The Cell in Development and Inheritance

nucleus Hke those described above, while the other assumes the structure of a radiating attraction-sphere containing a central granule (centrosome .''), and his observations suggest that the two bodies in question may have a common origin (Fig. 81). In none of these cases do the astral radiations, surrounding this body, seem to have any connection with cell-division, and it is probable that a careful comparison of their physiological significance here, in leucocytes, and in mitotic division, may give us a better under*^tand- ing of the general significance of astral formations in protoplasm.

The fate and physiological significance of the yolk-nucleus arc still to a considerable extent involved in doubt. In many cases i1 breaks up into smaller and smaller ^i^raniiles {Lunibricus, JlToIgida, Pliolciis, some myriapods, Antcdou), which scatter throu^^h the cyto- plasm and are believed by many observers ( Halbiani, Mertens, \Vill, Calkins, Crampton, Nemec), following the earlier views of Allen Thomson, to become directly converted into deutoplasm-spheres (Fig. 80). Other observers (Van Hambeke, Foot, Stiihlman, and others) adopt the original view of Siebold, that the fragments of the yolk-nucleus are absorbed or converted into protoplasmic elements and thus only indirectly contribute to the yolk. In still other cases {^c.g. the "vitelline body" of Lii)iiilus) the yolk-nucleus does not fragment, but seems to serve as a centre about which new deutoplasmic material is formed. A review of the general subject shows that we are justified only in the somewhat vague conclusion that the yolk-nucleus is probably involved in some manner in the general cytoplasmic growth ; and that the facts strongly suggest, though they hardly yet prove, that at least some forms of yolk-nuclei are products of the nuclear activity and form a connecting link between that activity and the constructive processes of the cyto- plasm. That the yolk-nuclei have no very definite morphological value, and that they are not necessary to growth, seems to be shown by Henneguy's observation, that in the eggs of vertebrates it is in some forms invariably present, in others only rarely, and in still others is quite wanting ('96, p. 162). If this be the case, we must conclude that the yolk-nucleus consists of material that contributes to the constructive process, but is not necessarily localized in a definite body.

As to its exact role we are, as Henneguy has said, reduced to mere hypotheses.^ The facts indicate that this material is a prod- uct of the nuclear activity, and that it may in some cases contribute directly to formed elements of the cytoplasm. It is probable, how- ever, that beyond this the yolk-nucleus may su])ply materials, perhaps ferments, that play a more subtle part in the constructive process, and of whose physiological significance we are quite ignorant. The whole subject seems a most interesting and important one for further study of the actions of the cell in constructive metabolism, and it is to be hoped that further research will place the facts in a clearer light.

(a) General. — The relation of the various parts of the sperma- tozoon to the structures of the spermatid is one of the most interesting questions in cytology, since it is here that we must look for a basis of interpretation of the part played by the sperma- tozoon in fertilization. Obviously the most important of the questions, thus suggested, is the source of the sperm-nucleus and centrosome, though the relation of the other parts to the spermatid- cytoplasm involves some interesting problems.

Owing to the extreme minuteness of the spermatozoon, the changes involved in the differentiation of its various parts have always been, and in some respects still remain, among the most vexed of cytological questions. The earlier observations of Kolliker, Schweigger-Seidel, and La Valette St. George, already mentioned, established the fact that the spermatozoon is a cell; but it required a long series of subsequent researches by many observers, foremost among them La Valette St. George himself, to make known the general course of spermatogenesis. This is, briefly, as follows : From the primordial germ-cells arise cells known as spennatogonia} which at a certain period pause in their divisions and undergo a con- siderable growth. Each spermatogonium is thus converted into a spermatocyte, which by two rapidly succeeding divisions gives rise to four spermatozoa, as follows.^ The primary spermatocyte first divides to form two daughter-cells known as spermatocytes of the second order or sperm-mother-cells. Each of these divides again — as a rule, without pausing, and without the reconstruction of the daughter-nuclei — to form two speimatids or sperm-cells. Each of the four spermatids is then directly transformed into a single sperma- tozoon, its nucleus becoming very small and compact, its cytoplasm giving rise to the tail and to certain other structures. The number of chromosomes entering into the nucleus of each spermatid and spermatozoon is always one-half that characteristic of the tissue-cells, and this reduction in number is in most, if not in all, cases effected during the two divisions of the primary spermatocyte. The reduction of the chromosomes, which is the most interesting and significant feature of the process, will be considered in the following chapter, and we are here only concerned with the transformation of the sper- matid into the spermatozoon.

