Talk:Paper - On the number of chromosomes and the type of sex chromosomes in man (1934)
472 Cytologia 5
On the Number of Chromosomes and the Type of Sex-chromosomes in Man “
by Osamu Minouchi and Takeo Ohta
Received December 19, 1933 . Introduction
Since Flemming (1882) described the chromosomes of man as about twenty-four in number, numerous investigators have studied the chromosomes in both somatic and germinal tissues. Various opinions have been put forward regarding the number of human chromosomes, the figures given ranging from eight to forty-eight. The majority of investigators in this field pointed to twenty-four as being about the diploid number in the male, the female number being either the same or one more, until Winiwarter (1912) published a very excellent work on this problem. He reached the conclusion that the chromosomes of spermatogonia are forty-seven in number, those of oogonia. forty-eight, those of spermatocytes twenty-four, and those of second spermatocytes twenty-three or twenty-four depending upon whether the X-element is present or not in the ‘cell, ‘and that the sex-chromosome goes undivided to a pole in the metaphase of the first division in advance of the autosomes. Painter (1922) observed forty-eight chromosomes in spermatogonia and twenty-four in the first spermatocytes. According to him the sexchromesomes of whites and negroes are composed of two elements, a rod-like X and a ball-like Y in the male. The elements X and Y are separated in the first maturation division, so that half of the spermatozoa will carry an X and the other half a Y element. These ‘two opinions about the number of chromosomes and the type of sexchromosomes in man are still strongly held by the respective parties, though within the last decade the difficulty has been largely removed from cytological technique. Oguma et Kihara (1923), Winiwarter
- This investigation was made possible by a grant from the Imperial Academy of
Japan, to which our thanks are due. 1934 On the number of chromosomes and the type of sex-chromosomes in man 473
et Oguma (1926) and Oguma (1930) support Winiwarter’s idea published in 1912, while Evans and Swezy (1929), Kemp (1929) who studied somatic cells, and recently Shiwago and Andres (1932) agree with Painter. The difficulty of obtaining suitable material for cytological purposes and of employing safe methods of preparation have been largely responsible for such differences in results.
Minouchi had long been of the opinion, based on his own work on mammalian chromosomes (1928 b, 1928 c, 1929), Nakamura’s on reptilian chromosomes (1928, 1931, 1932), and Iriki’s on amphibian and piscian chromosomes (1930, 1932 a, b, c, d, e, f) that every class of vertebrates should have a definite type of sex-chromosome. No such substantial difference should be expected between the sex-chromosomes of mammals and man, and nor such a marked difference between Belgians and Japanese in the size of the sex-chromosomes as reported by Winiwarter et Oguma (1926).
Since 1922, Minouchi had attempted to investigate the chromosomes in man, but failed to obtain suitable material for his purpose. In 1931, however, Minouchi and his colleague T. Ohta had the opportunity to obtain two fairly good testes by the courtesy of Prof. Kiyemon Isobé of the Surgical Institute, Department of Medicine, Kyoto Imperial University. They wish to express here their hearty thanks to Prof. K. Isobé. This work was carried out in the Zoological Institute, Department of Science, Kyoto Imperial University, and described under Prof. R. Goldschmidt, Kaiser Wilhelm Institut in Berlin-Dahlem. They are greatly indebted to Prof. R. Goldschmidt for his kindness and criticism.
It is a pity that they were obliged to use up almost all the material in obtaining metaphase plates of germ-cells; they had to give up their hope of accomplishing a complete study of human spermatogenesis, for lack of material. They think however the present work will suffice to determine the number of chromosomes and the type of the sex-chromosomes in man.
Material and method
The testes were removed by surgical operation from two different individuals, Japanese of about twenty-three and twenty-five years old. A great number of dividing spermatogonia, first spermatocytes and second spermatocytes, and of matured spermatozoa were present in some parts of the testis of the younger man. Some patho474 O. MINOUCHI and T. OHTA Cytologia 5
logical change, apparently a bud of some kind of tumor was found in connective tissues lying among seminal tubules of the testis. The wall of the tubules was thickened and their diameter reduced. These facts show that the tubules had began to degenerate and that the multiplication of germ-cells had been lessened. The other testis was taken from a beggar of about twenty-five years old who had eaten only once or twice every two days. There were plenty of dividing figures in the spermatogonia, but only a few in first spermatocytes, and very few in second spermatocytes, and fewer still of spermatozoa. The chromosomes in these cells tended to clump together. From these facts it is concluded that inanition affects the development of germ-cells in the order of spermatogenesis reversed, i.e. first spermatozoa, then spermatocytes, and so on. Poor somatic and psychic conditions seem to produce the same phenomena as Minouchi observed in the testis of a dog. For cytological purposes the material should therefore be taken out from healthy well-nourished individuals.
