VSRIATIOXS I N THE ACCNSSORY CHROllOSOAlES I N METAPHASE AND ANAPHASE O F THE

FIRST SPERMATOCYTES O F THE ALBINO RAT

EZRA4 ALLEN Univrt-sity of Pennsylvania, Philnilclphia

TWO PLATFS (THIRTY-THREE FIGURES)

I n my study of spermatogenesis of the albino rat (Allen, ’IS), only an X was reported. Subesquent examination of the material then used, together with some more recently pre- pared, has brought me to the conclusion that there is also a Y chromosome, as reported by certain other observers to be mentioned below. In connection with this study, variations in size and behavior were noted in the two accessories, and thcse are recorded in this paper. Hundreds of first spcrm- atocyte cells in metaphase and anapliase were studied.

MATERTAI, AND METHODS

The new material prepared mas from the testes of an adult albino rat from C. E. Tliaraldsen’s colony in the New York Medical College. Cell division was very active. All stages of meiosis were found. Nothing in the material indicated that it did not represent average conditions. Champy’s fluid made up bp the formula recommended by Minouchi ( ’28 a ) , B-15 (Allen, ’16), and R-20 (Allen, ’29) were the fixatives em- ployed. Cliampy’s seemed to penetrate better than the other fixatives, and produced equatorial plates with the tetrads widely separated. But the details of the chromosomes were difficult to study, due to an apparant shrinkage o r a conden- sation of the chromatids. While more metaphase complexes in polar views were obtained than by the other fixatives, great irregularity in fixation was present. No better complexes

453

454 EZRA ALLEX

were obtained than the best secured in my older material fixed in R-15. Most of tlie sections were cut at ‘7 or 8 micra; some at 10 and some at 3 and 5. Tlic thiniier sections yielded no better pictures tliaii those cut at 10 micra. Flemmiiig’s triple and iron liematoxylin were used for staining.

OBSERVATIOKS

Xo?-nzciZ 2ichCnvior. In fignrc I of mv 1918 paper tlie accessory is sliown in early pi-opliasc stages as a somewhat elongated body stained moix deeply at oiie eiid than at the other. hi figure 53 of that paper, a photograph of the zygoteiie stage, the ends are broader and much darker than tlie mid region. In fig~wc 27 of tlic 1918 papcr, a late propliase stage, tlie body is labellccl “X.” At this stage it is more elongated, tlie two compoiieiits stain more nearly alike, arid are inclined at a slight angle to each other. I n this form the duplex Body passes into metapliase uncliviclecl. It usually lags a little beliirid the autosomes in taking its place 011 the cviiiatorial

Before the autosomes hai-c full^ coiidenscd, the accessories usnally divide into a longer aiid a shorter dyad. They are hot11 sliglitly cni.ved rocls. Y is tlieii seen to i n o w i n just inside the outer ring of autosomes, or map locate just outside. It tends to remain close to t l ~ e iitncr encl of X. (Figs. <5-7, 21-25, 28.) S continnes in all cases to lie outside the antosomes, pro- jectiiig at riglit angles to the spindle, aiicl maiiitaiiis this position well into anaphase. Y tnriis a i d lies parallel with the spindle, and takes the stain sligIit1~- more deeply than X. These movements arc shown in figures 4-9, 21-28. At its distal wid, X a t this time may or may not show a slight splitting into ITTO chromatids. As Y is about the same size as tlie neigltbor*iiig small autosomes, aiid stairis similarlJ-, its iden- tification soon becomes difficult unless, as in figures 9, 10, 28, it lies just outside the antosomes. In figures 12, 14 aiitl 15 its identification is hriscd on size and relative position.

That X and Y move to opposite poles may be seen Prom figures 10-15 and 29-30. T a aiiapliase the two clironicitids of X

plate (figs. 1, 19, 20).

VARIATIONS IN THE ACCESSORY CHROMOSOMES 455

divide nearly in two and form the characteristic V-shape shown in figure 15. The only view of Y obtained in a similar condition at the same stage is shown in figure 14. Movement of all the chromosomes to the poles is very rapid, and con- sequently little opportunity is afforded to get clear views of the small chromosomes as they form V’s. I n general Y is nearly or quite two-thirds the length of X.