All observers are now agreed that the nucleus of the spermatid is directly transformed into that of the spermatozoon, the chromatin becoming extremely compact and losing, as a rule, all trace of its reticular structure. It is further certain that in some cases at least the spermatid-centrosome passes into, or gives rise to, a part of the middle-piece, and that from it the axial filament grows out into the tail. The remaining structures arise, as a rule, from the cytoplasm, and both the acrosome and the envelope of the axial filament otten show a direct relation to the remains of the achromatic figure ( " ar-

1 The terminology, now almost universally adopted, is due to La \'alette St. George. Cj. Fig. 1 1 8. 2 See Fig. 119. choplasm " or " kinoplasm ") which is found in the spermatid in the form of a sphere (sometimes an attraction-sphere) or *' Nebenkern " or both. Apart from the nuclear history, these facts have been definitely determined in only a few cases, and much confusion still exists in the accounts of different observers. Thus a number of investigators (r.^. Platner, I'^ield, l^enda, Julin, Prenant, Xiessinor) have asserted that the centrosome passes into the acrosome, instead of

Fig. 82. — Formation of the spermatozoon in an insect, Anasa. [Paulmier.] A. Telophase of secondary spermatocyte-division, showing extra chromosome (small dyad of Fig. 127) below. B. Reconstitution of the nuclei. C. Spermatid witli Nebenkern (A^) and acrosome ia). D. Nebenkern double, with centrosome between the two halves. E. F. G. Elon- gation of the spermatid, outgrowth of axial filament, migration of acrosome, H. Giant spermatid (double size) with two centrosomes and axial filaments. /. Giant spermatid (quadruple size) with four centrosomes and axial filaments.

the middle-piece — a result which .stands in contradiction with the fact that durinc; fertilization in a lar^^^e number of accurately known cases the centrosome arises from or in immediate relation to the middle- piece (Amphibia, echinoderms, tunicates, annelids, mollusks, insects, etc.; see p. 212). The clearest and most positive evidence on this question, afforded by recent observations on the spermatogenesis of insects, annelids, mollusks. Amphibia, and mammals, leaves, however, little doubt that the former result was an error and that, as the facts

of fertilization would lead us to expect, the centrosome of the sper- matid passes into the middle-piece. Accounts vary considerably regarding the origin of the acrosome, which according to most authors is of cytoplasmic origin, while a few describe it as arising inside or from the anterior part of the nucleus. (/7) Composition of the Spermatid. --1:\\q confusion that has arisen in this difficult subject is owing to the fact that the spermatid may contain, besides the nucleus and centrosome, no less than three addi- tional bodies, which were endlessly confused in the earlier studies on the subject. These are the Nebejikern} the attraction-sphere or idiozome (Meves), and the c/uvmatoid lYebenkorper {WQwd^).

The Nebenkern (Fig. 82), first described by Biitschli C/ijin the spermatids of butterflies, was afterward shown by La Valette ('86), Platner (^S6, '89), and many later investigators to arise wholly or in part from the remains of the spindle of the second spermatocyte division. Its origin is thus related to that of an attraction-sphere (which it often closely simulates), since the latter likewise arises from the achromatic figure. To the remains of the spindle, however, may be added granular elements, probably forming reserve-material C'centro-deutoplasm of Erlanger), that are scattered through the cyto- plasm or aggregated about the equator of the spindle (Fig. 126). Thus the Nebenkern may have a double origin, though its basis is formed by the spindle-remains. The Nebenkern sometimes takes a definite part in the formation of the tail-envelopes and of the acro- some (insects), but in many cases it seems to be wholly wanting. - The idiozome is in some cases an undoubted attraction-sphere derived from the aster of the last division and at first containing the centro- some, e.g. in the earthworm as shown by Calkins ('95) and Er- langer ('96, 4), in the salamander and guinea-pig, Meves ('96, '99), and in Helix according to Korff ('99), though in later stages the centrosomes usually pass out of the body of the idiozome. In some cases, however (in the rat, according to Lenhossek, '99), the idiozome seems to arise independently through condensation of the cytoplasmic substance into a sphere having no relation to the centrosomes. In some cases the idiozomes of adjoining cells remain for a time con- nected by bridges of material (Fig. 7) representing the remains of the spindle, and hence corresponding to a Nebenkern {e.g. salaman- der, Meves, '96), and the distinction between Nebenkern and idio- zome here fades away.

