| OF L

ORNL P
3066
.
:
7
1.0
EEEF EFE
-
|1:25 14 LE
w
MICROCOPY RESOLUTION TEST CHART
NATIONAL BUREAU OF STANDARD$-1963
exŁ
ansiosta
ORNw83066
Conf.670525--2
RASTE

-
-
-
-
-
JUN 1 3 1967
- .
-
.
.
CFSTI BRICIUS
..
..
..
..
..
.
HC12:00; mx_65
..
--...---
-
-
------
The use of sex-chromosome anomalies for measuring radiation
effects in different germ-cell stages of the mouse
Liane Brauch Russell
Biology Division, Oak Ridge National Laboratory
Oak Ridge, Tennessee
-------------------------
-
-
-
-
-
------
-------
The special features that distinguish meiotic systems from other dynamic
cell systems of higher organisms are, first, the fact that far more complex.
chromosomal changes are involved, and, second, the opportunity for studying
effects on such systems by genetic means. Mutagenesis studies in meiotic
systems of mommals began, strictly speaking, in the mid-1930's when ühe
pioneer workers in mammalian radiation genetics, Hertwig, Snell, and
Strandskov, were comparing progeny from matings made at various intervals
after irradiation of the male (see (1) fox review). However, detailed
determinations and interretations were not possible until much later, when
the relations in gametogenesis could be accurately established and the
direct effects of radiation on various cell stages studied (2-6).
----------.-.
-
.-
...
.
.
..
..
. .
.
.
. .
.
.
The two sexes of mammals offer different opportunities for comparing the
genetic effects of radiation in various germ-cell stages. In the male, all :
stages are continuously present throughout adult life. Genetic effecus on
the gonial stage can easily be studied separately by waiting appropriate
intervals after irradiation. Similarly, effects on mature spermatozoa' can
be studied separately in offspring conceived immediately after irradiation.
The intermediate stages. include the meiotic system which is the subject of
this symposium. For these intermediate stages, the timing for the mouse,
worked out by Oakberg and others [4; 7-9), tells us that they cannot be
represented in the ejaculate before certain intervals (e.g., spermatids not
before 7 1/2 days, spermatocytes not before 22 days, etc.). However, at
no time can one be sure of an ejaculate "pure" for specific irradiated
meiotic stages.
.
...
Research sponsored by the U. S. Atomic Energy Commission under contract
with the Union Carbide Corporation. .
LEGAL NOTICE
This report mo propared as an account of Government sponsored work. Neither the Valted
Buates, nor the Commission, nor any person acung on behalf of the Commission:
A. Makes any warranty or representation, expressed or implied, with respect to the accu-
racy, completeness, or usefulness of the information contained in this report, or that the we
of any information, apparatus, method, or procou daclound in that report may not latring.
printoly ownod righto; or
B. Assumos any liabilities with rospect to the un of, or for damagui romultos from the
use of any information, apparatus, method, or procon discloned in this report.
As used in the above, "person acting on behalf of the Commission" inclodou may on-
ploys or contractor of the Commission, or employne of much contractor, to the exteat that
such omployee or contractor of the Commission, or employw of much contractor preparo,
disseminates, or provides access to, any Information purnuant to No employment or contract
with the Commission, or his employment with much contractor.
-
-
-
DISTRIBUTION OF THIS DOCUMENT ES UNA MANERA
In the female, the situation is different. If there is a true oogonial
stage (which is disputed by some investigators), it is no longer present
in embryos older than 14 1/2 days post-conception. To get survival and
fertility from earlier embryos is difficut with anything but very low
doses of acute irradiation. The bulk of the meiotic prophase development
of the female occurs. during fetal life (10, 11). By birth, leptotene and
zygotene have been well completed; and even pachytene is found only with
low frequencies in the day-rld mouse. Diplotene is succeeded by dictyate,
a pseudo-resting stage that makes its first appearance around the time of.
birth. By adulthood, oocytes present in the ovary of the female are all
in dictyate (12), except every five days or so! (i.e., at each estrus) for
a few hours when a small number (arcund 10) continue into diakinesis and
metaphase-I and are ovulated. In the female, there is no haploid germ-
cell stage -- comparable to spermatid or spermatozoon -- since the second
meiotic division is not completed until after sperm entry 0:13, review).
