second polar body without previous reconstruction of the nucleus. Tlach dyad is halved to form two single chromosomes, one of which, again, remains in the egg while its sister pas.ses into the polar body. Both the <igg and the second polar bodv accordingly receive two single chromosomes (one-half the usual number), each of which is one-fourth of an original tetrad group. From the two remaining in the (t^)^ a reticular nucleus, much smaller than the original germinal vesicle, is now formed. ^

Division-period (the number of divi- sions is much greater). Spermatids. Spermatozoa. Fig. ii8. — Diagram showing the genesis of the spermatozoon. [After BOVF.RI.] Essentially similar facts have now been determined in a consider- able number of animals, though, as we shall presently see, tetrad- formation is not of universal occurrence, nor is it always of the same type. For the moment we need only point out that the numerical reduction of chromatin-w^ri-jTi" takes place before the polar bodies are actually formed, through processes which determine the number of tetrads within the germinal vesicle. The numerical reduction is therefore determined in the grandmother-cell of the Q,^g. The actual divisions by which the polar bodies are formed merely distribute the elements of the tetrads.

^ It is nearly certain that the division of the first polar body (which, however, may be omitted) is analogous to that by which the second is formed, i.e. each of the dyads is similarly halved. Cf. Griffin, '99. The researches of Platner ('89), Boveri, and especially of Oscar Hertwig ('90, i) have demonstrated that reduction takes place in the male in a manner almost precisely parallel to that occiirrin^; in the female. Platner first suggested ('89) that the formation of the polar bodies is directly comparable to the last two divisions of the sperm mother-cells (spermatocytes). In the following year Boveri reached the same result in Ascaris, stating his conclusion that reduction in the male must take place in the '* grandmother-cell of the sperma- tozoon, just as in the female it takes place in the grandmother-cell of the Qgg,'' and that the egg-formation and sperm-formation reallv agree down to the smallest detail ('90, p. 64). Later in the same year appeared Oscar Hertwig's splendid work on the spermato- genesis of Ascaris, which established this conclusion in the most striking manner. Like the ova, the spermatozoa are descended from primordial germ-cells which by mitotic division give rise to the spermatogonia from which the spermatozoa are ultimatelv formed (Fig. 118). Like the oogonia, the spermatogonia continue for a time to divide with the usual (somatic) number of chromosomes, i.e. four in Ascaris vicgalocephala bivalens. Ceasing for a time to divide, they now enlarge considerably to form spermatocytes, each of which is morphologically equivalent to an unripe ovarian ovum, or oocyte. Each spermatocyte finally divides twice in rapid succession, giving rise first to two daughter-spermatocytes and then to four spermatids, each of which is directly converted into a single spermatozoon. The history of the cJiromatin in these two divisions is exactly parallel to that in the formation of the polar bodies (Figs. 119, 120). From the chromatin of the spermatocyte are formed a number of tetrads equal to one-half the usual number of chromosomes.

Each tetrad is halved at the first division to form two dyads which pass into the respective daughter-spermatocytes. At the ensuing division, which occurs with- out the previous formation of a resting reticular nucleus, each dyad is halved to form two single chromosomes which enter the resj^ec- tive spermatids (ultimately spermatozoa). From each spermatocyte,, therefore, arise four spermatozoa, and each sperm-nucleus receives half the usual number of single chromosomes. The parallel with the egg-reduction is complete.

These facts leave no doubt that the spermatocyte is the morpho- logical equivalent of the oocyte or immature ovarian Q.gg, and that the group of four spermatozoa to which it gives rise is equivalent to the ripe ^g% plus the three polar bodies. Hertwig was thus led to the following beautifully clear and simple conclusion : '* The polar bodies are abortive eggs which are formed by a final process of division from the egg-mother-cell (oocyte) in the same manner as the spermatozoa are formed from the sperm-mother-cell (spermatocyte). Hut while in the latter case the products of the division are all used as functional spermatozoa, in the former case one of the products

Pig. 119. — Diapranis showing the essential facts of reduction in the male. The somatic num- ber of thromosomes is supposed to be four. . /. /?. Division of one of the spermatogonia, showing the full number (four) of chromosomes. C. Primary spermatocyte preparing for'division ; the chromatin forms two tetrads. D. It. F. First division to form two secondary spermatocytes each of which receives two dyads. G. H. Division of the two .secondary spermatocytes to form four spermatids. Each of the latter receives two single chromosomes and a centrosome which passes into the middle-piece of the spermatozoon.