The idiozome is usually concerned in the formation of the acrosome (Amphibia, mammals), but sometimes seems 1 The English equivalent of this should be paranucleus, but the latter word has already been used in various other senses, and it seems preferable to retain Piiilschli's original (ier- man word. to degenerate without contributing directly to the sperm-formation {Helix). The chromatoid Nebenkorper, finally, is a small rounded body, staining with plasma-stains, which appear always to degenerate without taking direct part in the formation of the spermatozoon. It is possibly an extruded nucleolus ( Lenhossek ). but its origin and meaning are not definitely known.

(r) Traiisforniation of the Spermatid into tJie Spennatozobii. — In the works of earlier authors it is often impossible to distinguish Pig, 3^. — Formation of the spermatozoon from the spermatid in the salamander. [HER- A. Young spermatid, showing the nucleus above, and below the colourless sphere, the ring, and the chromatic sphere. D. Later stage, showing the chromatic sphere and ring at the base of the nucleus. C. D. E. F. Later stages, showing the transformation of the chromatic sphere into the middle-piece (?«).

which of the various achromatic elements mentioned above have been under observation. We may therefore confine ourselves mainly to the latest works, in which these distinctions are clearly recognized. Owing to their great size, the spermatozoa of Amphibia have been the subject of most careful study; yet a clearer view of the subject may, perhaps, be obtained by taking the spermatogenesis of annelids and insects as a basis of comparison. In the insects (butterflies), Biitschli showed, in 1871, that the tail is formed by an elon^Mtion of the cell-body, into which extends the elongated Nebenkern, now divided into two longitudinal halves (Fig. 82). Platner ('89), confirm- ing this observation, further showed that the Nebenkern (in Pyi^icra) consisted of two parts, stating that one ("large mitosome") gives rise to the investment of the axial filament, the other ("small mitosome ") to the middle-piece ; while a third still smaller body, described as a " centrosome," passes to the apex. The later works of HenkingCgi) and Wilcox ('95, '96) render it nearly certain that Platner confu.sed the acrosome with the centrosome, the first-named observer finding in Pyrrhocoris and the second in Calopteniis that Platner's "centrosome" is derived from the Nebenkern, while Wilcox traced the centrosome directly into the middle-piece. Paulmier, finally, has shown in Auasa that the axial filament grows out from the centrosome,^ proving that such is the case by the highly interesting observation that in giant spermatozoa, arising by the non-division of the primary or secondary spermatocytes, either two or four centrosomes are present, each of which gives rise to a single axial filament, though only one Nebenkern is present (Fig. 82). (The bearing of this important fact on the centrosome-question is indicated elsewhere.) These observations, made on three widely different orders of insects, seem to leave no doubt that in insects the centrosome lies in the middle-piece {i.e. at the base of the nucleus), while both the acrosome and the inner tail- envelopes are derived from the Nebenkern. The outer envelope of the tail is derived from unmodified cytoplasm.

In the earthworm the phenomena are slightly different, the middle- piece arising from an idiozome or attraction-sphere (Calkins, 95), in which lies the centrosome (Erlanger, '96), while the Nebenkern seems to have no part in the formation of either acrosome or tail-envelopes.- We turn now to the Amphibia, elasmobranchs, and mammals, in which the same general result has been attained, though there is still some divergence of opinion regarding the exact history of the centro- some. Working on the basis laid by Flemming ('87, ^'^^X Hermann ('89) traced the middle-piece in the salamander to a " Nebenkorper," which he believed to be not a Nebenkern but an attraction-sphere.