Immediately after the testis was taken out from the body, it was cut into small pieces as fast as possible, and dropped into Flemming’s strong solution without any trace of acetic acid, or into Champy’s solution. The former gave better results than the latter in this case. The sections were cut 8,4 thick. After bleaching, the sections were left for twenty-four hours in a modified picro-acetic mixture (saturated solution of picric acid 6 parts and glacial acetic acid 4 parts) to differentiate the chromosomes. Then the slides were washed thoroughly with running water. Heidenhain’s iron-haematoxylin was exclusively used for staining. All of the figures were drawn at a level about 30 mm. below the stage, with Zeiss Apoch. 15mm. n. Ap. 1.38, comp. Oc. 18, and tube length 160 mm., and with the aid of an Abbe drawing apparatus.
Observations
1. The structure of the testis
The general structure of the human testis closely resembles that of mammals. The intertubular connective tissue is a little better developed than that of the albino rat, but not so much as in the case of other mammals. When a part of the Tunica albuginea of a human testis is cut off and the other part pressed from the surface, the seminal tubules are pushed out separately as in the case of the albino 1934 On the number of chromosomes and the type of sex-chromosomes in man 475
rat, if the testis is normally developed and has not undergone pathological changes. Every kind of germ-cell is found in the seminal tubules of younger material: in some parts they are arranged regularly, but in others spermatogonia occupy the portion where the first spermatocytes should be found. A number of very beautiful figures of division, not only of spermatogonia but also of both kinds of ‘spermatocytes are found in parts where the germ-cells are arranged regularly. In contradiction to this, various types of abnormal division, multipolar divisions, polymerisation of chromosomes or other disagreeable pathological changes occur in parts where the germ-cells are arranged abnormally.
2. The spermatogonia
Two kinds of spermatogonia, early and late in stage, or young and old, can be identified along the wall of the seminal tubules of man, just as in the mammals studied by Minouchi (1928, 1929). The early spermatogonia in metaphase show larger equatorial plates than those of the late ones. Their diameter becomes gradually reduced, division by division, so that a definite morphological difference is never found between them (Figs. 1—6).
Out of a large number of excellently preserved specimens of spermatogonia in the metaphase, only seven complete ones are shown in Figs. 1-7. Figs. 1-3 show early spermatogonia in the metaphase and Fig. 6 a late or ultimate one. Fig. 7 was obtained from material fixed with Flemming’s strong solution containing no trace of acetic acid. It is probably of the same kind as Figs. 4 and 5, which show the metaphase near the end of the spermatogonial stage. ‘As is shown in these figures, the chromosomes of human spermatogonia are forty-eight in number as reported by Painter (1923), Evans and Swezy (1929), and Shiwago and Andres (1932). After a careful observation of the figures given, we can find some pairs of chromosomes of similar size and shape. Such chromosomes are to be regarded as synaptic mates of homologous chromosomes. But it is very difficult to pair up all the chromosomes of a plate in these figures, and to point out the sex-chromosomes from such a serial arrangement of spermatogonial chromosomes as Painter (1923), Oguma et Kihara (1923), Winiwarter et Oguma (1926), and Shiwago und Andres (1932) have attempted.
Cytologia 5, 1934 82 A476 O. Minoucnt and T. OHTA Cytologia 5
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5 6 7 OF Figs. 1-3. The metaphase of younger spermatogonia treated with Champy-Minouchi, showing 48 chromosomes in each plate. Figs. 4-5, The metaphase of spermatogonia on the way to become ultimate, treated with the same, showing 48 chromosomes in each. Fig. 6. The metaphase of ultimate spermatogonia treated with the same, showing 48 chromosomes in each. Fig. 7. The metaphase of a spermatogonium corresponding to Figs. 4-5 treated with Flemming-Minouchi, showing 48 chromosomes.
