The Cell in Development and Inheritance

A. Primary spermatocyte in metaphase. B. Equatorial plate, showing ten large tetrads and one small one; "odd chromosome" at o. C. Separation of the dyads. D. Telophase, which is also a prophase of the second division. E. Secondary spermatocyte ; division of the dyads ; small dyad shown undivided. F. Final anaphase ; small dyad near the lower chromosome-group. (The figures are numbered from left to right. For later states, see ¥\g. 82.) become in part aggregated in a mass at one side of the nucleus ("synapsis," p. 276), from which delicate threads e.xtend througii the remaining nuclear space (Fig. 120, A). Even at this period the granules of the threads are divided into four parts. As the process proceeds the chromatin resolves itself into a single spircme-thread, consisting of four parallel rows of granules, which break in two to form the two tetrads (var. bivalcns\ or is directly converted into a single tetrad (var. luiivalcns) (Fig. 120). From these observations Brauer concludes that each tetrad arises from a rod, doubly split lengthwise by a process initiated at a very early period through the

double fission of the chromatin-f^ranulcs. U this be correct, there can be no reduction in Weismann's sense ; tor the four products of each primary chromati^-^^■ranule are equally distributed among the four daughter-cells. A similar conclusion, based on much more incomi)lete evidence, was reached by Hrauer (92) in tlic phyllopod Hrauer's evidently conscientious figures very strongly sustain his conclusion, which, reinforced by the earlier work of Ilertwig and Boveri, has until now seemed to rest upon an unassailable basis. The recent work of Sabaschnikoff ('97) nevertheless raises the possi- bilitv of a different interpretation. l^rauer himself justly urges that the essence of the process lies in the double fission of the chromatin- granules to which the formation of chromosomes is secondary.^ Kvervthing, therefore, turns on the manner in which the quadruple granules arise ; and Sabaschnikoff's work gives some ground for the view that they may arise, not by a double fission, but in some other way.

Accordin«r to this author there is a period (in the oiigcnesis) at which the nuclear threads wholly di.sappear, the entire chromatin Ijeing broken up into granules. From this state the granules emerge in quadruple form to arrange themselves in the doubly split spireme exactly as Brauer describes : and a few observations are given (regarding the size and arrangement of the granules) which suggest the possibility that the quadruple granules may arise by the conjugation either of four separate granules or of two pairs of double granules. Since there is ground for the view that tetrads may ari.se by the conjugation of chromosomes (see following section), there is no a priori objection to such a conclusion. Could it be sustained, the maturation- divisions Q>{ Ascaris would in fact involve a true reduction in Weismann s sense; for despite the fact that the chromosomes are only longitudinally divided, the four longitudinal constituents of each tetrad would not be equivalent with respect to the granules, and it is the reduction of the latter ('• ids'") that forms the essence of Weis- mann's hypothesis (p. 245). Another consideration, suggested to mc by Professor T. 11. Morgan, opens still another possibility, which .seems well worthy of test by further research. As already stated (p. 88), the long chromosomes oi Ascaris are plurivalent, since in all but the germ-cells each breaks up into a much larger number of smaller chromosomes (Fig. 73. p. U^)- If- therefore, the latter correspond to the chromosomes of other forms in which tetrads occur (i-.;'-.

Cyclops or Arteniia), the .so-called " tetrad " of Ascaris is a compound body : and the true process of reduction mu.st be .sought in the origin of the smaller elements of which it is com- posed, which are, perhaps, directly comparable with Sabaschnikoff's "granules." Until the questions thus opened have been further studied, the case iox Ascaris must remain open : and it is perhaps worth suggesting that a new point of view may here be found for further study al.so of reduction in the vertebrates. -

- Bodies closely resembling tetrads are sometimes formed in mitosis, where no reduction should occur. Thus, R. Hertwig ('95) has observed tetrads in the first cleavage-spindle of echinuderm-eggs after treatment with dilute poisons (p. 306). Klinckowstrom figures them in the .f.v^;/</ polar spindle of Prostheicrwus eggs, while Moore ('95) describes in the elasmo- branchs small ring-shaped chromosomes, not only in the first but also in the secotid sperma- tocyte-divisions, concluding that no reduction occurs in either division.