1 Moore ('95) seems to have been the first actually to describe the outgrowth of the axial filament from the centrosome, in the elasmobranchs. It has since been described by Meves ('97, 2) and Hermann ('97) in the salamander, by Lenhossek ('97), Meves ('98, '99), and Bardeleben ('97) in the rat, guinea-pig, and man; by Godlewski ('97) and Korff ('99) in Helix, and by several others. 2 Calkins's preparations, which I have carefully examined, seem to leave no doubt that the middle-piece arises from a true attraction-sphere derived from the spindle-poles; but Erlanger believes that the granular " centrodeutoplasm " also contributes to the sphere.

consisting of three parts, lying side by side in the cytoplasm (Fig. 83). These are {a)2i colourless sphere, shown by Meves's later researches to be probably an attraction-sphere ; (/^) a minute, intensely staining cor- puscle, and ((•) a small, deeply staining ring. The concurrent results of Hermann ('89, '92, '97), IkMula ('93), and Meves ('96, '97, 2) have shown that the small corpuscle (r) is one of the ccutrosoDies of tlie spcnnatiti, and all these observers agree that it passes into or gives

Fig. 84. — Formation of the spermatozoon in Amphibia. \^A-E. Salaviandra, MEVES; F-K. Aiiiphiiinta, McGkkijok,] A. Spermatid with peripheral pair of centrosomes lying outside the sphere, and axial filament. B. Centrosomes near the nucleus, outer one ring-shaped. C. Inner centrosome inside the nucleus, enlarging to form middle-piece. D. Portion of much older spermatid, showing divergence of two halves of the ring (/-). E. Portion of mature spermatozoon, showing upper half of ring at r, and the axial filament proceeding from it.

F. Spermatid of ^;;///////wa, showing sphere-bridges and ring-shaped mid-bodies. G. Later stage; outer centrosome ring-shaped, inner one double; sphere {s) converted into the acrosome. //. Migration of the centrosomes. /. Middle-piece at base of nucleus, y. The inner centrosome forms the end-knob within the middle-piece, which is now inside the nucleus. K. Enlargement of middle-piece, end-knob within it; elongation of the ring. rise to the middle-piece. According to Meves, who has most thor- oughly studied the entire formation of the spermatozoon, the history of these parts is as follows : In the young spermatids the two centro- somes lie quite at the periphery of the cell (Fig. 84),^ and from the outer one growls out the axial filament. The two ccntrosomes, leav- ing the idiozome by which they are first surrounded, now pass inwards toward the nucleus, the outer one meanwhile becoming trans- formed into the ring mentioned above, while the axial filament passes throusfh it to become attached to the inner centrosome. The latter pushes into the base of the nucleus and enlarges enormously to form a cylindrical body constituting the main body of the middle-piece. The ring meanwhile divides into two parts, the anterior of which gives rise to a small, deeply staining body at the posterior end of the middle-piece identical with the "end-knob." The other half of the ring w^anders out along the tail, finally lying at the limit between the main part of the latter and the end-piece. The envelope of the axial filament, here confined to that side opposite the marginal fin (i.e. the ** ventral " side of Czermak), is formed by an outgrowth of the general cytoplasm along the axial filament. The fin and marginal filament are beheved by Meves, as I understand him, to be formed from the axial filament ('97, 2, p. 127).'^ The acrosome, finally, is formed from the idiozome which wanders around the nucleus to its anterior pole. McGregor's results on Ampliiinmi (99) agree in their broader features with those of Meves, but differ on two points, one of which is of great importance. The acrosome here arises from only a part of the sphere (idiozome), while a second smaller part passes to the base of the nucleus and forms the main part of the middle-piece.

The inner centrosome passes into the middle-piece to persist as the end- knob from which the axial filament passes out into the tail (Fig. 84). The history of the sphere thus recalls the phenomena seen in the Xe- benkern of the insect-spermatid ; though the posterior moiety does not contribute to the tail-envelope, while the history of the inner centro.s<Mnc is somewhat like that observed in the mammals, as described beycaui. In the elasmobranchs Moore ('95), Hermann ('98), Suzuki ("98). and Benda ('98) likewise traced the spermatid-centrosome into the middle- piece (Fig. 85), and Moore first showed that from it the axial filament grows out. 3 Moore derived both middle-piece and acrosome ixom the

- Hermann ('97) gives a somewhat different account of the process, believing- that the ring is derived from the mid-body of the last mitosis. Meves and McCIregor have, however, shown that the ring and mid-body coexist in the early spermatids (Fig. 84), which seems decisive against Hermann's conclusion. 3 Hermann finds also the ring ol)served in the salamander, and believes it t.. be the mid- body. The middle-piece is regarded by him as a product of the spimlle-remams, but on both these points he is contradicted by Suzuki.