We shall illustrate this difficulty. The chromosomes of a Japanese grass-hopper Trixalis nasuta, are so large and simple that we can easily recognize them even under the low magnification of 30%. They are also easy to observe in vivo as well as in fixed material. It is therefore easy to control here the effect of fixatives. Two equatorial plates given in Figs. 8 and 9, seem to have undergone no 1934 On the number of chromosomes and the type of sex-chromosomes in man AT7
evident change due to the fixative, except shrinking to some extent; they have lost neither relative size nor structure in comparison with those in vivo. Lining up the chromosomes as shown in Fig. 10 according to their length as represented in Figs. 8 and 9, we find that chromosomes 1, 2, 15, 18, 19, 20, and 23 remain unpaired in Series A, and chromosomes 5, 14, 17, 18, 19, 20 and 21 remain in Series B.
SCZ Fr Ws, OP ae
Fig. 8. A polar view of the spermatogonial metaphase in Trixalis nasuta, a Japanese grasshopper, treated with diluted Flemming-Minouchi (Flemming’s strong solution
with no trace of acetic acid is diluted with three parts of water) showing 28 chromosomes. Fig. 9, The same showing 25 chromosomes, of which two are supernumeraries.
OCI.
1 2 34 78 910 11121314 15 1617 18 19 20 2122 23
CUciatitaraa Vavaaee
89 1011 1213 14 1516 171819 20 21 2223 2425
Series A
Series B
Fig. 10. Series A. The chromosomes in Fig. 1 are lined up, according to their length. 1, 2, 15, 18, 19, 20 and 23 remain unpaired. Series B. The chromosomes in Fig. 2 are lined up. 5, 14, 17, 18, 19, 20 and 21 remain unpaired.
No one could find out which is the true unpaired sex-chromosome in such a case. In a side view of the first division it becomes clear that the true unpaired chromosome is the one which goes undivided to a pole in advance of the others (Figs. 11 and 12). From its dual nature and from the information obtained by tracing it back to the growth period, it is evident that this one is the real sex-chromosome
32* 478
O. MINOUCHI and T. OHTA
Cytologia 5 K j / Fig. 11. A side view of the anaphase in Fig. 12. The same showing 11 chromothe first meiosis showing 11 chromo- somes in a daughter set and 12 in the somes in a daughter set and 12 in the other.
The supernumeraries remain at the equator.
4 ( 0 NANA‘ Awar
other. X=sex-chromosome.
‘yuyyie we
- boda () 0f\ (Sane
(\OuKyuyur
7 8 9 10 11 #12 18
Fig. 13. Series A: Two daughter sets of chromosomes of Fig. 4 are lined up in two rows, according to their length. No. 8 is the sex-chromosome. Series B: Two daughter sets of chromosomes of Fig.5 are lined up in two rpws
. No.6 is the sex-chromosome. 1934 On the number of chromosomes and the type of sex-chromosomes in man 479
derived from the nucleolus in the growth period. To find out the position of the sex-chromosome in the serial arrangement we tried to line up the two daughter sets of chromosomes in different rows respectively according to their length (Fig. 13), because the homologous pairs of chromosomes are easily and surely found in the side view of the anaphase of the first meiosis, their mutual length relationship being not yet lost. The chromosomes of Fig. 11 are lined up in Series A, and those of Fig. 12 in Series B. Then it is found that the sex-chromosome is No.8 in Series A, while it is No.6 in Series B. The difference comes from the fact that the sex-chromosome in Series A lies obliquely to the optical plane and the length represented here is therefore too short. If the sex-chromosome lies parallel to the optical plane, it appears longer than in the case of Series B, so that in the spermatogonial series (Fig. 10, A) the one which lies at number 11, should be the sex-chromosome, although it appears to be homologous with number 12. It is very difficult even in Orthoptera to find out the sex-chromosomes from lining up the spermatogonial chromosomes as shown in these examples. Much more is this the case with man in which the chromosomes are smaller, more complicated and more difficult to preserve than those of the grasshopper.
As shown in Figs. 1-6, the human spermatogonial chromosomes appear as V’s, J’s and rods of various sizes in the metaphase. We could not find such a small unpaired chromosome as that given by Painter (1923) and Shiwago und Andres (1932).
