The Cell in Development and Inheritance

Boveri himself has suggested ^ that the ^gg may be incited to development by a specific chemical substance carried by the sperma- tozoon, and the same view has been more recently urged by IMead,- while Loeb's recent remarkable experiments on sea-urchins ('99) show that the ^^g may in this case {Arbacia) undergo complete parthe- nogenetic development as the result of artificial chemical stimulus.-"^ Assuming such a substance to exist, by what part of the spermato- zoon is it carried .-* It is possible that the vehicle may be the nucleus, which forms the main bulk of that which enters the Q.gg ; and this view seems to be supported by what is at present known of fertiliza- tion in the plants (p. 221). Yet when we regard the facts of fertili- zation 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 Qgg by the middle-piece, and is localized in the sperm-centro- somes which are conveyed to the nuclear poles during the am phi- aster-formation. Accepting such a view, we could gain an intelligible view of the genetic relation between spermatid-centrosome, middle- piece, sperm-centrosome, and cleavage-centrosomes, without commit- ting 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 Boveri's theory while leaving room for the abandonment of the too simple morphological form in which it was originally cast.

The investigation of fertilization in the plants has always lagged somewhat behind that of the animals, and even at the i)resent time uur knowledge of it is rather incomplete. It is, however, sufficient to show that the essential fact is everywhere a union of two germ- j^^jclci — 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. Manv earl v observers from the time of Pringsheim ('55) onward described a conjugation of cells in the lower plants, but the union of germ-Huclci, as far as I can find, was first clearly made out in the Howering plants by Strasburger in xZ'j'j-'j'^, and carefully described by him in 1884. Schmitz observed a union of the nuclei of the

A. /?. Early stages in tlie 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 spermatozoJds of ferns and of Marsilia, Fig. 71). D. Archegonium during fertilization. In the centre the ovum containing the apposed germ-nuclei (d", 9 ). conjugating cells of Spirogyra in 1879, and made similar observations on other algx in 1S84, Among other forms in which the same phenomenon has been described may be mentioned Gidigoniuin (Klebahn, '92), Vauchcria (Oltmanns, '95), Cystopus (Wager, '96), Splurrothcca and /:";;;'.v////r (Harper, '96), /v/^/m' ( Farmer and Williams, '96, Strasburger, '97), Inisidioboliis (Fairchild, '97), Pilularia (Fig. 105, Campbell, '88), Onoclca (Shaw, '98, 2), Zamia (Webber, '97, 2), and Lilitim (Guignard, '91, Mottier, '97), Ginkgo (Hirase, '97).^ 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

plants. In Albugo bliti (one of the Peronosporece), 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 proto- plasmic mass derived from the anthcridium, each nucleus of the latter conjugating with one of the egg-nuclei. But although the conjugat- ing bodies are here multinucleate, the germ-nuclei conjugate two and two (as is also the case in the multinucleate cysts of ActinospJuEriuDi, p. 279); and the case therefore forms no real exception to the general rule that one paternal nucleus unites with one maternal.

Fig. 106. — Formation of the ovum and penetration of the pollen-tube in flowering plants. [Strasburger.] A. Embryo-sac of Monotropa, showing the division that follows the two maturation-divisions and produces the upper and lower " tetrads." B. The same, ready for fertihzation. sliowing ovum {0), synergidae {s), upper and lower polar cells (/), and antipodal cells (<j). C. I'enetration of the pollen-tube (/./.) in Orchis ; o. ovum, with synergidas 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. ^ Such i)lural 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 Fiicus, Pilularia, and the ferns and cycads, the germ-nuclei differ

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 cchinoderm-egg. In the case of angiosperms all earlier observers, including Strasburger ('78, '84), Guignard ('91. i ), and Mottier (97, i ), found the conjugating nuclei to be closely similar at the time of union. The recent obser- vations of Guignard ('99) and Nawaschin (99) show, however, that even here the sperm-nucleus is smaller, more compact, and of differ- ent form (spindle-shaped) from the egg-nucleua (Fig. 107).