.
.
.-..
---
The contrast between the sexes is thus marked. Genial and mature serm-cell
stages are easy to segregate for study in the male. These are inaccessible
in the female. On t'ie other hand, certain of the intermediate events can
be studied in rather pure cell populations in the female. These are the
dictyate and man with appropriate synchronization -- some of the stages be-
between diakinesis and anaphase II.
..-.. -----------
.
.
.
.
.
.
w imminestones
The germ-cell system also includes events not commonly thought of in this
connection, namely post-copulation stages up to syngamy preceding the first
cleavage. Following sperm entry into the oocyte and the completion of the
second meiotic division, the male and female pronuclei are separate genomes
lying in a common cytoplasm. The pronuclear stage, which is of extremely
short duration in many of the organisms used in mutagenesis studies !e.g.,
insects), is quite long in mammals and thus provides favorable material for
sensitivity comparisons.
. .
.
a
t o
Several indices of genetic change have been employed in comparing germ-cell
stages for radiation effects. These include dominant lethals in both sexes
(14-16, reviews; 17, 18), translocations in the male (14-16), and specific-
locus mutations [19-21).
stalatie de alimentatore skispos
The dominant-lethal category comprises a heterogeneous group of changes of
unknown composition and can, moreover, be studied only with special pre-
cautions in females because of the complications of induced superovulation
and indirect effects [47, 31). The scoring of translocations requires
large-scale fertility testing and is subject to some error. Because of
these considerations, we embarked a few years ago on a program of making
all possible germ-cell stage comparisons in the mouse by using sex-
chromosome aberrations as an index of genetic damage.
I
METHODS
The discovery that the xo mouse is a viable, fertile female (22, 23] and
that XXY is a viable male (24), both types being phenotypically recognizable
in the presence of appropriate X-linked markers, led directly to experiments
in which sex-chromosome aberrations could be used as an index to genetic
damage (25-29, 14-16, 30).
.
.....
..
...............
...
............
The various cell stages investigated so far are summarized in Fig. 1.
Meiotic events remaining to be surveyed are metaphase I, II, and immediately
adjacent stages in both sexes. In the male, these are too short t:) be
recovered in the necessarily mixed sperm samples. In the female, however,
it is quite feasible to isolate some of them for study and this has been
done in dominant-lethal experiments (31, 17). We have recently attempted to
do so for sex-chromosome anomalies, using hormone-synchronized ovulation,
but various practical problems ascribable to this technique have temporarily
delayed the collection of samples of sufficient size.
Some of the marker schemes that have been used in various of our experiments
are shown in Figs. 2-4. In the irradiation of male germ-cell stages (Fig.
2), the use of a mate carrying two closely linked markers in repulsion is
a useful device, since females homozygous for most sex-linked markers have
reduced vigor. It can be seen that, with this scheme, all exceptional
progeny resulting from loss or first-division nondisjunction is pheno-
typically detectable except in the relatively rare crossover classes.
Experiments on predictyate prophase stages in the female require two
generations of marked matings (Fig. 3). The first produces the hetero-
zygotes that must be irradiated in utero (in various stages of development)
or as newborns. These are then allowed to grow up and are mated to males
carrying a different marker. For irradiation of dictyate, the same scheme
applies, except that the heterouygous females are irradiated as adults.
Mates are not added until 24 hours after irradiation in order to avoid
contamination from post-dictyate stages. It may be noted that the mating
scheme in Fig. 3 allows detection not only of maternal 1988 and nondis-
junction but also o. paternal loss (not shown in figure). The latter
would presumably be spontaneous, but could be mimicked by certair, induced
aberrations in the female (see below).
When "zygote" stages are irradiated (Fig. 4), the expected results would
vary according to whether the damage induced was chromosome 1088, chromatid
loss, or nond!! sjunction. Only the first category is phenotypically detect-
able with certainty.
It should be noted that some of the expected exceptional phenotypes can
also be produced by mechanisms other than those shown. The X/0 phenotype
f(marker)
can be the result of an )
genotype, or of a deficiency in the
Y involving male-determining regions. Various chromosome aberrations
leading to non-random X inactivation, as in Searle's translocation (48),
could mimic an X/0 phenotype. Finally, there could be misclassification .
as a result of having a chance preponderance of cells in which the same X
differentiated as the active one. Genetic and/or cytological tests of
exceptional progeny are thus most desirable, whenever they can be made.
