INDUCTION O F PATERNAL SEX CHROMOSOME LOSSES
BY IRRADIATION OF MOUSE SPERMATOZOA
LIANE BRAUCH RUSSELL
AND
CLYDE LEE SAYLORS
Biology Division, Oak Ridge National Laboratory,l Oak Ridge, Tennessee
Received November 10, 1961
N experiments already reported (RUSSELLand SAYLORS
1960; RUSSELL1961)
I we were able to induce loss of a sex chromosome by irradiating during the
interval between sperm entry into the oocyte and the first cleavage. This interval
is relatively long, and during a 4x-hour span within it, there is a change in
sensitivity to sex-chromosome loss and to lethal effects. Since the chromosomal
state of the haploid pronuclei within the zygote thus appears to change with time,
it seemed of interest to investigate the haploid sperm nucleus prior to its entry
into the oocyte with respect to frequency of total or partial sex-chromosomeloss
1961.)
from irradiation. (For preliminary summary see RUSSELLand SAYLORS
Furthermore, sex-chromosome loss induced in spermatozoa may be useful as a
measure of chromosome damage in comparative experiments (e.g., dose rates,
protective treatments). Finally, the present results will be of interest in comparison with data now being collected on nondisjunction frequencies from the irradiation of meiotic stages in both males and females.
While the present experiment succeeded in providing frequency estimates for
induced sex-chromosome “ ~ o s s that
’ ~ are useful for certain comparisons, it must
be considered preliminary in that the exact nature of the losses has not yet been
analyzed.
METHOD
Wild-type males [C3H/R1 and (101/R1 x C3H/R1)F1] were irradiated with
600r X rays (250 kvp, 15 ma, anterior two thirds of body shielded) and mated for
two weeks immediately following irradiation to females bearing dominant sexlinked markers. Since almost all matings in the present experiment occurred in
the first week following irradiation, we feel that the results obtained concern
mostly irradiated spermatozoa. To keep numbers of progeny approximately equaI
in irradiated and control groups, about twice as many matings were set up in the
former than in the latter to allow for loss due to dominant lethals.
Markers used were Ta = Tabby and BZo = Blotchy, two sex-linked dominants,
viable in the male. Crossover frequency Ta-BZo is about four percent (RUSSELL
1960). Phenotypic relations involving T a may be summarized as follows:
T a n 8 = Ta/O 0 = Ta/TaP distinguishable from Ta/+ 0 = Ta/+/Y 8 distinguishable from
P = +/O 0 = +/Y 8 . The situation for BZo is analogous. Mates
+/+
loperated by Union Carbide Corporation for the U. S. Atomic Energy Commission.
Genetics 47 : 7-10 January 1962.
8
L. B.
RUSSELL A N D C . L. SAYLORS
+/+
for the wild-type males were Ta/+, or B l o / f , or T a
Blo. Irradiated and
control groups were closely matched for distribution of these various genotypes
as well as for age of mates. The exceptional animals scored were daughters which,
on the basis of phenotype, were presumed to be hemizygous or homozygous for
the sex-linked marker. The probability of homozygosity seems so small that it
can be ignored for practical purposes. Hemizygosity of the marker in a female
could result from loss of an entire paternal sex chromosome, either X or Y; or
from X deficiencies involving the loci in question; or, from Y deficiencies involving all male-determining regions. The distinction between monosomy and deficiency can be made cytologically, and preliminary analysis, by DR. E. H. Y.
CHU, of a random sample of the exceptional animals indicates that both conditions are represented (two females with counts of 39,XO; one female with a
count of 40, probable deficiency in X ) . Consequently, the phrase “paternal sexchromosome loss” as used may include either total or partial (of the types
specified). The symbol “0” in Ta/O aEd BZo/O and in X’/O is used in a similar
sense.
In the case of Ta/+ and Blo/+ mates, the incidence of T a l 0 or Blo/O daughters
represents 50 percent of the progeny with paternal sex-chromosome ‘‘loss.’’ The
other 50 percent, which are S/O, are not detectable by phenotype, since they are
indistinguishable from the normal segregant
When the mates used are
Ta
Blo, detectability of XM/O progeny is 100 percent, providing that the
rare wild-type crossover females are further tested to determine whether they are
or +/O. All offspring were examined on the day of birth and at least
once a week thereafter until the time of weaning.
+/+.
+/+
+ +/+ + +
RESULTS
Table 1 lists results to date. On combining the data from all types of matings
used, a statistically significant increase in estimated frequency of paternal sexchromosome loss is obtained as a result of irradiation. (P = 0.01 1, using FISHER’S
exact test; we wish to thank DR.M. A. KASTEKBAUM
of the ORNL Mathematics
Panel for this calculation.) For calculation of X”/O frequencies, as well as for the
probability computation, only one half of the total classified progeny of Ta/+
and N o / + mates was used, in order to make allowance for the fact that only 50
percent of XIcI/Odaughters from such matiiigs were detectable.
The estimated frequencies, from all types of mating, of daughters hemizygous
for the maternal-X marker were 0.14 percent for controls and 1.32 percent for
males that had received 600r to sperm. Because of viability problems, these percentages may underestimate the actual frequencies, at least as far as contribution
from whole-chromosome loss is concerned. Monosomy for X is known to produce
a slight decrease in viability, even when the X is wild-type (RUSSELL,
RUSSELL
and GOWER1959). While Ta/O, as shown by independent data, is probably not
markedly more inviable than +/0 (at least up until the age at which exceptionals
are detected), hemizygosity for Blo may cause additional inviability (note relatively lower frequency of Blo/O exceptionals in Table 1 ) .