3. The first spermatocyte
When the nuclear membrane disappears in the late prophase of the first meiosis the bivalent chromosomes or tetrads gather towards the center of the nucleus. Then they are arranged in a plane, at a certain distance from each other, to form the equatorial plate of the metaphase. Fig.14 shows the stage before the metaphase. The process of condensation of the ground substance is not yet finished in this stage and we can already observe the structure of individual chromosomes, in which deeply stained spirals are imbedded in the lightly stained ground substance. Whether these spirals are of a single or double nature cannot be discriminated owing to the smallness of their size. Sometimes, however, four spirals can be observed in a tetrad. In animals four spermatids 480 O. Minoucu! and T. OHTA Cytologia 5
are derived from a first spermatocyte. These spermatids develop to spermatozoa without any other division, so that the spiral cannot be expected to be double in a chromatid of a tetrad. It is perhaps single and a tetrad is probably composed of four spirals.
The chromosome labelled XY is composed of two unequal parts, the anterior a short rod and the posterior an elongated rod, differing from the others which are made up of two equal parts. A spiral turn is found in the ground substance of the anterior part, while
a
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oS » me et do . o, *~ 14 15a 156 Fig. 14. The stage prior to the metaphase of the first division treated with ChampyMinoucni. XY = sex-chromosome. Fig.15a. A side view of the metaphase in the
first meiosis treated with the same, showing the sex-chromosome. Fig. 156. The photomicrograph of the same material as that of Fig. 15a.
there are three turns in the posterior. These short and long spirals should theoretically be of double nature, but it was difficult to observe in the present material whether they are of single or double nature. Considering the number of turns, the Y-element is about one third of the X in length and the X about one half of the large V-chromosome which Winiwarter et Oguma (1926) have described as the sex-chromosome in Belgian material.
The mode of conjugation of these two elements differs from that of the autosomes, the polar end of X being connected lineally to the distal end of Y. In a side view of the metaphase the Yelement stands vertically to the equatorial plane, while the X one lies horizontally as shown in Fig. 15a (Fig. 15b in the photomicrograph). The spindle fibers are attached to Y at its anterior end that is more distant from X, and to X also at its anterior end where it connects with Y. Thus in the first meiotic division the autosomes are found in the form of parasyndesis and the sex-chromosomes in the form of telosyndesis with the polar end of the X attached to the distal end of Y. This mode of conjugation has been described by 1934 On the number of chromosomes and the type of sex-chromosomes in man 48}
Minouchi (1928) as asymmetrical synapsis, which is a fundamental and characteristic feature of the sex-chromosomes in the first maturation division of mammalian spermatogenesis. That is to say, the poles of both elements, X and Y, are not arranged in mirror image form, while those of the autosomes show the mirror image (symmetrical synapsis) arrangement. There are therefore found both modes of conjugation, symmetrical and asymmetrical synapsis, in the human spermatogenesis as is the case in the mammals reported by Minouchi (1928, 1929).
Figs. 16-21 represent the polar views of the metaphase in the first meiotic division showing twenty-three tetrads, each of which is made up of two homomorphic univalents and a tetrad of hetero
Fig. 16a. A polar view of the metaphase in the first meiosis treated with Flemming
Minouchi, showing 24 chromosomes. Fig. 165. The photomicrograph. Figs. 17~21.
Polar views of the metaphase in the first meiosis treated with Champy-Minouchi. Twenty-four chromosomes are shown in each plate. 482 O. MINOUCHI and T. OHTA Cytologia 5
morphic ones in every plate. The chromosomes of first spermatocytes are therefore twenty-four in number including the sex-chromosomes. The tetrads of autosomes take various forms of rings and V’s. The rings are single and horizontal, double, and triple. In the normal condition, as shown in Fig. 16a, twelve large tetrads, of which two are triple rings, three double rings, two 7’s, three horizontal single rings and two rods, and a sex-chromosome encircle the other small ones so as to form a rosette. The sex-chromosome lies at the periphery of the equatorial plate and not in the center as stated by Painter (1923). Painter states that polar views of equatorial plates are of little or no service from the standpoint of counting, because the large elongated tetrads which typically lie on the outside of the spindle, tend to obstruct the view of the smaller bodies lying within. But in our material the polar views of equatorial plates are the best for determining the number of chromosomes. In such metaphase plates as shown in Figs. 16~21, individual chromosomes being arranged on a plate, there is no chromosome tending to obstruct the view of the smaller ones. Judging from the morphology of these tetrads, two triple rings seem to be derived from the homologous chromosomes of submedian fiber attachment by coupling, because the horizontal rings lying at both ends of the tetrad are different in size from each other, three double rings and two 7’s are subterminal; and two rods are terminal. When the largest tetrad of triple rings is deformed it looks like a W, which Oguma et Kihara (1923) have erroneously interpreted as the sex-chromosome of XO type.