{c) The Forviation of Tetrads by Conjugation. — A considerable number of observers have maintained that reduction may be effected by the union or conjugation of chromosomes that were previously separate. This view agrees in princii)le with that of Ruckert, Hacker, and Vom Rath ; for the bivalent chromosomes assumed by these authors may be conceived as two conjugated chromosomes, ft seems to be confirmed by the observations of Born and Pick on Amphibia and those of Ruckert on selachians {Pristiunis)\ for in all these cases the number of chromatin-masses at the time the first polar body is formed is but half the number obser\^ed in younger stages of the germinal vesicle. In Pristinrus there are at first thirty- six double segments in the germinal vesicle. At a later period these give rise to a close spireme, which then becomes more open, and is found to form a double thread segmented into eighteen double seg- ments ; i.e. the reduced number. In this case, therefore, the prelimi- nary pseudo-reduction is almost certainly effected by the union of the original thirty-six double chromosomes, two by two. The most specific accounts of such a mode of origin have, however, been given by Calkins (earthworm) and Wilcox (grasshopper). The latter author asserts ('95) that in Caloptenus the spireme of the first sj^erma- tocyte gives rise without longitudinal division to twenty-four chromo- somes (double the somatic number). These then become associated in pairs, and still later the twelve pairs conjugate two and two to form six tetrads. There is, therefore, no longitudinal splitting of the chro- mosomes. The a priori improbability of such a conclusion is in- creased by the studies of Paulmier on the Hemiptera, which demon- strate the occurrence of a longitudinal division in a number of these forms and confirm the original studies of Vom Rath on Cryllotalpa}

The second case, which is perhaps better founded, is that of the earthworm {Linnbriciis terrestris), as described by Calkins ('95, 2), whose w^ork was done under my own direction. Calkins finds that the spireme splits longitudinally and then divides transverselv into 32 double segments. These then unite, two by two, to form 16 tetrads. The 32 primary double segments therefore represent chromosomes of the normal number that have split huigitudinally,

I.e. -, etc., and the formula for a tetrad is I , or — i— Such a tetrad, therefore, agrees as to its composition with the formulas of Hacker, Vom Rath, and Ruckert, and agrees in mode of origin with the process described by Ruckert in the eggs of Pristinnis. While these observations are not absolutely conclusive, they never- ^ Montgomery, who has denied the occurrence of a longitudinal division in roitotoma ('98, i), has subsequently found such a division in the nearly related if not identical genus Euchistis ('99).

theless rest on strong evidence, and they do not stand in actual con- tradiction of what is known in the copepods and vertebrates. The possibility of such a mode of origin in other forms must, I think, be Under the same category must be placed Korschelt's unique results in the egg-reduction of the annelid Opliryotrocha {'<^^\ which are verv ditficult to reconcile with anything known in other forms. The typical somatic number of chromosomes is here four. The same number oi chromosomes a})pear in the germinal vesicle (Fig. 125, D). Thev are at first single, then double by a longitudinal split, but after- ward single again by a reunion of the halves. The four chromo- somes group themselves in a single tetrad, two passing into the first polar body, while two remain in the ^^g, but meanwhile each of them again splits into two. Of the four chromosomes thus left in the ^gg two are passed out into the second polar body, while the two remain- ing in the ^'g^^ give rise to the germ-nucleus. From this it follows that the formation of the first polar body is a reducing division — a result which agrees with the earlier conclusions of Henking on Pyrroclioris, and with those of Paulmier on the Hemiptera.

As already stated (p. 246), the formation of actual tetrads is of relatively rare occurrence, being thus far certainly known only in the arthropods, nematodes, and some annelids. In the greater number of cases the two divisions of the primary chromatin-masses {i.e. of the primary oocyte or spermatocyte) are separated by a considerable inter- val, during which the first maturation cell-division takes place or is ini- tiated, and hence no actual tetrads are formed. This obviously differs onlv in degree from tetrad-formation, the latter occurring only when the two divisions are simultaneous or occur in rapid succession.