The ovum or oosphere of the flowering ])lant 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 morphologi- cally represents the female prothallium or sexual generation of the j)teridoj)hyte and is itself embedded in the ovule within the ovary. ^ The male germ-cells are represented in the cycads by two ciliated spcrmatozoids (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. ^

The formation of the pollen-tube, and its growth down through the tissue of the ])istil to the ovule, was observed by Amici ('23), Ikongniart ('26), and Robert Brown ('31); and in 1833-34 Corda was able to follow its tip through the micropyle into the ovule.'^ Stras- burger first demonstrated the fact that the generative nucleus, carried at the tip of the pollen-tube, enters the ovum and unites with the egg- nucleus, and the facts have been since carefully studied by himself, by Guignard, Mottier, Webber, Ikeno, Mirase, and a number of others. In the cycads, according to the last-named two observers, a single spermatozoid enters the egg, its nucleus soon fusing with that of the

' The eight cells are at first arranged in an upper and a lower " tetrad " of four cells each, the former including the ovum, two synergida-, and an " upper j)olar cell," the latter a "lower |X)lar cell" and three antipodal cells (Figs. io6, 107); cj. p. 263. ' It is interesting to note that the botanists of the eighteenth century engaged in the same fantastic controversy regarding the origin of the enibryo as that of the zoologists of the time. Morcland (1703), followed by Etienne Francois Geoff roy, Needham, and others, placed himself on the side of Leeuwenhoek and the spermatists, maintaining that the pollen 5,.. .,1. .1 ji^p embryo which entered the ovule through the micropyle (the latter had been «!■ - ; '1 by CIrew in 1672) ; and even Schleiden adopted a similar viey'. 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 trachea of the pistil.

egg (Fig. 108); and the earlier observers of the angiosperms, includ- ing Strasburger ('84, '88) and Guignard ('91, i), likewise found that only one of the generative nuclei entered the embryo-sac. Guignard Fig. 107. — Fertilization in the lily. \^D from MOTTIEK, the others from GflGNARD.] A. Embryo-sac, ready for fertilization. B. Both generative nuclei have entered the embrvo- sac ; one is approaching the egg-nucleus, the other uniting with the upjier polar nucleus. C'. Union of the germ-nuclei ; below, union of the second generative nucleus and the two polar nuclei, D. 'I"he fertilized egg, showing fusion of the germ-nuclei. E. The fertilized egg dividing; below, division of the endosperm-nuclei, a. antipodal cells ; e. endosperm-nuclei; t'. the oosphere or ovum; /. polar nuclei ; /. t. pollen-tube.

and Nawaschin have, however, recently made the remarkable dis- covery that in Liliuni and Fntillaria both generative nuclei enter the embryo-sac. One of these conjugates with the egg-nucleus and thus effects fertilization ( Fig. 107). The other coujugatcs with one of the poiar uuciei {\is\\2l\\\ the upper), which then unites with the other polar nucleus (</. p. 264). By division of the tertiHzed egg arises the embryo ; while hv division of the compound nucleus resulting from the

fusion of the polar nuclei and the second sperm nu- cleus are formed the endo- sperm-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 to produce the embryo.^ 1 As in the case of animals (p. 176), the germ-nuclei of phanero- gams also show marked differ- ences in structure and staining-reac- tion before their union, though they ultimately become exactly equiva- lent. Thus, according to Rosen ('92, p. 443), on treatment by fuchsin-methyl-blue the male germ- nucleus is " cyanophilous," the female " erythrophilous,'' as de- scribed by Auerbach in animals. Strasburger, while confirming this observation in some cases, finds the reaction to be inconstant, though the germ-nuclei usually show marked differences in their staining-capac- ity. These are ascril^cd by Stras- burger ('92, '94) to differences in the conditions of nutrition ; by /acharias and Schwarz to corre- sponding differences in chemical composition, the male nucleus i)eing in general richer in nuclcin,and the female nucleus poorer. This dis- tinction disappears during fertiliza- liun, and Strasburger has observed, in the case of gymnosperms (after treatment with a mixture of fuchsin-iodine-green), that the paternal nucleus, which is at first " cyanophil- ous," .becomes " er)throphilous," like the 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.