Painter (1923) believed it to be a characteristic feature of the first maturation division that there is a lack of regularity in the time when the individual chromosomes divide, and that one frequently finds spindles in which the tetrads have separated and either the two halves are found at opposite poles or one half is still lying in the equatorial plate of the spindle. Such phenomena arise from faulty fixation. Figures of the same kind are also given by Winiwarter et Oguma (1926). All the chromosomes in man divide with regularity in the first maturation division, as is the case in mammals and insects.
4. The second spermatocyte
It is very hard to obtain an accurate count of the chromosomes in the second maturation division of man, because of the difficulty 1934 On the number of chromosomes and the type of sex-chromosomes in man 483
of obtaining suitable material for cytological study, the second spermatocytes tending to be influenced more easily than the first ones by pathorological or other changes of conditions. Winiwarter (1912), Winiwarter et Oguma (1926), Shiwago und Andres (1932) succeeded in preserving second spermotocytes in the metaphase and anaphase, while Painter (1932) and Oguma et Kihara (1923) failed to count the number of chromosomes in these stages. In the present material many dividing figures of the second spermatocytes were observed, but to our regret we could not distinguish morphologjeally individual chromosomes
in these stages and compare them ~ ww J with those found in the sper- A728, shyt matogonia. v=, ~& Svv 2 woe ~ a O%. 4 Vv Out of many second division Eon et, s & t<|} figures only four figures of the te @ ie Ma metaphase in polar view are 22 +
given in Figs. 22-25. Every 23
equatorial plate shows twentyfour chromosomes, in which ys double V’s, double J’s, and single Sy Liv V’s are included. It is clear that avg F dyn” ae ye double V’s come from the pairing A "cg qa no a up of chromosomes of submedian 94 a 25 *
fiber attachment in the spermato- Figs. 22-25, Polar views of the metagonia, double J’s from sub- phase in the second meiosis treated with terminal ones and single V’s the same, showing 24 chromosomes. from terminal. The number of
each form of chromosome should therefore be half that of the spermatogonia. We could not find any cell containing twenty-three chromosomes as reported by Winiwarter (1912) and Winiwater et Oguma (1926), although we carefully examined the dividing cells lying near each other which seem to be derived from the same
spermatocyte. 5. Abnormal division
Abnormal divisions are often found even in normal tissue, but much more in pathological tissues. The present material is not perfectly normal as already noted. It is therefore natural that many abnormally dividing cells should have been found in this material. Pathological condition, inanition, despondency of the individuals ete. 484 O. MINnoucHI and T. OHTA Cytologia 5
may be the cause of various kinds of abnormal] division. What the changes are that occur in the cell, it is very difficult to say, although many examples of abnormal divisions have already been produced experimentally,
Daleq (1921) denoted various kinds of abnormal divisions in the spermatogenesis of lizards, not only in the spermatogonia but also in both meiotic divisions, and even of abnormal spermatozoa. We observed such kinds of abnormal division principally in spermatogonia, not so often during meiosis, in which cells produced by abnormal division or dividing abnormally seem to degenerate soon after. Why such differences between man and lizard are found is difficult to explain. We know only that the cells of lower vertebrates are more resistant than those of higher forms to reagents, temperature differences or other bad conditions.