In the cases now to be considered the length of the pause between the maturation-divisions varies considerably, and in some forms (verte- brates, flowering plants) it is so prolonged that the nucleus is partially reconstructed. In all, or nearly all, these cases the first maturation- division is of the heterotypieal form, the chromosomes having the form of rings and arising by a process that agrees in most of its features with that leading to tetrad-formation. There is here, however, exactly the same contradiction of results as in the case of tetrad-formation described at page 247, and a bewildering confusion of the subject still exists. In brief, it may be stated that most observers of reduction of this type in the lower animals (flat-worms, annelids, mollusks) have found one transverse and one longitudinal division ; most of those

who have studied the vertebrates find two longitudinal divisions ; while opinion regarding the plants is still divided. {a) Animals. — In the gephyrean Tlialasscvia and the mollusk Z/;///^<^ (Figs. 128-130) Griffin ('99) finds that the rings, arising as described above, place themselves in the equator of the spindle with the longitudinal division in the equatorial plane. They are then drawn out toward the spindle-poles from the middle point, first assuming the form of a double cross, then of elongated ellipses, and finally break into two daughter-U's or -Vs. The first division is therefore longitudinal. During the late anaphase the V's break at the apex, the two limbs come close together, so as to give the decep-

Fig. 128. — Diagrams of reduction in the types represented by Thalassema (.-/) and SaU- mandra {B). In both the first division is heterotypical. The second division (6) is transverse in the first and longitudinal in the second. tive appearance of a longitudinal split, and are separated by the second division (following immediately upon the first without inter- vening resting stage). The latter is therefore a transverse division (Fig. 130). An essentially similar result, though less comi)letely worked out, is independently reached by Holies Lee (97) in Hf/ix .- by Klinckowstrom ('97) in the turbellarian ProstJuxcnrus : and by Francotte ('97) and Van der Stricht ('98, i ) in Thysanzoon. Klinckow- strom shows that there is much variation in the way in which the rings open out and break apart, though the result is the same in all. In case of the vertebrates, Flemming {'%7) long since described and figured typical tetrads in the salamander, but regarded them as "anomalies." Vom Rath's later conclusion (93, 95) that they are

normal tetrads has not been sustained by the still more recent work of Meves ('96). whose careful studies, together with those of Moore, Lenhossck, and others, thus far give no evidence of tetrad-formation, and seem oi)posed to the occurrence of reducing divisions in the vertebrates. Meves's work in the main confirms the earlier results of Flemming, except that he shows that, as in so many other animals, onlv two generations of spermatocytes exist. At the first division the' nuclear reticulum resolves itself into twelve (the reduced num- ber) segments, which split lengthwise, the halves remaining united to form elongated rings (Fi©-^- -7' ^:>7 )■ These do not, however, con-

Fig. 129— Maturation and fertilization in an annelid (armed gephyrean) Thalassema. [(IklKIIN.j A. .\ few moments after entrance of the spermatozoon, showing accessory asters; tetrads forming. />'. T^-irly prophase of first polar mitosis with centrosomes. C. In-pushing of nuclear w;i!l. D. Central spindle established; elimination of nucleolus and nuclear reticulum. E. Slightly : ,t.r stage viewed from above. t\ First polar spindle established, cross-shaped tetrads, crossing of astral rays; sperm-head at j.

dense into tetrads, but break apart during the first division at the points corresponding with the ends of the united halves. The first division is therefore an equation-division. As the V-shaped halves sep- arate they again split lengthwise ( Fig. 131), each of the secondary sper- matocytes receiving twelve double V's or dyads. In the telophases and ensuing resting stage, however, all traces of this splitting are lost, the nuclei partially returning to the resting stage, but retaining traces of a spireme-like arrangement (Fig. 131). In the second division twelve double V's reappear, showing a longitudinal division which Flemming and Meves believe to be directly related to that

seen during the foregoing anaphases. There is therefore no evi- dence of a transverse division. McGregor ('99) describes a nearly sniiilar process in Amphiuma, where the longitudinal division of the Fig. 130. — Maturation in the lamellibranch Zirp/uca and in I/taiasst'/nii. [GRIFFIN.] A. Unfertilized ^gg, ring-shaped and cross-shaped chromosomes. D. Prophase of first polar mitosis. C. First polar spindle ; double crosses. D. Slightly later stage. E. The double crosses have broken apart (equation-division). G. Ensuing stage; daughter-V's broken apart at ihe apex. H. Telophase of first, early prophase of second, division ; limbs of tlie V's separate but closely opposed. F. Later prophase of second division. /. Second polar spindle in mctaphase.

daughter-V's is seen with the greatest clearness throughout the anaphases. The weak point in both the foregoing cases is the fact that all traces of the second longitudinal division are lost during the ensuing resting period ; and I do not think that even the observations of Flemming (97), who has pubHshed the fullest evidence in the case, completely establish the occurrence of a subsequent longitudinal divi- Fig. 131. — (Compare Fig. 27). Maturation-divisions in Salanjatidra. [E from FlemMING, the others from Meves.]

