View source for 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

Si ff
aa (UL
~ ohne @ s* ZU2 4
DIDS me MASS
Y Pe ING F Peg e "we
= ty ee (Sd SD
ow? () C7 ams - mu! 4
BR a
<a 7) 2 ) c)
SF G aa Net
023 FIG Gum TW. CO,
HDD AZ LZ SUV (Ale
CREA Gye
ef) ey alt » ZiRb ~ ee
SG ~ a? CX
3 Q CoP , “78H
qe
ofthe IN) -e
ate4 4 Ss aT) &
at Tbh Sele Sy" Ce
pee MALE SNe) Sao
SIEM IS SASS Sa Gy FA
Says Gere Bvixx
Aes =* oye
olen TA Hal

e

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

; hes 9 apes
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.

Literature cited

Iriki, S. 1980: Studies on amphibian chromosomes. I. On the chromosomes
of Hyla arborea japonica. Mem. Coll. Sci. Kyoto Imp. Univ.,
Series B. 5.

1932a: Studies on amphibian chromosomes. IV. On the chromosomes
of Rana rugosa and R. nigromaculata. Sci. Rep., Tokyo
Bunrika Daigaku. Section B, I.
—___ 1932b: Studies on amphibian chromosomes. V. Sex-chromosomes type
of Hyla arborea japonica from the standpoint of spiral strueture. ibid.
1932c: Studies on amphibian chromosomes. VI. On the chromosomes
of Diemyctylus pyrrhogaster. ibid.
1932d: Studies on amphibian chromosomes. VII. On the chromosomes
of Megalobatrachus japonicus. ibid.
1932e: On the fixation of the chromosomes of Hyla arborea japonica
and some other animals. ibid.
1982f: Studies on the chromosomes of Pisces. On the chromosomes
of Aplocheilus latipes. ibid.
Kemp, T. 1929: Uber das Verhalten der Chromosomen in den somatischen
Zellen des Menschen. Z. Zellf. Mikr. Anat. 16.
Minouchi, O. 1928a: On the fixation of chromosomes in mammals and some other
animals. Jap. Jour. Zool., I.

1928b: On the spermatogenesis of the albino rat. ibid.

1928¢e: On the spermatogenesis of the dog, with special reference to
meiosis. ibid.
A new conception of the mode of synapsis. ibid.

—__—_. 1929: On the spermatogenesis of the racoon-dog. Cytologia I.

Minouchi, O, ous

and Iriki, S, 1931: Studies on amphibian chromosomes. II. On the chromosomes
of Bufo japonicus. Mem. Coll. Sci., Kyoto Imp. Univ. Series
B. 6.

Nakamura, K, 1928: On the chromosomes of a snake, Natrix tigrina (Studies on re
ptilian chromosomes I). Mem. Coll. Sci., Kyoto Univ., Series

B. 4.
Nakamura, K, 1981:
————— «1982:

Oguma, K.
Oguma, K.

et Kihara, H, 1923:
19238 :

Painter, T.

Shiwago, P.

u. Andres, A, 1932:

Stohler, R.

Winge, O.

Winiwarter, H, 1912:

Winiwarter, H.

et Oguma, K. 1926:
1924:

Witschi, E.

1933;

1930 :

1928 :

1930:

Q. Minoucu! and T. OHTA Cytologia 5

Studies on reptilian chromosomes. II. On the chromosomes of
Eumeces latisculatus (Hallowell), a lizard. Cytologia 2.
Studies on reptilian chromosomes. III. Chromosomes of some
Geckos. Cytologia 3.

A further study on the human chromosomes. Arch. Biol., 40.

Etudes des chromosomes chez l'homme. Arch. Biol., 33.
Studies in mammalian spermatogenesis. II. The spermatogenesis in man. J. Exp. Zool., 37.

Die Geschlechtschromosomen in der Spermatogenese des
Menschen. Z. Zellf. mikr. Anat., 16.

Zytologische Untersuchungen an den Keimdriisen mit’ eleuropaischer Kréten. Z. Zellf. mikr. Anat., 7.

Zytologische Untersuchungen tiber die Natur maligner
Tumoren. II]. Teerkarzinome bei Madusen. Z. Zellf. mikr.
Anat., 10.

Etudes sur la spermatogénése. Arch. Biol., 27.

Nouvelles recherches sur la spermatogenése humaine. ibid., 36.
Die Entwicklung der Keimzellen der Rana temporaria L, Z.
Zellf. mikr. Anat. I.

Contribution to the cytology of amphibian germ cells. I.
Chromosomes in the spermatocyte divisions of five Nortb
American species of Toads. Cytologia 4.