Spermatogonia with balled chromosomes are also found in this case. The cytoplasm of such cells is always disintegrated, so that it is plausible to conclude that they are in course of disintegration. A kind of giant cells, which contain a large number of chromosomes —two hundred or more,—are observed in this material. The form and the size of polymerised chromosomes closely resemble those of normal ones, Whether the number increases always in multiples of the haploid set or not, is a very interesting problem. In some giant cells the chromosomes lie parallel two by two. This seems to show that the prophase nucleus has returned to the resting stage without division. The chromosomes which have split into two enter the resting stage, without losing their relationship to one another. The daughter chromosomes remain parallel in the nucleus as they were in the late prophase. In the next prophase the distance between the daughter halves widens when the chromosomes are fully formed. Because the skeletal portions of the chromosome, the chromonemata, lie much more separated from each other in the resting stage owing to the swelling of the ground substance than in the last prophase, condensation of the ground substance takes place around the widely separated chromonemata in the next prophase. The behavior of chromosomes from anaphase to the next prophase has already been reported by a number of investigators, and it is a well known fact that the chromosomes appear in the prophase as they were in the last anaphase, but separated more from each other. In the next prophase the individual chromosomes split again into two as in the last prophase. As a result their number becomes quadrupled. This 1934 On the number of chromosomes and the type of sex-chromosomes in man 45
explanation, however, does not apply to all the supernumeraries which we have met with. The chromosome number increases sometimes without any definite rule, as has been reported in the case of cancer cells by Winge (1929) and Andres (1932). Ohta” has carried out an investigation on the chromosomes in cancer of the human uterus. He observed many cancer-cells containing a large number of chromosomes of normal size and form, but the number does not always increase in multiples of 24. No adequate explanation has yet been found as to the manner in which such irregular increasing comes about, although multiplication of chromosomes, nén-disjunction of chromosomes, transverse division of chromosomes, multipolar division, etc. are, thus far, considered to be the causes. This is one of the most important but most difficult problems in karyology at present.
Multipolar division was rarely observed in the present material. The nuclei produced by such divisions contain an abnormal number of chromosomes, because not only does the chromosome-set divide according to the number of poles, but also there are always some chromosomes connected by spindle fibers to three or more poles which will be cast out of the daughter nuclei in the anaphase.
During meiosis abnormal divisions were scarcely found in this case, although in the early stage of meiosis there are large nuclei which seem to contain a large number of chromosomes. Cells of this kind were never observed in the metaphase. They probably had disintegrated before dividing. Sometimes a large number of cells, both first and second spermatocytes, show equatorial plates in which the chromosomes lose their individuality or clump together, and their cytoplasm shows also an appearance of disintegration. Such cells probably degenerate.
Abnormal spermatozoa were never found in the present material so far as our observation went. This fact shows that the germ-cells derived from abnormal divisions rarely continue to develop.
Discussion Winiwarter et Oguma (1926), and Oguma (1930) have published several papers to show that the number of chromosomes of man
is forty-seven and the sex-chromosome consists of only an element X. To determine whether the chromosomes are forty-seven or forty
1) The work will shortly be published.
486 O. MINOUCHI and T. OHTA Cytologia 5
eight is a delicate and difficult problem in the spermatogonia, and also even in the first and second spermatocytes it is difficult to obtain an accurate count when the material has been preserved in such fluids as Bouin-Allen, Flemming, Carnoy-Flemming or Hermann. Even if cells are well preserved, it is hardly possible to avoid making a mistake of one or two in counting forty-eight chromosomes; this we think was the case with the above quoted investigators. To determine the basal diploid number of chromosomes it is therefore necessary to consider not only the number of spermatogonia but also that of the maturation divisions. Our results agree with Painter’s (1923), and Shiwago and Andres’ (1932) though we could not recognize the specially small chromosome in the spermatogonia noted by them.
The method of arranging the chromosomes serially tried by Painter (1923) and Oguma et Kihara (1923) has since been generally adopted by students of human chromosomes to control their observations of the first meiosis. But as we have stated, this method does not provide any crucial evidence for the possibility of arranging the chromosomes in homologous pairs. This is also endorsed by the fact that the conclusion of Oguma et Kihara (19238) is very different from Painter’s (1923) with regard to the size of the X-chromosome. If the method of arranging the chromosomes in pairs gives significant results for the determination of unpaired chromosomes, the opinions of these investigators should be in accord with regard to the morphology of the X-element, whether the Y-element is present or not. And if Oguma et Kihara had kept in mind that the morphology of chromosomes in the metaphase of the first meiosis is decisive for the identification of the sex-chromosomes, Oguma (1930) might have avoided such a conclusion as that the Japanese and Belgians differ in the size of the X-element.
Shiwago and Andres (1932) failed to observe the heteropycnosis in the growth period in material preserved with Champy-Minouchi. But the heteropycnosis of sex-chromosomes is clearly seen in our Champy-Minouchi material though the nucleolus sometimes remains unstained. It may have been overlooked on account of unsatisfactory technique. This follows from their figures of the metaphase of the first division. If they had differentiated the chromosomes with Chura’s solution, they would have been able to obtain crucial evidence of the presence of the XY-chromosome in the first meiosis. In their figures 10-12 the chromosomes are preserved well separat1934 On the number of chromosomes and the type of sex-chromosomes in man 487
ed, while their structure is imperfectly displayed on account of insufficient differentiation. It is very difficult to identify the sex-chromosomes in such figures.
