The Cell in Development and Inheritance

It is an important question whether the axial filament actually arises from the substance of the centrosome or is formed by differ- entiation from the cytoplasmic substance, after the fashion of an astral ray or spindle-fibre. Meves ('97, p. 117) accepts the latter alternative ; but the observations of Korff on Ifclix and of Suzuki on elasmobranchs seem to show clearly that, in these cases at least, the inner centrosome elon<;ates bodily to form an extremely long fila- ment traversing the greater part of the flagellum, and apparently of the same nature as the true axial filament developed from the outer or distal centrosome. This seems to establish a probability in favour of the first of the above alternatives, and to show that contractile elements may be directly derived from the centrosome-substance. If this be true, this substance is itself nearly related with " archo- plasm " ; and the origin of a centrosome dc 7iovo may be brought under the same category with the formation of archoplasm.^

While the origin of the spermatozoids has not yet been as fully investigated as that of the spermatozoa, recent researches have given good ground for the conclusion that essentially similar phenomena are involved in the two cases. All recent observ^ers are acfreed that the nucleus of the spermatozoid is directly derived from that of the spermatid, while the cytoplasm of the latter gives rise to the cilia and to certain other structures. The ])rincipal interest of the subject now lies in the origin of the cilia and their relation to the ** archoplasmic " or " kinoplasmic " structures of the mother-cell. Belajeff ('92, '94) found that in Cliara the cilia grow forth from a small, highly refract- ing body, taking an intense plasma-stain, that lies in the cytoplasma beside the nucleus. He afterward found the same body " which reminds one of a centrosome " in the developing spermatozoids of ferns and Equisetace^e (Fig. ^^), where it grows out into a band, lying in the anterior part of the spermatozoid, from which the cilia grow forth. Comparing these results with those of Hermann, Bela- jeff concluded ''that the deeply staining corpuscle" {i.e. the cen- trosome) "in the spermatids of the salamander and the mouse corresponds completely ta the deeply staining corpuscle in the sper- matogenic cells of the Characere, ferns, and Kquisetacece " ; that, furthermore, " the middle-piece of the spermatozoon represents the band which bears the cilia of the plant spermatozoid, while the tail- like flagella^ of the salamander or mouse represents the cilia." '^

1 QC p. 321. For the function of the centrosome in fertilization, see p. 208. 2 In the original " Faden " perhaps meant to designate the axial filament. This tallies with Strasburger's earlier conclusion that the cilia-bearing region consists of " kinoplasm " and corresponds to the middle-piece ('92, p. 139), but gives a still more definite basis of comparison. ^ The history of the centrosomc-like hod:\^?. {hlcpharoplasts of Web- ber, '97, 3) has been carefully followed out in Zcwiia and Giugko by Webber ('97), and in Cycas by Ikeno ('97, '98) with nearly similar results. In all these forms (Fig. Zj) the blepharoplasts appear in the

A. Developing pollen-tube, showing stalk-cell {s), vegetative cell {v) and generative cell (^). the latter with two blepharoplasts. B. Generative cell, somewhat later, with blepharoplasts and asters. C. The same in the prophases of division, showing breaking up of blepliaroplasts. D. The two spermatids formed by division of the generative cell ; blepharoplasts fragmented ; from these fragments arises the cilia-bearing band. E. Blepharoplast of Cycas, at a stage some- what later than Fig. C; cilia developing. F. Later stage; ciliated band (derived from tlie last stage) attached to a prolongation from the nucleus. G. Cilia-bearing band continuous. //. Nearly ripe spermatozoid with nucleus in the centre; ciliated band, sliown in section, forming a spiral. /. Slightly later stage, viewed from above, showing the spiral course of the band (cilia omitted).

penultimate cell-generation lying one on cither side the nucleus, and in earlier stages surrounded by a.stral radiations very closely resem- bUng those of a typical mitotic aster, and they lie opposite the poles 1 The "anterior" region of the spermatozoid thus corresponds to the "posterior" region of the spermatozoon, the confusion of terms having arisen from the fact that the former svi^ims with the ciUa-bearing region in front, the latter with the flagellum directed backward.