"archoplasm" of the spermatid. Suzuki's studies clearly show, how- ever, that the entire axial filament of the long middle-piece arises by the elongation of the inner centrosome, while the outer centrosome, from which the axial filament of the tail grows out, lies at the pos- terior limit of the middle-piece (Fig. 85). A nearly similar result is reached by Korff ('99) in the case of llclix. It was shown by God- lewski ( '97) that in this form the axial filament likewise grows out

Fig. 85. — Formation of the spermatozoon in elasmobranchs. [.-/-C SuzUKi; D, MoORE; and in Helix, E-G, KoRFF.] A-lJ. Outgrowth of axial filament from peripheral centrosome (r^), which persists at the posterior limit of the middle-piece or connecting-piece (w). Elongation of inner centrosome (<:'-) to form the axial filament of the latter. E-G show similar phenomena in Helix, with casting off of the sphere (j). a. Acrosome; c'^. peripheral, and c"^. inner centrosome; /. flagellum ; k. end-knob, derived from inner centrosome.

from the centrosome. Korff's later studies show that here, exactly as in the elasmobranch, the axial filament grows out from the periph- eral centrosome and is afterward transformed into a ring (Fig. 85). The inner centrosome elongates to form a rod, which afterward becomes a long filament traversing the elongated middle-piece and terminating in front in an end-knob at the base of the nucleus, while the ring lies at its posterior limit. The idiozome (a true attraction- sphere) degenerates without taking part in the formation of an aero-

some. The envelope of the middle-piece is here formed out of the general cytoplasm. In the mammals the recent work of Lenhossck on the rat ('98) and Meves on the rat, guinea-pig, and man ('98, '99) gives a result agree- ing in its broader features with the forms already considered. In all these mammals the young spermatids are closely similar to those of the salamander, containing two peripherally placed centrosomes, from the outer one of which the axial filament grows out (Fig. 86). Mcves

Fig. 86. — Formation of the spermatozoon in mammals. [Meves.] A. Spermatid of man, showing centrosomes and axial filament. /A Spermatid of guinea-pig, with acrosome. C. Nearly mature spermatozoon, showing backward migration of the ring. a. Acrosome surrounded by cytoplasm of the cell-body, most of which is after\vard thrown off; c. centrosomes; c.p. connecting-piece; / flagellum ; k. neck, containing end-knobs; and Lenhossek differ somewhat in their accounts of the later history of these centrosomes, though agreeing that both contribute to the formation of the middle-piece. Lenhossek states that in the rat both centrosomes persist at the base of the nucleus to form the end-knob, which, as Jensen showed {"^jX is double in this animal. Meves finds the process to be more complicated, agreeing in the main with that observed by him in the salamander. In man and the rat the inner centrosome passes to the base of the nucleus and flattens against it to form a small disc-shaped body. The posterior centrosome divides

into two parts, of which the anterior gives rise to the end-knob, while the posterior is transformed into a rin;^, which wanders back to its final position at the posterior end of the so-called "connecting-piece." From this it follows that the latter body ( VerbindimgsstUck) does not correspond to the middle-piece of the salamander (here represented by the small disc-shaped body at the base of the nucleus), but belongs to the flagellum ]:)roper. The origin of the axial filament and end- knob is, however, nearly the same in the two cases. In the guinea- pig the process is somewhat more complicated and is not quite cleared up bv Meves ; but the origin and fate of the ring is the same, and the end-knob passes into the neck of the spermatozoon as in the rat. Taken together, these observations conclusively show that in mam- mals and Amphibia the end-knob is a derivative of the centrosome, thus sustaining, though with some modifications, Hermann's earlier conjecture ('92) as to the nature of this body; and they overturn Niessing's result ('96) that the centrosome passes into the acrosome. As in the salamander, the acrosome is formed from an idiozome derived in the guinea-pig from the remains of the attraction-sphere (Meves), while in the rat, according to Lenhossek, it is independently formed in the cytoplasm without relation to the preceding mitotic figure or the centrosomes. Within the sphere appears a small, deeply staining body, resembling a centrosome, yet staining differently from the true centrosome, which enlarges to form the acrosome, while about it is formed a clear substance forming the " head-cap " (p. 139). In the rat the acrosome remains small (" Spitzcnknopfchen " of Mer- kel;; in the guinea-pig it becomes nearly as large as the nucleus itself (Fig. 86).