Winiwarter et Oguma (1926) have pointed out with some examples that the so-called XY-chromosomes of Painter as pictured in side views of the first meiosis, are not always the true sex-chromosomes; we think also that such figures appear occasionally when the chromosomes of the first meiosis have not been well preserved. No one who bears in mind the degeneration processes of chromosomes demonstrated in the experiments of Chambers could be sure whether the chromosomes in question are a group of two unequal elements, X and Y, or an artifact. Iriki (1932a, b) also made it clear that Witschi’s XY chromosomes in Rana (1929) come from a technical mistake. Therefore, the figures presented by Painter (1923) seem to be insufficient as crucial evidence for the presence of a Y-element.
Winiwarter et Oguma (1926) have pointed to the anaphase of the first meiosis, in which one of the daughter halves shows twentythree chromosomes, the other twenty-four including the unpaired one, as the most powerful evidence in support of their idea. There is a peculiar form of chromosome (8), bifurcated at its anterior end, in Fig. 26a (after Winiwarter et Oguma). Such bifurcation shows that this chromosome is a double V, while the corresponding one (8) in Fig. 26b is a single V and the unpaired X lies near it. It is impossible to consider that a double V and a single V should be homologous in the autosomes, because the double V comes from a V-shape in the spermatogonia, while the single V comes from a rod-shape. Such a conflict with a general rule seems to speak in favor of the presence of a Y-element in the daughter cell, and the double V chromosome might be in fact the overlapping of a Y-element above the other single V. The same peculiar form of chromosome is given in spermatogonia by Oguma et Kihara (1923) as shown in Fig. 27-28 (after Oguma et Kihara). Such a figure can arise from overlapping of two chromosomes and should be counted as two elements. Winiwarter et Oguma (1926) show the chromosomes of the second division, the numbers being twenty-three and twenty-four. But in this case all of the chromosomes were so deformed that no one could identify them properly. Therefore, we think, we have no evidence that man is of XO type. The human sex-chromosomes show heteropycnosis in the growth period; in well preserved material they lie at the periphery of the equatorial plate; 488 O. MINOouCcHI and T. OHTA Cytologia 5
265 , 28
Fig. 26. Polar view of the anaphase in the first meiosis (after Winiwarter et Oguma,
1926). P= peculiar chromosome bifurcated at the polar end. Figs. 27-28. Two polar
views of spermatogonial metaphase (after Oguma et Kihara, 1923). P = peculiar chromosome bifurcated at the polar end.
in the first meiosis composed of unequal two parts, they are short and long rods, and separate from each other in the anaphase, so that the second spermatocytes receive twenty-four chromosomes, of which half contain the Y-element and the other half the X-element. These features are common to all mammals.
Summary
1. The spermatogonia of a Japanese man show forty-eight chremosomes. But it is difficult to locate the unpaired chromosomes X and Y in the spermatogonial chromosome-complex. The serial arrangement is of no service, as is illustrated by the example of Trixalis nasuta in which the chromosomes are much larger in size and much fewer in number than in man.
2. In the first maturation division the chromosomes are twentyfour in number. The sex-chromosome and twelve large tetrads encircle the small ones so as to form a rosette in the metaphase of the first division. 1934 On the number of chromosomes and the type of sex-chromosomes in man 489
3. The sex-chromosome is composed of two heteromorphous elements, a long and a short rod. It lies at the periphery of the equatorial plate in the normal condition. Sometimes it is displaced into the centre of the equatorial plate by the effects of the fixation. There is no evidence that the sex-chromosome is of V-shape as reported by Oguma et Kihara, and Winiwarter et Oguma.
4. The X and Y elements conjugate lineally in the first meiosis as is the case in all mammals. This mode of conjugation has been reported by Minouchi as asymmetrical synapsis.
5. These elements separate in the first meiosis; as a result, one of the daughter cells receives the X-element and the other the Yelement, so.that in the metaphase of the second division each of the germ-cells contains twenty-four chromosomes.
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