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Nuclear sex in species showing male homogamety.

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Nuclear Sex in Species Showing Male Homogamety
DAVID 3. B. ASHLEY AND ERICH A. THEISS
A r m e d Forces Institute o f Pathology a n d Veterans Administration Central
Laboratory f o r Anatomical Pathology a n d Research,
W a s h i n g t o n , D. C .
Barr and Bertram (’49) introduced the
valuable technic of nuclear sexing to the
cytological study of problems of sex differentiation. Their method involves the
recognition of a small mass of chromatin
in the nucleus of the resting somatic cell
in one sex only. The original observations,
made in the cat, have been extended to a
wide variety of other species; Grumbach
and Barr (’58) state that a nuclear sex
difference can be demonstrated in cells
of the somatic tissues in representatives
of the mammalian orders, Marsupalia, Artiodactyla, Carnivora and Primate but that
such differentiation is not possible in
rodents.
In mammals, the class to which all these
orders belong, the sex chromosomes are
distributed in the manner of female homogamety, i.e., the female has the pair
of X chromosomes and the male has a dissimilar X and Y, and the “sex chromatin”
of Barr is seen in the female but not in
the male. There is a considerable body
of genetic evidence that the mass of chromatin seen in the cells of the females of
these species is the heterochromatic form
of the pair of X chromosomes, although the
point has not yet been proved cytologically.
If the technic of nuclear sexing could
be used in species in which there was male
homogamety, i.e., in which the male carries the pair of X chromosomes and the
female the unpaired X and Y, the sex
chromatin mass should be seen in the
male and not in the female. Such an observation would provide evidence of the
identity of the sex chromatin body of the
resting cells with the pair of sex chromosomes. Only one attempt to demonstrate
sex chromatin in a species belonging to a
lower order of animals has so far been
reported. Brusa (’52) examined the nerve
cells of the pigeon and was unable to
detect any sex difference.
Male homogamety is seen in vertebrates
of the class Aves (Birds), in which all
members show this chromosome constitution, and in the classes Reptilia, Amphibia
and Pisces. In the latter three classes the
chromosome constitutions are not constant; some species show male homogamety and some female (deRobertis, Nowinski and Saez, ’54).
We decided to undertake an investigation of the nuclear appearance of cells
from birds and reptiles in an attempt to
find a species in which it was possible to
determine the sex by examining nuclei
of the resting somatic cells. Specimens
were obtained from a series of 20 domestic
fowl; 10 male and 10 female; 20 “ducks.”
10 male and 10 female; 8 parrots and
parakeets, 5 male and three female, and
from a group of 15 snakes, 7 male and 8
female. The group of snakes included
members of several families.
Sections of various tissues (including
epidermis, liver, heart, epithelium and
smooth musculature from the alimentary
tract) from these animals were prepared
and stained by the routine hematoxylin
and eosin method, which is usually adequate for the detection of sex chromatin,
and also by the Feulgen technic (Pearse,
’ 5 3 ) , which is a specific histochemical
method for the identification of desoxyribonucleic acid.
Sections from specimens of known sex
were examined to try to detect the nature
of the sex difference, if any. Search was
made for chromatin masses similar to
those seen in the human and in many
mammalian species. No such body was
found. The paranucleolar satellite seen
in the cat nerve cells was also sought;
again no such body could be found. Finally, the cells were studied individually
* Dillon
Research Fellow.
115
116
DAVID J. B . ASHLEY AND ERICH A. THEISS
in an effort to establish some other criterion on which to base cytological differentiation of the sexes in these species.
We were unable to find any method of
distinguishing between the cells of the
male and female in any of these groups
of animals. In both sexes the cells contained many condensed masses of chromatin, some of which bore a close resemblance to the sex chromatin of man, but
it was not possible to demonstrate that
any one of these was to be found in only
one of the sexes (figs. 1 to 5). Counting
the chromatin condensations and comparing the numbers in the two sexes did not
add any further information; in both sexes
the number of chromatin bodies was inconstant; the wide range seen was similar
in males and in females and sex differentiation by this method was impossible.
The sections were studied by methods
designed to show true associations between
the cytological appearances and the sex
of the individual, if such an association
existed. Several times a sex difference
was thought to be recognizable, but when
the slides were examined “blind” and the
results compared with the known anatomical sex it was found that no clearcut difference existed. In all cases the final test
was the ability to diagnose the sex of the
individual using the appearance of the
cells alone.
DISCUSSION
The results of the investigation reported
here are negative. It was impossible to
find criteria by which the technic of nuclear sexing could be extended from the
mammalian species, in which it is so
useful, to certain species of the lower orders of animals. The reasons for this
failure are, however, worthy of comment.
The cells examined in all cases contained
many clumps of chromatin; some of these
conformed with the solitary mass seen in
the cells of the human female, but in no
case could an association with the functional sex of the individual be detected.