Spontaneous frequencies of various sex-chromosome anomalies in the mouse
are quite unequal (26, 16). By far the most common is 1088 of a paternal
sex chromosome (x or Y), and frequencies in various stocks and experiments
vary from about 0.1 to 1.1%. Non-disjunction of paternal X and Y leading
to XXPY ( 24 ) 18 very much rarer, the ratio of XMO to XMXY being about
40:1 where the two could be compared in equivalent situations. Spontaneous
meternal X aberrations are extremely rare, with ox even less frequent than
x"xPY, and XMxMy never having been reported.
i
,"
.
RESULTS
P
The results for various irradiated germ-cell stages in male gametogenesis,
female gametogenesis and the zygote are summarized in Tables I, II, and III,
respectively. In all cases, the last columns have been calculated on the
same basis in order to facilitate comparison between Tables. The adjusted
r R involves subtraction of the control frequency which
is always based on a strictly contemporary and otherwise comparable control
group. This is considered necessary because of the rather wide variation in
spontaneous XO frequency (26).
--
.
..
-
..-
--
Not all phenotypically exceptional animals could be verified by genetic or
cytological tests (see, e.g., some of the presumed Xho animals in Tables I
and II). Siace, however, among these tested, the proportion misclassified
was relatively small, the untested group also was included in calculations
of frequencies. Among the 26 tested phenotypically XO animals in Table I
are 81x animals that had 40 caramosomes but bred like Xo. These are pre-
sumed to have X or Y deficiencies. Two of them, in fact, had a recognizably
short chromosome (16).
--
-
-
-
---
-*--,'trace ....
Trisomies that would result from induced meiotic non-disjunction are detect-
able for only some of the possible genotypes. Thus, none of the XXX's
(Figs. 2 and 3) or XYY's (Fig. 2) can probably be recognized phenotypically:
and the same applies to a fraction of the XXY's, namely those where there
has been a crossover in the female. Moreover, one cannot expect to find
induced trisomies from post-divisionally irradiated stages, i.e., spermatids
and spermatozoa. However, even allowing for all of these factors, it is
evident that induced non-disjunction is quite negligible in comparison with
induced 1088: trisomy was never found after irradiation of females (Table
II); and the single case observed after irradiation of males (Table I) couid
conceivably have been spontaneous. The subsequent discussion 18 thus based
on induced sex-chromosome 1088e8.
**
*
.
.
.
Dettarta
.-
of male germ-cell stages investigated (Table I), spermatids are most sensi-
tive. The chromosome-1088 frequency for all spermatocyte stages takon
together is approximately equivalent to that for spermatozoa (on & per-R
basis). Such a comparison should, however, be made with caution since the
XO yield in the former case is measured in a selected cell population, in
the latter case in an unselected one (15). The first week of the spermato-
cyte sampling (possibly representing mostly post pachytene stages) gave
a lower frequency of exceptions than the following two weeks.
-
2
Irradiation of female germ-cell stages was successful in producing 0
type that almost never occurs spontaneously. The dictyate stage in mature
follicles was found to give a .onsiderably higher yield than did pre-
dictyate prophage stages (Table II). As a matter of fact, dictyate oocytes
in mature follicles appear to be at least as sensitive as are spermatids --
if not more 80. For both of these stages, there is no direct germ-cell
killing with the doses used, and one is therefore comparing chromosome-1088
rates in presumably unselected populations. For earlier stages in both
sexes, the possibility of selection does exist, and it may thus be fortuitous
that leptotene-through-diplotene oocytes, as a group, yield approximately
the same loss frequency as do late spermatocyte stages.
.