A. Spermatozoid. B. The same after entrance into the egg. showing nucleus («) and cilia-bearing Ijand (^r). D. Union of the germ-nuclei, cilia-bearing band near periphery (V). 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.^ In the cycads Zamia and Cyras, Webber and Ikeno ('98) agree that the entire spermato- zoid enters, but only the nucleus appears to be concerned in fertiliza- tion. 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-peripherv, gives rise to no astral or other fibrillar formations, and apparentl)' remains quite passive (Fig. 108).

In angiosperms, too, the evidence seems to show that no centro- somes are concerned in fertilization. Guignard ('91, i ), in a very detailed and clearly illustrated paper, gave an account of the centro- somes in the lily agreeing almost exactly with the " quadrille of centres" as described by Fol,^ paternal and maternal centrosomes conjugating two by two. The later and very careful studies of Mot- tier and others have, however, entirely failed to confirm Guignard 's results, the germ-nuclei fusing without the participation of centro- somes or astral formations, and after a time dividing, without centro- somes, in the manner characteristic of the higher plants.'^ Neither in the cryptogams has any one thus far succeeded in finding fertiliza- tion-centrosomes or asters at the time the germ-nuclei unite. Stras- burger contributes, however, the interesting observation that in Fncns 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 Qgg by the spermatozoid.'* Combining these facts with the phenomena involved in the origin of the spermatozoids, Strasburger suggests that the sperm-nucleus may import into tlie Qgg either a formed centrosome (probably thus in Finns) or a cer- tain quantity of ** kinoplasm," which incites the mitotic phenomena in the absence of individualized centrosomes.^ This view harmo- nizes with that suggested at pages in 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 develojiment is given by the paternal germ-nucleus. Nevertheless, the analogy of animal fertilization would lead us to expect that the fertilizing sub- 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 I keno'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 ^^^, and the ccntro- sonie is a body far more minute. The possibility has been admitted that this centrosome may be formed dc novo under the influence of the middle-piece, which itself ]:)erishes. In like manner it may also be possible that the primary stimulus in Zai)iia 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.

The conjugation of unicellular organisms possesses a peculiar inter- est, 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 pro- duced 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 multi- cellular body. The validity of this comparison, in a morphological sense, is generally admitted.^ 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 jiidicc? 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 Hutschli, ICngclmann, Maupas, and Richard Hertwig, though many valuable observations on the -conjugation of unicellular plants have been made by De Hary, Schmitz, Klebahn, and Overton. All these obser\-ers have reached the same general result as that attained through study of the fertilization of the o.^^, ; namely, that an essen- tial phenomenon of conjugation is a luiion of tJic nuclei of the conju- gating^ cells. Among the unicellular plants both the cell-bodies and the nuclei completely fuse. Among animals this may occur ; but in

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 Fig. 109. — Diagram showing the history of the micronuclei during the conjugation of Para- moecium. [Modified from Maupas.]

♦ -V and F represent the opposed macro- and micronuclei in the two respective gametes; circles represent degenerating nuclei ; Vjlack dots, persisting nuclei. of the species runs in cycles, a long period of multiplication by coil- division being succeeded by an *' epidemic of conjugation." which inaugurates a new cycle, and is obviously comparable in its physio- logical aspect with the period of sexual maturity in the Metazoa. If conjugation does not occur, the race rapidly degenerates and dies out ; and Maupas beheves himself justified in the conclusion that conju-

o-ation counteracts the tendency to senile degeneration and causes rejuvenescence, as maintained bv Hiitschli and Minot.^ In StvloHVi/iia pustulata. which Miiupas followed continuously from the end of February until Juh", the first conjugation occurred on April 29th, after 128 bi-parti- tii.ns; and the epidemic reached its iieight three weeks later, after 175 bi-partitions. 'I'he descendants of individuals prevented from conjugation died out through '-senile iKueneracy." after 316 bii)artitions. 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 ihxradation of the nuiUar apparatus. I n Stylonycliia pustulata and Onyiliodromus ^i^randis this process especially atTects 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 aflects the macronucleus, which may lose its chromatin, undergo fatty degeneration, and may finally disappear altogether {StylonycJiia tnvtilus), af'ter 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 macnmuclcHS and one or more small micronnclei. During conjuga- tion the macronucleus degenerates and disappears, and the micronu- cleus alone is concerned in the essential part of the process. The latter divides several times, one of the products, the gcrm-imclcus, conjugating with a corresponding germ-nucleus from the other indi- vidual, while the others degenerate as "corpuscules de rebut." The dual nucleus thus formed, which corresponds with the cleavage- nucleus 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 Paramaxium caiidatiim, 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 macronu- cleus of each begins to degenerate, while the micronucleus divides twice to form four spindle-shaped bodies (Fig. i 10, A, />). 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. 1 10, C-H ). Each animal now contains a cleavage- nucleus equally derived from both the conjugating animals, and the latter soon separate. The cleavage-nucleus in each divides three