The spindle fiber attaches to the central end of each dyad. This brings the fiber for X at the region where it was formerly attached to Y (figs. 9, 10, 11).

Vnriatiozzs in size a w l behavior. X seems to vary in size more than Y. In figures 6, 7, 22 and 24, X is considerably smaller than in figures 4, 5, 8, 25 and 27. These figures represent the extremes observed in size. Unless X is presented in side view its shape is difficult t o determine accurately. The degree of curvature of X varies; also the number of curves. I n some cases there is only a single curve, as in figures 4, 5, 21 and 26. An elongated S-shape is frequent (figs. 1,s and 27). In all cases X shortens and thickens in metaphase. It is prob- ably at its shortest in late anaphase (fig. 13). From the time X reaches the metaphase stage it stains more faintly tliaii the autosomes.

Thickness may or may not correlate directly with length. Compare figure 4 with figures 6 and 7. I n the complexes with a small X, the autosomes may be small also. Normally its chromatids are equal in size (fig. 15). A marked difference appears in figures 16, 17, 31 and 32. I n view of the inequality of the two chromatids in these dyads, it would appear that one has lost a part. In figures 17 and 32 both chromatids are very short, and their shape indicates a loss in the mid region of each. The shorter chromatid in figure 16 indicates a similar loss. The structure of the smaller chromatid in each cell does not resemble Y. Y has not been identified in either cell. The autosomes in figure 16 are normal. Those in figure 17 are not so distinct.

A peculiar variation appears in figures 11 and 29. The fiber attachments seem to be reversed, and the oorresponding

456 EZRA ALLEN

positions of X and Y. This cell is so well fixed and stained that it is easily studied and accurately drawn.

I n figures 18 and 33 a supevnumcrary X is slio~vn. Y lies between two X dyads. Although the autosomes are in early anaphasc, the accessories have retained the structure and staining reaction of an early metaphase. It 11-ould appear that the presence of the supernumerary is at least one cause fo r this extreme lag.

A. common variation in behavior is an exceptional lazging Iwliinil the autosomes in equatoi*ial plate formation. Fipnres 1, 19 m c l 20, reprcscnt sucli a lag. (Compare autosomes in fig. 1 with those in 4 to 7).

DISCU8810N

11:- observations on the earlp history of the accessories in tlic filest speumatocytes agree on the whole with the full description given by hliiiouchi ( ’28b). hlinouchi states that the two appear “in tlie albino rat from the outset as a single heterochromosome during the growth period, so that no con- jugation can be found in its history” (p. 249). Koller ancl Darlington ( ’34) differ in regard to conjugation. The:- m i t e 011 page 165: “At pachytene the X and Y chroinosomes liavc~ been seen paired throughout the length of the shorter P except for a rcry small end segment corresponding with the very large unpaired segment of the X.’,

l lwre is agreement among all observers that the two acces- sories separate a t about the time of formation of the meta- phase plate. There is also agreement that Y is shorter than X, and that they both vary in length. Painter (’26, fig. 2, anaphase of first spermatocyte) shows Y about one-fourth the lcngth of X. Pincus (’27) figures great variation in shape and size of X and Y in spermatogonia. In the description of his fig;nrcs 13 to 16, lateral views of the first spermatocyte in cw1’1~- anapliase, he states that Y is about two-thirds tlic lciigtli of X. This seems to he the case in his figure 15, but in figure 16 Y appears twice as large as in figure 15, with X also some- d i a t larger. In his alignment of first spermatocyte chromo-

r i

VARIATIONS IN TIIE ACCESSORY CHROMOSOMES 4.5 7

somes (figs. 67 and 68) Y shows size differences. Swezy (’28) does not figure the two sex chromosomes but describes them as J-shaped, “the turned-up portion at the base forming a small Y chromosome. ” Minouchi ( ’28b) draws different lengths f o r each, X varying more than Y, and Y varying in relative length one-fourth to one-third of X. (See his figs. 23 and 36.) Xy figures indicate less relative variation. Y is consistently longer than in Rlinouchi’s figures except in my figure 2, where the ratio is quite similar to that in Rlinouchi’s figure 23. His drawing in figure 23 and mine in my figure 2 are almost identical, a significant observation in relation to the subject under discussion.