A. First division in mctaphase, showing heterotype rings. B. Anaphase; longitudinal split- ting of the daughter-loops. C. Telophase. D. Ensuing pause. E. Early prophase of second division with longitudinally divided segmented spireme. F. Later prophase. G. Metaphase of second division. sion of the chromosomes in the second mitosis. In DesmognatJins, however, where the resting stage is less complete, Kingsbury ('99) finds the longitudinal split in the persistent chromosomes of the

pause following the first division; and he believes this to be the same division as that seen during the anaphase. Carnoy and Le Hrun ('99) reach the same result in the formation of the polar bodies in Triton, though their general account of the heterotypical mitosis differs very considerably from that of other authors, the rings being stated to arise by a double instead of a single longitudinal split These observers describe the rings of the early anaphase as having almost exactly the same double cross-form as those in Thahisscma ox Ztrphcea (Griffin, '99), but believe them to arise in a manner nearly in accordance with Strasburger's abandoned view of 1895,1 and with Guignard's ('98, 2) and Gregoire's ('99) latest results on the flowering plants, the ring being stated to arise by a double longitudinal split""- ting, as explained at page 265.

In the elasmobranch Scy Ilium Moore ('95) finds twelve (the re- duced number) ring-shaped chromosomes at the first division. These closely resemble tetrads ; but a resting stage follows, and the second division is likewise stated to be of the heterotypical form, l^oth divi- sions are stated to be equation-divisions — a conclusion well sup- ported in case of the first, but so far from clear in the .second that a careful reexamination of the matter is highly desirable.

In mammals the first division is of the heterotypical form (Her- mann, '89, Lenhossek, '98), though the rings are much smaller than ni the salamander, recalling those seen in arthropods. No true tetrads are, however, formed, and the two divisions are separated by a resting period. The character of the second division is undeter- mined, though Lenhossek believes it to be heterotypical, like the first. (b) Plants. — It is in the flowering plants, where reduction likewise occurs, as a rule, without true tetrad-formation, that the contradiction of results reaches its cHmax ; and it must be said that until further research clears up the present confusion no definite result can be stated. The earlier work of Strasburger and Guignard indicated that no reducing division occurred, the numerical reduction being directly effected by a segmentation of the spireme-thread into half the somatic number of chromosomes. Thus these observers found in the male that the chromosomes suddenly appeared in the reduced number (twelve in the lily, eight in the onion) at the first division of the pollen-mother-cell, and in the female at the first division of the mother-cell of the embryo-sac. The subsequent ])henomena differ in a very interesting way from those in animals, owing to the fact that the two maturation-divisions are followed in the female by one and in the male by two or more additional divisions, in both of which the reduced number of chromosomes persists. In the male the two maturation-divisions give rise to four pollen-grains, in the female to

the four primary cells of the embryo-sac (Fig. 132); and these two divisions undoubtedly correspond to the two maturation-divisions in animals. In the female, as in the animals, only one of the four resulting cells gives rise to the (i<^^, the other three corresponding to the polar bodies in the animal (t'^^^j:,, though they here continue to divide, and thus form a rudimentary prothalHum.^ The first-men- Fig. 132. — General view of the maturation-divisions in flowering plants. [MOITIER.] A-C, in the male; D-F, in the female. A. The two secondary spermatocytes (pollen-mother- cells) just after the first division (Liliurn). B. Final anaphase of second division {Podophyllum^.