The earlier meiotic prophase stages in the female were studied in eight
separate age-groups, namely days 13 1/2, 14 1/2, 15 1/2, 16 1/2, 17 1/2,
18 1/2, 19 1/2, 20 1/2 postconception, the last two of these being post-
natal with the stocks used. The following fertility picture emerged in
the course of this experiment. Pemales irradiated as 13 1/2-day embryos,
and those treated as newborns or day-olds were very severly affected. Only
& small percentage was productive at all; and even those animals that were.
yielded very few young. On the other hand, among females irradiated with
250 R at intermediate stages, days 14 1/2 to 18 1/2 postconception, the
percentage of productive ones was little, if at all, reduced from control;
although the number of offspring per female was only about halt normal,
due to early cessation of breeding. At these intermediate stages, the
bulk of the oocytes are probably in pachytene (11). The very sensitive
stages at each end may represent prepachytere on the one hand, and
diplotene on the other. In newborn mice, Peters, on the basis of histo-
logical studies, considered pachytene and late diplotene quite sensitive
and early diplotone resistant (49). Strain differences very probably hayo
& marked effect on the developmental schedule of the ovary.
As far as induced chromosome loss is concerned, the frequencies are not
high enough to allow a meaningful comparison of different predictyate
stages. The ox is obtained to date came from females irradiated days
13 1/2, 17 1/2, and 18 1/2 postconception.
17 1/2, and obtained to det. comparison of
Irradiation of penetrated oocytes (Table III) can yield frequencies of
sex-chromosome losses that are much higher than those obtained for any
germ-cell stage irradiated in the gonads of either sex. Thus, peak
incidences during that period are 3-4 times as great as those for irradi-
atea spermatid or mature dictyate stages. Comparison of maternally and
paternally contributed chromosomes indicates that sensitivity to X 1088
may be highest during final phases of the second meiotic division of the
oocyte, while sensitivity to X or Y 1088 does not reach its peak until
carly pronuclear stage. (We are now making this comparison more accurately
by using synchronized material.) A very clear result is the sharp drop in
total XO's between early and late pronuclear stage. Loss of a maternal X,
in fact, can apparently no longer be induced after ll a.m. by 100 R (Table
III) or even by 200 R given at 12:30, 2:00, or 3:00 p.m. (15). Paralleling
the drop in total sex-chromosome loss is a decrease in mortality, as
measured either by littersize at birth or by fetal survival (15, 29).
It may be seen from Fig. 4 that irradiation of zygotes could, under certain
circumstances, result in the formation of various mosaics. So far, no
positive evidence for this has been obtained (15), and it has been
tentatively concluded that irradiation of these stages 18 more likely to
cause chromosome loss than chromatid 1088 or nondisjunction at cleavage.
DISCUSSION
Several types of anomaly that would be lethal if they involved antosomes
(14) can be recovered in viable offspring iſ the chromosomes affected are
the X and/or y. It is this circumstance and the fact that many of the
aberrant types can be recognized phenotypically by the use of appropriate
markers that render induced sex-chromosome anomalies particularly
suitable for studying certain genetic effects of radiation and of other
mutagens.
The question of whether sensitivity of sex-chromosomes 18 representative
of chromosome sensitivity in general must, of course, be considered. For
zygotes irradiated in early pronuclear stage, it can be calculated that
the mortality results are ir. good agreement with induced XO frequency,
if one assuues that the probability of eliminating autosomes and sex
chromosomes is, on the average, the same and that 1088 of one or more
autosomes is lethal. (15). For a few other cell stages, the ugreement
was not quite as good, mortality being always slightly greater than
expected on the basis of XO frequency (15, 29). None of the disagreements
within a given stage were, however, major. Morecver, comparison of
relative sensitivities of different cell stages to the induction of sex-
chromosome losses and of dominant lethale (presumed autosomal 1088es) in
general gives good parallelism (see below). If truly substantiated cases
of disagreement should eventually be found, these could be due to various
causes. Thus, at certain stages, sex chromosomes might present an .
atypical proportion of the total chromosomal target, e.g., during male
meiosis, when there is asynchrony of condensation of the sex bivalent.
Alternatively, mortality after irradiation of certain stages might be due
to causes additional to aneuploidy; or there might be a class of aber-
rations which would produce dominant lethality if they involved the
autosomes but would be nondetectable (with the marker setup used) if they
involved the sex chromosomes.
In the mouse, there is no good evidence that nondisjunction is induced by
irradiation of meiotic stages. Despite the relative ease with which the
XO type can be obtained, only one XXY animal was observed in all the
various experiments. This is in contrast to the situation in Drosophila,
where meiotic prophase irradiation has been shown to induce nondisjunction.