of the ensuing division-spindle. They seem, however, to have no part in the formation of the mitotic fic^ure or in division, and both .-/. Primary spermatogonium (two generations before the primary spermatocytes) in diyision showing centrosomes. B. Primary spermatocyte with pair of " blepharoplastoids " (centrosomes).' C. i^pindle of primary spermatocyte (f^rst maturation-division). D. Four of the eight secondary spermatocytes w,th blepharoplast. Z^-^ Prophase of second maturation-division. H Pair of spermatids (Gymno^^ram,„e) with blepharoplasts. /-J. Formation of the ciliated bandVrom the Dlepnaroplast A. Nearly ripe spermatozoid, showing ciliated band {b), nucleus, and "cyto- plasmic vesicle (the latter is ultimately cast off). E M. Spermatids of Equisetum. N Ripe spermatozoid from above, showing spiral ciliated band. O. Ripe spermatozoid of MarsiUa with very long spiral ciliated band. ". wm

Webber and Ikeno have produced apparently stron^r evidence ^ that they arise separately and de novo in the cytoplasm. After the ensu- ing division (by which the two spermatids are formed) the astral rays disappear, and the blepharoplast gives rise by a peculiar process to a long, spiral, deeply staining band, from which the ciha grow forth. The later studies of Shaw ('98, i) and Helajeff ('99) on the blepharo- plasts in Onoclea and Maisilia leave no doubt that these bodies are to be identified with centrosomes. In Marsilia Shaw first found the blepharoplasts lying at the poles of the spindle during the anaphase of the first maturation-division and very closely resembling centro- somes. Each blepharoplast, at first single, divides into two during the late telophase, and during the prophases of the second division the halves diverge to opposite poles of the nucleus and lie at the respec- tive spindle-poles. This account is confirmed by Belajeff, who shows further that during the prophases astral rays surround the blepharo- plasts, and a central spindle is formed between them (Fig. 88). Belajeff also finds centrosomes in all of the earher spermatogenic divisions. The blepharoplasts are thus proved to be, in one case at least, dividing organs w^hich in every way correspond to the centro- somes of the animal spermatocytes; and the justice of Belajeff's comparison is demonstrated. Shaw believed that the primary blepha- roplast, which by division gives rise to those of the two spermatids, arose de novo. He made, however, the significant observation that in Marsilia *' blepharoplastoids," exactly like the blepharoplasts, ap- pear at the spindle-poles of the preceding (antepenultimate) division, and that each of these divides into two in the late telophase.

These are said to disappear, without relation to the blepharoplasts which at a slightly later period are found at the spindle-poles of the first matu- ration division ; but in view of the demonstrated continuitv of the blepharoplasts during the second division we may well hesitate to accept this result, as well as Webber's conclusion regarding the independent and separate origin of the blepharoplasts in Zauiia. In any case the facts give the strongest ground for the conclusion that the formation of the spermatozoids agrees in its essential features with that of the spermatozoa, and for the expectation that the history of the achromatic structures in fertilization will yet be found to shnw an essential agreement in plants and animals.

It was pointed out by Ryder in 1883 that in the oyster the germ- nuclei stain differently in the two sexes; for if the hermaphrodite 1 Dr. Webber has kindly given me an opportunity to look through his beautiful prepa- rations. gland of this animal be treated with a mixture of saffranin and methyl- green, the egg-nuclei are coloured red, the s]:)erm-nuclei bluish green. A similar difference was afterward observed by Auerbach ('91) in the case of many vertebrate germ-cells, where the egg-nucleus was shown to have a special affinity for various red and yellow dyes (eosin, fuchsin, aurantia, carmine), while the sperm-nuclei were esj)e- cially stained with blue and green dyes (methyl-green, aniline-blue, hematoxylin). He was thus led to regard the chromatin of the ^'^^^^ as especially " erythrophilous," and that of the s])erm as " cyanophi- lous." That the distinction as regards colour is of no value has been shown bv Zacharias, Heidenhain, and others ; for staining-agents can- not be logically classed according to colour, but according to their chemical composition ; and a red dye, such as saffranin, may in a given cell show the same affinity for the chromatin as a green or blue dye of different chemical nature, such as methyl-green or hoema- toxylin. Thus Field has shown that the sperm-nucleus of Astcrias may be stained green (methyl-green), blue (haematoxylin, gentian violet), red (saffranin), or yellow (iodine), and it is here a manifest absurdity to speak of '' cyanophilous " chromatin ((/. p. 335). It is certainly a very interesting fact that a difference of staining-reaction exists between the two sexes, as indicating a corresponding difference of chemical composition in the chromatin ; but even this has been shown to be of a transitory character, for the staining-reactions of the germ-nuclei vary at different periods and are exactly alike at the time of their union in fertilization.