INDUCED SEX-CHROMOSOME LOSSES
9
TABLE 1
Frequency of partial or total paternal sex-chromosome loss following irradiation of spermatozoa
Progeny
Treatment
4
e
*
Mate
Exceptional* Other classified
females
females
Classified
males
Percent
daughters hemizygous
for marked X” locust
TQ/+
1%
318
335
0.31
BW+
0
0
249
46
263
73
...
1
613
671
0.14
...
0
Tu+/+ Blo
Total
E%
Tu/+
5t
1.77
1s
259
216
30 1
BW+
226
0.46
Ta+/+Blo
Total
2%
8
57
532
45
5 72
1.92
1.32
I/)
0
*
i,
* Hemizygous for marker on XM. This can be the result of loss of the entire paternal sex chromosome or of certain
deficiencies in XP or Y (see text).
t Estimated frequency qf occurrence calculated by taking account of detectability which, in the groups where To/+
or Blo/+ mates are used, is only one half (see text).
Z TdO.
$! Blo/O.
Dominant lethal incidence in this experiment may be estimated by reduction
in litter size and in average number of litters. The average numbers of young
born per litter in control and irradiated groups were 5.63 (= 1363/242) and 3.12
(= 1242/398), respectively; the average numbers of litters per female mated
were 0.81 and 0.66, respectively. If the relatively smaller average number of
litters in the irradiated group were due entirely to fewer conceptions, then dominant lethal incidence, calculated on the basis of young per litter, would be 44.6
percent. If it were due entirely to some litters with 100 percent death, dominant
lethal incidence, calculated from young per female mated, would be 56.1 percent.
The true value is undoubtedly somewhere in between but probably closer to the
former estimate.
DISCUSSION
Comparison of frequencies of paternal sex-chromosome “loss” and dominant
lethals is of interest since most losses of the type scored in the present experiment
would, if they affected autosomes, probably lead to dominant lethality. ‘On the
other hand, only a portion of the type of aberrations that can lead to dominant
lethality (deficiencies, asymmetrical interchanges) would, if they involved the
sex chromosome, be detectable with the present marker setup. Nondetectable
would be deletions of X not involving the marked loci, or deletions of Y not
involving male-determining regions. I n addition, asymmetrical interchanges
involving a sex chromosome would be added to the dominant lethal category.
Thus, unless the sex chromosomes were differentially sensitive to breakage, the
ratio of frequencies of sex-chromosome “loss” (scored by the present method)
to dominant lethals should be smaller (by an unknown quantity) than the ratio-
10
L. B. R U S S E L L A N D C. L. SAYLORS
of average length of X and Y to total length of haploid autosomes. The former
ratio is about 1/40 in the present experiment. Nothing is known concerning
relative chromosome lengths in sperm, but rough measurements from a mitotic
metaphase karyotype prepared by CHU (RUSSELLand CHU 1961) yield 1/24 as
the ratio of average length of X and Y to total length of haploid autosomes. In line
with the above argument, the former ratio is indeed somewhat smaller than the
latter. However, in view of the fact that the expected quantitative relation between these two ratios is unknown, it would be premature to draw any conclusion
regarding relative sensitivities of sex chromosomes and autosomes other than if
an inequality does exist, it is probably not a very great one.
A comparison may be made between the induction of paternal sex-chromosome
loss in spermatozoa and in the male pronucleus formed shortly after sperm entry
into the oocyte. With 600r to spermatozoa, the induced rate (experimental minus
control) is 1.2 percent (Table 1). With only 100r delivered after sperm entry
( 11:00 AM postcopulation), the induced rate for paternal losses is 2.3 percent
(RUSSELL1961). It, therefore, appears that irradiation of the male pronucleus
may be more effective by an order of magnitude than irradiation of sperm in
causing paternal sex-chromosome losses.
SUMMARY
Paternal sex-chromosome loss, both partial and complete, can be induced by
irradiation of mouse spermatozoa, 600r giving an induced frequency of at least
1.2 percent of daughters hemizygous for a marker on the maternal X. Comparison with dominant lethal frequency indicates that sensitivity of the sex chromosomes is probably not too unlike that of autosomes. Comparison between the induction of paternal sex-chromosome loss in spermatozoa, on the one hand, and in
the male pronucleus shortly after sperm entry into the oocyte, on the other,
indicates the latter stage to be more sensitive by approximately one order of
magnitude.
LITERATURE CITED
RUSSELL,LIANEB., 1960 Mouse News Letter 23: 58-59.
1961 Genetics of mammalian sex chromosomes. Science 133: 1795-1803.
RUSSELL,LIANEB., and E. H. Y. CHU, 1961 An XXY male in the mouse. Proc. Natl. Acad.
Sci. U. S. 47: 571-575.
RUSSELL,LIANEB., and C. L. SAYLORS,
1960 Factors causing a high frequency of mice having
the XO sex-chromosome constitution. Science 131 : 1321-1322.
1961 Spontaneous and induced abnormal sex-chromosome number in the mouse. Genetics
46: 894.
RUSSELL,W. L., LIANEB. RUSSELL,and J. S. GOWER,1959 Exceptional inheritance of a sexlinked gene in the mouse explained on the basis that the X/O sex-chromosome constitution
i n female. Proc. Natl. Acad. Sci. U.S. 45: 554-560.