(32-36] and where, therefore, a portion of the XO's must be thought of as
complementary to the sex-chromosome trisomics. Because of this situation,
the fact that the highest XO frequency in Drosophila is found following
irradiation of spermatocytes does not necessarily indicate that this
stage is most sensitive to chromosome breakage. Not only nondisjunction
in the purest sense, but perhaps other types of meiotic misdivision
leading to lo88 only (e.g., "anaphase lagging") may contribute to the
high XO yield from premeiotic irradiation.
-
Whether the latter occurs in the mouse, or whether all cases of sex-
chromosome 1088 represent breakage phenomena is not know. Thus, various
stages can be compared only in terms of the effect -- chromosome loss --
rather than the underlying cause for this effect.
-
-
-
-
The finding that the highest frequency of loss following irradiation of
the male mouse occurs after treatment of spermatids parallels results
obtained in dominant lethal studies (37-39, 18) and translocation
experiments (40). Studies of irradiated spermatocytes are complicated
by the relatively high sensitivity to cell killing of these stages.
Thus, even with a dose as low as 200 R, from 13 to 48% -- depending on
exact prophase stage irradiated -- never reach the spermatid stage of
development (9), and some of those that do, go on to form abnormal
sperm. This is a particularly serious complication for dominant-lethal
experiments, since the usual method of calculating preimplantation death
cannot be applied. The easiest way out (and one used by most authors),
that of merely ignoring this component of the total effect, may lead to
results of doubtrud validity. This particular difficulty 18 avoided . .
when the index of genetic damage is sex-chromosome 1088. However, there
is still the possibility that the frequency determination is being :
carried out on a selected population.
The same is true of early oocyte stages, both predictyate and dictyate in
carly follicles. Oocytes in mature follicles, however no both dictyate and
post-dictyate -- are presumably not subject to selection by direct effects.
In fact, it has been shown that arter treatment of the stage most sensitive
to the induction of dominant lethals, meiotic metaphase I, the syerage
number of eles normally ovulated and fertilized (as measured by viable
two-cell embryos formed) was normal, even though only 0.1 offspring per
female survived to fetal life [41).
It is unfortunate that technical difficulties to date have prevented the
accumulation of good sex-chromosome-1088 data for the oocyte stages that
intervene between late dictyate and meiotic anaphase II. Dominant-lethal
studies have shown metaphase I to be very much more sensitive not only than
preceding steges (42, 31, 17) -- paralleling results on insects (43, 44) --
but also slightly more sensitive than the subsequent stages of anaphase I
and metaphase II (17). Metaphase II S probably included in the population.
of zygotes irradiated at 8:30 a... (29). This population is highly sensitive
to induced sex-chromosome loss (Table III);. and if the parallelism with
dominant-lethal results should hold, it might be expected that metaphase I
would give an even higher yield of Xo's. In the comparison between metaphase
II and what was considered an early pronuclear stage, the dominant lethal
data of Edwards and Searle showed a sharp drop in frequency (17]. The XO
data show no drop between 8:30 and 11:00 a.m. [29). At 8:30, most of the
zygotes were already past metaphase II. It is also quite likely that what
was considered early pronuclear stage in the dominant-lethal experiment (17)
actually corresponds to our 3:30 p.m. irradiation, since over 12 hours hard
elapsed since metaphase II.
In speculating on the factors that determine differential sensitivity, the
only clear fact that seems to emerge is that these are probably manifold
and that the answer will be most complex. In one of the most favorable
systems or study, that of the mammalian zygote, it appears that sensitivity
changes may be related to nuclear growth and/or DNA synthesis (29, 45).
The relation between nuclear volume and sensitivity, however, can work in
opposite directions. Thus, as the sperm head swells to become the early
pronucleus, sensitivity increases greatly, but with further volume increase,
it drops again sharply. This may point to DNA synthesis as a more important
factor in the sensitivity change between early and late pronucleus, the
presynthetic or synthetic phase being the sensitive one. High sensitivity
in zygote stages, however, can be the result of other factors as well, since
it is also found at the time of second-polar-body formation. This latter
fits with Magni's suggestion that chromosomes just emerging from meiotic
division are particularly vulnerable (46). However, time relation to
meiosis cannot be a major determinant, for the early paternal pronucleus,
which is post-meiotic by several weeks, 18 as sensitive as the maternal
pronucleus, postmeiotic only by a matter of hours; and it 18 much more
sensitive than the spermatid which is the direct product of male meiosis.