Thus Hermann has shown that when the spermatids and immature spermatozoa of the salamander are treated with saffranin (red) and gentian violet (blue),^ the chromatic network is stained blue, the nucleoli and the middle-piece red ; while in the mature spermatozoon the reverse effect is produced, the nuclei being clear red, the middle-piece blue. A similar change of staining- capacity occurs in the mammals. The great changes in the staining- capacity of the egg-nucleus at different periods of its history are de- scribed at pages 338-340. Again, Watase has observed in the newt that the germ-nuclei, which stain differently throughout the whole period of their maturation, and even during the earlier phases of fertilization, become more and more alike in the later phases, and at the time of their union show identical staining-reactions. ^ A very similar series of facts has been observed in the germ-nuclei of plants by Strasburger (p. 220). These and many other facts of like import demonstrate that the chemical differences between the germ-nuclei are not of a fundamental but only of a secondary character. They are doubtless connected with the very different character of the meta- bolic processes that occur in the history of the two germ-cells ; and

the difference of the staining-reaction is probably due to the fact that the sperm-chromatin contains a higher percentage of nucleinic acid, while the egg-chromatin is a nuclein containing a much higher percentage of albumin. Ballowitz, E. — Untersuchungen tiller die Struktur der Spermatozoen: i. {birds) vtals) Zeit. wiss. Zool.. LII. 1891. Belajeff, W. — Uber die Centrosomen in den spermatogenen Zellen : Ber. d. deuisch. bot. Ges., XVII., 6. 1899. Boveri, Th. — Uber Differenzierung der Zellkerne wahrend der Furchung des Eies

von Ascaris meg.: Anat. Anz. 1887. Id. — Die Entwicklung von Ascaris megalocephala mit besonderer Rucksicht auf die Kernverhaltnisse : Festschr. fur C. v. Knpffer. Jena, 1899. Brunn, M. von. — Beitrage zur Kenntniss der Samenkorper und ihrer Entwickelung bei Vogeln und Saugethieren : Arch. mik. Anat., XXXIII. 1889. Hacker, V. — Die Eibildung bei Cyclops und Camptocanthus : Zool. Jahrb.. \. 1892. (See also List V.) Hermann, F. — Urogenitalsystem : Struktur und Histiogenese der Spermatozoen :

Merkel nnd Bonnefs Ergebnisse, II. 1892. Ikeno, S. — Untersuchungen Uber die Entwickelung der Geschechtsorgane, t'/t:., bei Cycas : Jahrb. wiss. Bot.., XXXI L, 4. 1898. Kolliker, A. — Beitrage zur Kenntniss der Geschlechtsverhaltnisse und der Samen- fliissigkeit wirbelloser Tiere. Berlin. 1841. Leydig, Fr. — Beitrage zur Kenntniss des thierischen Eies im unbefruchteten Zu- stande : Zool. Jahrb., III. 1889. Moves, F. — Uber die Entwicklung der mannlichen Gescheclitszellen von Salaman-

dra : Arch. mik. A?iat.j XLVIII. 1896. Id. — t'ber Struktur und Histogenese der Samenfaden des Meerschweinchens : Arch. mik. Anat., LIV. 1899. Schweigger-Seidel, F. — Uber die Samenkorperchen und ihre Entwicklun-^ : Arch. und die Befruchtungsvorgange bei den Gymnospermen, Schwarmsporen, prianz- liche Spermatozoiden und das Wesen der Befruchtung. Fischer, Jena, 1892. Thomson, Allen. — Article '' Ovum,'' in Todd's Cyclopedia of Anatomy and Physi- ology. 1859. Van Beneden, E. — Recherches sur la composition et la signification de I'd-'uf : .Mem.