Koller and Darlington ( ’34) state on page 166 : “The sex cliromosomes seem to vary considerably in size and staining capacity independently of any variation in autosomes.” This agrees with my observations. The exceptional reversal with respect to spindle fiber attachment and position of the two shown in figure 11 has already been mentioned. I hare found no similar condition in other papers.

For the most part my observations agree with those given 13~- 3linouchi ( ’28b) of the two chromosomes in metapliase and anaphase. However, I have not found that after they have moved apart they still remain attached, as he states, by “a slender chromatin connection. ” Painter ( ’26, fig. 2) , Pincus ( ’27, figs. 26 and 25), and Koller and Darlington ( ’34, text- figures 4, 5, 13, 15 and 16) picture a somewhat similar condi- tion. In Painter’s figure, an anaphase, the thread is very long, nearly twice the length of X. Both Pincus and Xinouchi figure it much shorter. Bryden (’32a) shows none. In his 1933 paper he shows a sliort one in one figure but none in four other figures of the same stage. Koller and Darlington (’34) state on page 166: “ In about nine-tenths of the nuclei the s e s chromosomes are paired by a single chiasma in the short arm of X. . . In the remainder different associations occur, sometimes appearing in whole groups of nuclei. ” These authors show a variety of such tlii*ead-like attachments, and treat each as a chiasma. They discuss various possible chias-

458 EZRA ALLEN

mata. In several figures Y is shown in the shape of a ring attached directly to the end of X, and is interpreted as “chiasmata in both arms” of the Y (their figs. 7 and 16D). I have seen no ring form of Y or any other of the forms shown in their figure 16. I n my material no connecting thread has been observed, although in some cells (figs. 1 and 3) there seems a t tlie time of separation to be a tendency for each dyad to become somewhat pointed at its junction end. If a thread is present it should appear in my figures 9 and 10, which show the two dyads shortly after separation. I n the case of the supernumerary X, the more distal X seems to be still attached to Y by a faintly-staining more or less granular substance, which is represented in the drawing by small dots.

The supernumerary dyad pictured in figure 18 must find cLsplanation in the previous Iiistory of the chromosomal con- stitution of this cell.

Lagging of the accessories in moving to the poles is noted by Bryden ( ’32s) in a late anaphase (his fig. lo), when all the autosomes are assembled at the poles. I have seen no lagging after the two accessories have separated. I n this connection Bryden ( ’32a) writes :

“Owing to their pairing properties the sex chromosomes can separate precociously (see fig, 5). I n some cases the opposite behaviour of the sex-chromosomes was observed; lagging in metaphase plate (fig. 10). This could be produced if the pairing of the two unequal chromosomes took place by interstitial chiasmata as was observed by Crew and Koller ( ’33). This unusual behaviour of these chromosomes cannot be due entirely to inequality in size, but is possibly also con- nected with qualitative differences between them and the auto- somes.” (P. 404.)

Koller and Darlington ( ’34) in their lengthy account of the accessory chromosomes discuss theoretical aspects of the phenomena under discussion, and name four mechanical prop- erties that distinguish X and Y from the autosomes. It is beyond the scope of this paper to discuss theoi-etical questions,

VARIATIONS I N THE ACCESSORY CHROMOSOMES 459

interesting as they are, but those who are interested will find Koller and Darlington's contributioii worthy of consideration.

LITERATURE CITED

ALLEN, EZRA 1916 Studies on ccll division in the albino rat. T I . Experiments in technique. Aiiat. Rec., vol. 10, pp. 565-589.