An irregular chromatin condensation is
also seen in rodents and in the rabbit and
is the reason why nuclear sexing cannot
be used in these groups. The reason for
this irregularity is not clear. In those spe-
cies in which nuclear sexing is possible the
amount of heterochromatin is small and
appears to be concentrated in the sex chromosomes; the chromatin of the autosomes
is widely dispersed throughout the nucleus
in the intermitotic phase of the life of
the cell, and any condensation which occurs is of submicroscopic size. The chromosome number does not seem to be
relevant in this matter. The species in
which nuclear sexing is possible have chromosome numbers ranging from 22 in
the case of the opossum to 78 in the case
of the dog; in the mammalian species
in which nuclear sexing is not possible the
chromosome number ranges from 40 to 64
(Spector, ’56). In the class Aves, from
which most of the specimens in this study
were taken, the chromosome number varies from 72 to 84 and it might be concluded that the large number of presumably small chromosomes is responsible
for our inability to detect the sex chrcmosome complex. However, the chromosome numbers in the reptiles are much
smaller, 22 to 56; the range in rodents,
which are also unsuitable for this investigation, is also low, 40 to 64, and the
absolute number of chromosomes does not
appear to be the critical factor. The conclusion which we have reached is that
the feasibility of the use of the technic
of nuclear sexing depends on the cytological characteristics of the species concerned, the size and shape of the sex chromosomes, the amount of heterochromatin
in the whole of the chromosomes and its
distribution, and the nature of the mitotic
cycle.
It is unfortunate that almost all of the
species studied in this connection show
female homogamety and that i t has been
impossible to distinguish between the male
and female on the basis of the cytological
appearance of the somatic nuclei in species of the lower orders. We consider that
further efforts should be made to find
animals in which the nuclear sex can be
determined and in which there is male
homogamety. Proof of the identity of
the pair of X chromosomes with the sex
chromatin particle would be of invaluable
assistance in understanding the errors of
NUCLEAR SEX I N MALE HOMOGAMETY
sex development which have been uncovered by the application of this new technic.
SUMMARY
It has not proved possible to extend the
technic of nuclear sexing to cells from
three groups of birds and a group of
snakes. Probably the individual cytological
characteristics of the cells of a species determine whether or not it is possible to
carry out nuclear sexing. It is concluded
that the chromosome number, per se, is
not the critical factor.
It is urged that fresh efforts should be
made to find a species showing male
homogamety which is susceptible to this
type of investigation in order to provide
evidence as to the nature of the sex chromatin body.
ACKNOWLEDGMENT
We are indebted to Dr. G. S . McKee of
the U. S. Department of Agriculture who
obtained for us the specimens of fowl
and duck tissue.
117
ADDENDA
Since the manuscript was completed,
Kosin and Ishizaki (Science 130, 43-44,
1959) have claimed that sex chromatin
can be demonstrated in the cells of the
female domestic fowl (Gallus domesticus).
We have no evidence from our studies that
this is the case.
LITERATURE CITED
Barr, M. L., and E. G. Bertram 1949 A morphological distinction between neurones of the
male and female, and the behavior of the
nucleolar satellites during accelerated nucleoprotein synthesis. Nature, 163: 676-677.
Brusa, A. 1952 A propos de la structure du
noyau de la cellule nerveuse. Anat. Anz., 98:
343-352.
Grumbach, M. M., and M. L. Barr 1958 Cytologic tests of chromosomal sex i n relation
to sexual anomalies i n man. Rec. Progr. Hormone Res., 14: 255-334.
Pearse, A. G. E. 1953 Histochemistry, Theoretical and Applied. Churchill, London, p. 425.
deRobertis, E. D. P., W. W. Nowinski and F. A.
Saez 1954 General Cytology. W. B. Saunders, Philadelphia, ed. 2, p. 325.
Spector, W. S. 1956 Handbook of Biological
Data. W. B. Saunders, Philadelphia, p. 93.
PLATE 1
EXPLANATION OF FIGURES
1 Hepatic cells from a male fowl showing numerous chromatin condensations within the
nuclei. Several of the granules resemble sex chromatin i n size and location. Neg.
58-14428. Feulgen. x 1750.
118
2
Heart cells from female fowl. The nuclear chromatin condensations are qualitatively
similar to the ones seen i n male tissues. Neg. 58-14431. Feulgen. x 1750.
3
Hepatic cells from a drake showing numerous nuclear chromatin condensations, many
of which resemble sex chromatin. Neg. 58.14429. Feulgen. X 1750.
4
Heart cells from a duck. The nuclei contain many chromatin granules which resemble
sex chromatin in size and location. Neg. 58-14430. Feulgen. X 1750.
5
Squamous epithelium (epidermis) from a female rattlesnake. The nuclei contain
numerous irregular chromatin condensations. Many of the granules are indistinguishable
from sex chromatin. Neg. 58-14432. Hematoxylin and eosin. x 1750.
NUCLEAR SEX IN MALE HQMQGAMETY
David J. B. Ashley and Eric A. Theiss
PLATE I
119
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