" 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, regardcil 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." IIUXLEY.i 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 exact dupli- cate 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 prob- able, 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 mat- ter derived from another eel I. This operation, known as fertiliza- tion 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 fer- tilization 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, Biitschli, Hertwig, and others, which assume that protoplasm tends gradually to pass into a state of increasingly sta- ble equilibrium in which its activity diminishes, and that fertilization restores it to a labile state, and hence to one of activity, through mix- ture with protoplasm that has been subjected to different conditions. BiitschU ('76) pointed out that the life-cycle of the metazoon is com-

parable to that of a protozoan race, a long series of cell-divisions being in each case followed by a mixture of protoplasms through conjuga- tion; and he assumed that, in both cases, conjugation results in reju- venescence 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. Mau- pas i^'^Z, '89), in his celebrated researches in the conjugation of Infu- soria, attempted to test this conclusion by following out continuously the hfe-history of various species through the entire cycle of their exist- ence. Though not yet adequately confirmed, and indeed opposed in some particulars by more recent work,^ 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 parthe- nogenesis the ^^% may develop without fertilization, and we do not know whether the tendency to " senescence " and the need for fer- tilization are primary attributes of living matter.

The foregoing views maybe 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 con- cerned with the process of variation. Long since suggested by Tre- viranus and more lately developed by Brooks ^ and Weismann '^ is the hypothesis that fertilization is a source of variation — a conclusion sug- gested by the experience of practical breeders of plants and animals. Weismann brings forward strong arguments against the rejuvenescence- theory, and regards the need for fertilization as a secondaiy acquisi- tion, the mixture of protoplasms to which it leads producing variations — or rather insuring their "mingling and persistent renewal"-' — 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

It is remarkable that we should still remain uncertain as to the physi- ological 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, espe- cially in the unicellular forms, where the key to the ultimate problem is undoubtedly to be sought.

Among the unicellular plants and animals, fertilization is effected bv means of conjugation, a process in which two individuals either fuse together permanently or unite temporarily and effect an exchange Fig. 89. — Fertilization of the egg of the snail, P/iysa. [Kostanecki and Wierzejski.] A. The eniire spermatozoon lies in the egg, its nucleus at the right, flagellum at the left, while the minute sperm-amphiaster occupies the position of the middle-piece. The first polar body has been formed, the second is forming. B. The enlarged sperm-nucleus and sperm-amphiaster lie near the centre; second polar body forming and the first dividing. The egg-centrosomes and asters afterward disappear, their place being taken by those of the spermatozoon.

of nuclear matter, after which they separate. /;/ (7// the higher forms fertilization consists in the permanent fusion of tivo germ-cells, one of paternal and ofie of maternal origin. We may first consider the fer- tilization of the animal Qgg, 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. Leeuwenhoek, whose pupil Hamm discovered the spermatozoa (1677), put forth the conjecture that the spermatozoon must pene- trate into the egg; and the classical experiments of Spallanzani on the frog's egg (1786) proved that the fertihzing 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 Qg^\ and it was described by Pringsheim a year later in one of the lower plants, Gldigoniuui. The first adequate description of the process was given by Hermann Fol, in 1879,^ though many earlier observers, from the time of Martin Barry ('43) onward, had seen the spermatozoon inside the egg-enve- lopes, or asserted its entrance into the ^^g.

In many cases the entire spermatozoon enters the (^^^^^ (mollusks, insects, nematodes, some annehds, Petromyson, axolotl, etc.), and in such cases the long flageilum may sometimes be seen coiled within the ^gg (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 Qgg. At or near the time of fertilization, the Qgg successively segments off at the upjxT pole two minute cells, known as t\\Q polar bodies {¥\gs. 89, 90, 116) or directive corpuscles, which degenerate and take no part in the subse- quent development. This phenomenon takes place, as a rule, imme- diately 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 ^gg is prepared for fertilization, and we may defer its consideration to the following chapter.