C. Resulting telophase, which by division of the cytoplasmic mass produces four pollen-grains. D. Embryo-sac after completion of the first nuclear division {Liliuni). E. The same after the second division. F. The upper four cells resultin.; from the third division {c/. Fig. io6) : o, ovum; /, upper polar cell ; s, synergida;. (For further details, see Figs. 133, 134.) 1 Of these three cells one divides to form the "synergidre," the other two divide to form thriee "antipodal cells" (which like the synergida:; finally degenerate) and a "lower polar cell." The latter sooner or later conjugates with the " upper polar cell " (the sister-cell of the egg) to form the " secondary embryo-sac-nucleus," by the division of which the endo- sperm-cells arise. Of the whole group of eight cells thus arising only the egg contributes

tioned cell, however, does not directly become the e^^g, but divides once, one of the products being the ^gg and the other the '' upper polar cell" (Fig. 132, F), which contributes to the endosperm-forma- tion (see footnote, and compare page 218). In the male the two maturation-divisions are in the angiosperms followed by two others, one of which separates a "vegetative" trom a "generative" cell, while the second divides the generative nucleus into two definite germ-nuclei. In the gymnosperms more than two such additional divisions take place. In these later divisions, both in the male and in the female (with the exception noted in the footnote below), the reduced number persists, and the principal interest centres in the first two or maturation-divisions. Strasburger and Guignard found in Lilium that while both these divisions differed in many respects from the mitosis of ordinary vegetative cells, neither involved a transverse or reducing division, the chromosomes under- going a longitudinal splitting for each of the maturation-divisions. Further investigations by Farmer ('93), Belajeff ('94), Dixon (96), Sargant ('96, '97), and others, showed that the first division is often of the heterotypical form, the daughter-chromosomes in the late-meta- phase having the form of two V's united by their bases (<>). Despite the complication of these figures, due to torsion and other modifications, their resemblance to the ring-shaped bodies observed in the first maturation-division of so many animals is unmistakable, as was first clearly pointed out by Farmer and Moore ('95).

Botanists have differed, and still differ, widely in their interpreta- tion both of the origin and subsequent history of these bodies upon which the question of reduction turns. According to Strasburger's ('95) first account their origin has nothing in common with that of the tetrad-rings, since they were described as arising by a double \o\\- gitudinal splitting of a primary rod, the halves then separating first from one end along one of the division-planes, and then from the other end along the other plane, meanwhile opening out to form a ring such as is shown in Fig. 133. (This process, somewhat difficult to understand from a description, will be understood from the dia- gram. Fig. 135, E~I^ The four elements of the ring are then distrib- uted without further division by the two ensuing maturation-divisions ; and the process, except for the peculiar opening out of the ring, is

to the morphological formation of the embryo. It is a highly interesting fact that the nuni- ber of chromosomes shown in the division of the lower of the two nuclei {i.e. the mother- nucleus of the antipodal cells and lower polar-cell) formed at the first division of the embryo-sac-nucleus is inconstant, varying in the lily from 12, 16, 20, to 24 (Cluignard, '91, l), in which respect they contrast with the descendants (egg, synergidx) of the upper nucleus, which always show the reduced number (Mottier, '97, i), i.e. in l.ilium twelve. This exception only emphasizes the rule of the constancy of the chromosome-number in general; for these cells are destined to speedy degeneration.

essentially in agreement with the facts described in Ascaris, and involves no reduction-division. Essentially the same result is reached by Guignard (98) in his latest paper on Naias, and by Gregoire ('99) Strasburger twice shifted ground in rapid succession. First (97, 2), with M()tticr('97, i ). he somewhat doubtfully adopted a view agreeing Fig. 133. — The first maturation-division in flowering plants. [/>", STRASBURGER and MOT- TlKk , the others from MorilER.]

. /. Mother-cell of the embrvo-sac in Lilium ; early prophase of first division : chromatin- threads already longitudinally divided. B. Slightly later stage (split spireme) in the nucleus of the poUen-mo'ther-cell. C. h slightly later prophase (pollen-mother-cell. Podophyllum) with twisted split spireme. D. Earlier prophase (ZL/V/ww, female) ; split twisted chromosomes. E. Equatorial plate {Lilium, male). F.. First maturation-spindle {Fritillaria, male). G. Diver- gence of the daughter-chromosomes {Lilium, male).

essentially with the interpretation of Vom Rath, Riickert, etc. (p. 247). The primary rods split once, and bend into a V, the branches of which often come close together, and may be twisted on themselves, thus giving the appearance of the second longitudinal split described in Strasburger's paper of 1895. The two halves of the split U then separate, opening out from the apex, to form the 0 -figure. In the second division the limbs of the daughter-V's again come close together, remaining, however, united at one end, where they were believed finally to break apart during the second division. The latter was, therefore, regarded as a true reduction-division, the apparent longitudinal split being merely the plane along which the halves of the V come into contact (Fig. 134, C, D).