Fig. no. — Conjugation of Paramascium caudatum. [.-/-C, after R. Hertwic. ; D-K, after Maupas.] (The macronuclei dotted in all the figures.) A. Micronuclei preparing for their first division. R. Second division. C. Third division; three polar bodies or " corpuscules de rebut," and one dividing germ-nucleus in each animal. D. Exchange of the germ-nuclei. E. The same, enlarged. F. Fusion of the germ-nuclei. G. The same, enlarged. H. Cleavage-nucleus, {c) preparing for the first division. /. The cleavage- nucleus has divided twice, y. After three divisions of the cleavage-nucleus; macronucleus breaking up. K. Four of the nuclei enlarging to form new macronuclei. The first fission soon

times successively, and of the eight resulting bodies four become macronuclei and four micronuclei (Fig. no, H-K). By two suc- ceeding fissions the four macronuclei are then distributed, one to each of the four resulting individuals. In some other species the micro- nuclei are equally distributed in like manner, but in J\ caudatnni the j)rocess is more complicated, since three of them degenerate, and the fourth divides twice to produce four new micronuclei. In cither case at the close of the process each of the conjugating individuals

Fig. III. — Conjugation of Vorticellids. [MaL'TAS.] A. Attachment of the small frec-swimining microgamete to the large fixed macrogamete ; micronucleus dividing in each (Carc/iesiuin). B. Microgamete containing eight micronuclei; ; • imete four ( I'orticella). C. All but one of the micronuclei have degenerated as polar , . t>r"corpuscules de rebut." D. liach of the micronuclei of the last stage has divided into . 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 divid- ing at each fission, until, after many generations, conjugation recurs. Fssentially similar facts have been observed by Richard Hertwig and Maupas in a large number of forms. In cases of permanent conjugation, as in l\)rticclla, where a smaller microgamete unites with a larger ^nacrogamcte, 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

one-eighth of the original micronucleus (Fig. i 1 1 ). Each germ- nucleus 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 ^g^^. 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 pro- genitor of all the nuclei of the daughter-cells arising by subsequent divisions. In both cases, moreover (if we confine the comparison to the ^gg), 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

A. Union of the gametes, apposition of the nuclei. D. Complete fusion of the gametes. Above and below the apposed nuclei are the centrosomes. C. Cleavage-spindle, consisting of two 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 tJie germ-nuclei ujiite ivJien in tJic form of spimiies or mitotic figures. These spindles consist of achromatic fibres, or *'archoplasm," and chromosomes, but no asters or undoubted cen- trosomes have been thus far seen in them. During union the spindles join side by side (Fig. lio, G\ and this gives good reason to believe that the chromatin of the two gametes is equally distrib- uted 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 Xocti- luca, which has been studied with some care by Cienkowsky and Ishikawa (Fig. 112). Here the conjugating animals completely fuse, but the nuclei are merely apposed and give rise each to one-half of

the mitotic ^^wxo.. At either pole of the spindle is a centrosome, the orif(in of which remains undetermined. It is an interestini; fact that in Xoctilnca, in the gregarines, and probably in some other Protozoa, conjugation is followed by a very rapid multiplication of the nucleus followed, by a corresponding divi- sion of the cell-body to form "spores," which remain for a time closely aggregated before their liberation. The resemblance of this A. Union of the conjugating cells {S. communis). B. The typical, though not invariable, mode of fusion in S. Weber i ; the chromatophore of the "female" cell breaks in the middle, while that 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.