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 sperma- tozoon within the ^gg. Earlier observers had, it is true, paved the way by showing that, at the time of fertilization, the Q.gg contains two nuclei that fuse together or become closely associated before development begins. (Warneck, Biitschli, Auerbach, Wan IkMieden. Strasburger. ) Hertwig discovered, in the iigg of the sea-urchin {Toxopnenstes lividius\ that one of these nuclei belofii^s to the a^g, 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. /// every known case an

1 See Pllenogenie, pp. 124 ff., for a full historical account. essoitial pJuiiovioiou of fcrtilizatioi is tJic union of a spciin-uucItHSy of paternal origin, ivith an cgg-nuclcns, of maternal origin, to form tJie primary niielens of the emluyo. TJiis nucleus, knozi'n as tJie cleavage- or segmentation-nucleus, gives rise by divisioii to all t/w nuclei of the body, and hence ever]' nucleus of the child )nay contain nuclear substance y' derived from both parents. And thus Hcrtwi<^ was led to the conclu- sion ('84), independently reached at the same time by Strasburger, Kolliker, and Weismann, that the nucleus is the most essential cle- ment concerned in hereditary transmission.

This conclusion received a strong suj^port in the year 1883, through the s]:)lendid discoveries of Van Heneden on the fertilization of the thread-worm, Ascaris megalocephala, the <t^g of which has since ranked with that of the echinoderm as a classical object for the study of cell- problems. Van Beneden's researches especially elucidated the struc- ture 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 totallv disappears during fertilization, but that the two nuclei undergo a parallel series of structural changes which demonstrate their precise morj:)hological 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 bivalens) are essentially as follows (Fig. 90): After the entrance of the spermatozoon, and during the for- mation 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 chromo- somes (Fig. 90, D, E).

Meanwhile an amphiaster has been devel- oped which, with the four chromosomes, forms the mitotic figure for the first cleavage of the ovum, the chronmtic portion of zuhicJi lias been synthetically formed by the unioii of two equal germ-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. EacJi of these nuclei, tJici'cfore, receives exactly equal amounts of paternal and maternal chromatin.

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 Corouilla 4, in OpJiiostomiini 6, and in Filaroidcs 8. A little later Boveri ('90) showed that the law of numerical equality of the paternal and maternal chromosomes held ^ood for other groups of animals, being in the sea-urchin Echijius 9, in the worm Sagitta 9, in the medusa Tiara 14, and in the mollusk Ptcrotmclica 16 from each sex. Similar results were obtained in other animals and in j^lants, as first shown by Guignard in the lily ('91 ), where each sex contributes 12 chromosomes.

Fig. 91. — Germ-nuclei and chromosomes in the eggs of nematodes. [Carnov.] A. Egg of nematode parasitic in ScylUum ; the two germ-nuclei in apposition, each containing four chromosomes; the two polar bodies above. B. Egg of Filaroidcs; each germ-nucleus with eight chromosomes ; polar bodies above, deutoplasm-spheres below. In the onion the number is 8 (Strasburger) ; in the annelid OpJiryo- trocJia it is only 2 from each sex (Korschelt). In all these cases the number contributed by each is one-Jialf the number eliaracteristie 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 tJie number of chromosomes in sexually produced ori^anisms is akuays eve7i}

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 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 transmis- sion.

It is generally agreed that the amphiaster of the primary mitotic figure of the fertilized ovum arises from the egg-substance precisely Fig. 92. — Maturation and fertilization of the egg of the mouse. [SoHo ita.] A. The ovarian Qgg still surrounded by the follicle-cells and the membnuie (c./. zona pel- lucida) ; the polar spindle formed. D. Egg immediately after entrance of the spermatozoon (sperm-nucleus at cf). C. The two germ-nuclei (c?, 9) siill unequal; polar bodies above. D. Germ-nuclei approaching, of equal size. E. The chromosomes forming. F. The minute cleavage-spindle in the centre; on either side the paternal and maternal groui^s of chromosomes.