An interesting feature in the formation of the mammalian spermatozoon is the casting off of a portion of the spermatid-cytoplasm in the form of a ''cytoplasmic vesicle" or ** tail- vesicle," which degenerates without further use (Fig. ?)G). This pro- cess, described by Meves ('99) in the guinea-j^ig, is closely similar to that which occurs in the spermatozoid-formation in ferns (p. 144). Resume. In reviewing the foregoing facts we find, despite many variations in detail, three points of fundamental agreement, namely : ( I )the origin of the sperm-nucleus from that of the spermatid ; (2) the origin of a part at least of the "middle-piece" from the spermatid- centrosomes; and (3) the outgrowth of the axial filament from one of the spermatid-centrosomes. It is clear, however, that the term middle- piece has been applied to structures of quite different morphological nature, which agree only in lying behind the nucleus. Thus in the salamander the inner centrosome gives rise to the main body of the middle-piece ; in the rat or in man it gives rise only to the small disc- shaped body lying in the "neck" in front of the so-called middle-

piece ; while in Helix or the elasmobranch it is transformed into a long filament traversing a cytoplasmic "middle-piece" which forms a considerable part of the flagellum. The term viiddlc-piccc has thus become highly ambiguous and should only be employed, if at all, as a convenient descriptive term which has no definite morpholo'^ncal meaning. A very striking fact in the origin of the spermatozoon is the promi- nent part played by the " archoplasm," i.e. substance in the form of idiozome or Nebenkern derived from the mitotic figure, l^oth the source and the fate of this material seem, however, to vary in differ- ent cases, the acrosome now arising from the Nebenkern (insects), now from the idiozome (salamander), the envelope of the flagellum being formed in some cases from the Nebenkern (insects;, in others from unmodified cytoplasm (salamander, snail), while the idiozome may form the acrosome (salamander, mammal) or degenerate without apparent use (snail). We find here, I think, additional reason for regarding " archoplasm" not as a distinct and permanent form of protoplasm, but only as a phase in the general metabolic transfor- mation of the cell-substance, which may or may not persist and jdUiv a definite morphological role in the cell according to circumstances. The close relation of this substance to the motor phenomena of the cell cannot, however, be overlooked.^

The outgrowth of the axial filament from the centrosome is a highly interesting fact, whether we compare it with the analogous phenomena in plants (p. 172) or with the facts observed in ordinary ciliated cells. In the latter case (Fig. 17), as has long been known, each cilium is attached to a small, highly refracting body known as the " basal knob" lying near the cell-periphery. These bodies stain intensely in iron hsematoxyhn, and it has been recently suggested by Henneguy ('98) and Lenhossek ('98) that they are of the same nature as centro- somes. The truth of this surmise must be tested by further study ; but it seems highly probable that they are at least analogous to the spermatid-centrosome. Ishikawa ('99) has clearly shown that in the formation of the swarm-spores of Noctiluea the flagellum grows out from that end of the cell at which the centrosome lies, its substance apparently arising from the central spindle, while the centrosome lies at its base. A very interesting fact discovered by Moore ("95) in elasmobranchs, and confirmed by Meves ('97, 5) and Henneguy (98) in the insects, is a more or less abortive attempt to form a fiagellum by the spermatocytes, i.e. one or two generations before the sper- matozoon. In the insects (Fig. 166) Henneguy has found the cilia actually attached to the centrosomes of the mitotic figure, thus remov- ing every doubt as to their nature. -