1918 Studies on cell divisioii i n the albino rat. 111. Spermat- ogenesis. Jonr. Morph., vol. 31, pp. 133-185.

_- 1929 In McClung 's JTandbook of Microscopical Technique. P. B. Hoeber, New York.

BRYDEN, W1\.r. 193% Cytogenetic studies on the rat. Somatic chromosome coln- plexes, meiosis, and chiasma frequency. Jour. Genetics, vol. 26, pp. 395-415.

193Db A comparisou of the chromosomes of the rat and the mouse. Jour. Genetics, vol. 27, pp. 421-431.

KOLLER, P. CH, AXD C. D. DARLINGTON 1934 The genetical and mechanical properties of the sex-chromosomes. I. Rattus iiorvegicus, male. Jour. Genetics, vol. 29, pp. 160-173.

XINOUOHI, 0. 1928a On the fixation of the chroinosonies in mainiiials and some othcr animals. Jap. Jour. Zool., vol. 1, pp. 219-234.

1928b Spermatogenesis of the albino rat (Mus iiorvegicus albus) Jap . Jour. Zool., vol. 1, pp. 235-254.

PAINTER, TH. S. 1926 PINCUS, G.

The chromosomes of rodents. Sci., vol. 64, p. 336. 1927 A comparative study of the chromosoines of the Norway rat

(Rattus iiorvegicus Rrxl) and the black rat (Rattus r;tttus). J. Morph.,

swEZY, 0. 1928 On the existriice of t n o chromosonie numbers in a mixed r01. 44, pp. 515-527.

rat strain. J. Exp. Zool., vol. 51, pp. 135-155.

PLATES 1 and 2

EXPLANATION OF FIGURES

Figures 1 to 18 were in:id(~ 1)) tracing ~ l l o t n l n i ~ ~ o g r a p l ~ ~ of cells focussed for X and Y. The antosomes were then drawn i n by aid of the microscop. Figures 19 to 33 arc reproduced directly from plroton~ierographs. For all the photomicro- graphs, a Leitz plintoniirrogral~liic camera X Z I S used in coinbinatjoii with a 1.9 fluorite oil immersion objective aiid a 2Ox ocular except for figure 20, which w a b takeii with an 8x ocular. Figuies 1 to 18 were enlarged to 3750 diameters for dtnning puiposcs. I n 1 eprndnction tliey lixve been reduced to 3500. All the photoniicrographs a re at x SO00 except figure 80, i T h i c h is about X 1800. The cytop1:tsni i n the photomiciograplis uas washed out of the prints to a greater or less extrnt by n reducing ngent, in order that X and Y might stand out more clearlr. Figures 2 and 20, 4 and 21, 8 and 27, 11 and 29, 15 and 30, 16 and 31, 1 7 and 3.‘. 18 aiid 33 are from the same cells respectiTely.

1 to 3, 19, 20

4 to 7, 21 to 27

S aiid Y still joined before taking their position on the

Early nietaphase complexes; X and Y i n tFpieal positions; cquatoiial plate. 111 each case tliey a ic lagging moic than coninionlg.

6 and 7 nitli X sinallc~ than in 4, 5, 8. 8 and 27 9 to 18,,09 to 33

11 and 29 E-13.

13 one of the largrst cliioniosonics.

15 and 30 16,17, 31, 32 X \\ith abiiornial chromatids. Fixative, B 20. 18 and 30 Snpernumerar~ X. Y slio-\\ s 110 spindle filwr uttaclinient. Fixative,

La t r r metapliase comples; X X-shaped :cad large.

X and Y rcvcrsed in spindle attaclimrnt nii i l po\ilioil. Early and late anaphases.

Fixative,

Polar view of part of cliromosomes in latc aiiRpliase: B compared with

S normally split at mid-anaphase to form V.

B-20.

460

VARIATIONS IN THE ACCESSORY CHROMOSOMES EZRA ALLEN

PLATE 1

/ f i / / I \ ' / I \ d"d t> 'a v

461

VARIATIONS I N T H E ACCESSORY CHROMOSOMES EZRA ALLIFIN

P L A T E 2

462