Tae similarity of dictyate oocytes in mature follicles to spermatids,
both with respect to frequency of induced sex-chromosome loss and the
absence of direct radiation killing, is difficult to explain in view of
the fact that one stage is premeiotic and the other postmeiotic. Finally,
the exceedingly high sensitivity of metaphase I, which seems indicated by
dominant-lethal data, cannot be accounted for by any of the interpretations
suggested so far.
SUMMARY
Several types of anomaly that would be lethal if they involyed autosomes
can be recovered in viable offspring if the chromosomes affected we the
X and/or Y. Marker systems have been developed for recognizing a high
percentage of the possible anomalous types that would result from nondise
junction, monosomy, deficiency, and perhaps, certain rearrangements.
Because sex-chromosolie anomalies are thus sspecially suitable for studying
effects of radiation and other mutagens, we have used them in extensive
comparisons of various germ-cell stages in the mouse, including meiotic
systems.
No good evidence has been obtained for X-ray induction of nondisjunction.
On the other hand, the XO phenotype is easily produced, The great.
majority of these cases is the result of simple monosomy, but a few XO"
animals have also been observed that probably carry sex-chromosome
deficiencies or, perhaps, other aberrations.
By far the highest yields of sex-chromosome losses are obtained by irradiat-
ing zygotes, from the time of sperm entry (second meiotic division, through
early pronuclear stage. XM may be relatively more sensitive than XP or I.
during the first part of this period; and there is reason to think that ox
yield for irradiated metaphase-I of the female, when finally measured, may
turn out to be even higher. There is a sharp drop in sensitivity between
early and later pronuclear. stages.
Among gonadal germ cells tested, the ones yielding the highest XO frequencies
are the dictyate in nature follicles of the female, and the spermatid in the
male. · Leptotene-through-diplotene oocytes, and spermatocytes, taken as a
group, give a lower, and roughly equal, yield. Among spermatocytes, post-
pachytene stages give the lowest frequency of xo. These comparisons must,
however, take account of the fact that to yield from irradiation of
spermatocytes and predictyate oocytes is presumably being measured in
selected cell populations.
The factors that determine differential sensitivity are probably manifold
and complex. It is very likely that they vary from one comparison to the
next.
REFERENCES
-
.
..
-
-
.
-
-
-
-
--
-
-
-
-
--
-
,
-
..
.
.
..
.
..
.
..
--
-- --
..
--.
.
-
-
..
.
-
(1) RUSSELL, W. L., "Genetic effects of radiation in mammals", Cn. 12,
Radiation Biology 1 (HOLLAENDER, A., Ed.), McGraw-Hill, New York.
(1954).
OAKBERG, E. F., A description of spermiogenesis in the mouse and its
use in analysis of the cycle of the seminiferous epithelium and gern
cell renewal. Am. J. Anat. 99 (1956) 391.
OAKBERG, E. F., Duration of spermatogenesis in the mouse and timing
of stages of the cycle of the seminiferous epithelium. Am. J. Anat.
99 (1956): 507.
OAKBERG, E. F., Initial depletion and subsequent recovery of spermato-
gonia of the mouse after 20 R gamma- and 100, 200, and 600 R X-rays.
Rad. Res. 11 (1959) 700.
OAKBERG, E., CLARK, E., "Species comparisons of radiation response of
the gonads", pp. 11-24, Effects of Tonizing Radiation on The
Reproductive System (CARLSON, Wi D., GASSNER, F. X., Eds.), Pergamon
Press, O.xford (1964).
EDWARDS, R. G., SIRLIN, J. L., The effect of 200 R of X-rays on the
rate of spermatogenesis and spermiogenesis in the mouse, Exptl. Cell
Res. 15 (1958) 522.
OAKBERG, E. F., Duration of spermatogenesis in the mouse, Nature 130
(1957) 1137, 1497.
SIRLIN, J. L., EDWARDS, R. G., Sensitivity of immature mouse sperm to
the mutagenic effects of X-rays, Nature 179 (1957) 725.
OAKBERG, E. F., DIMINNO, R. L., X-ray sensitivity of primary spermato-
cytes of the mouse, Intern. J. Rad. Biol.2 (1960) 196.
[10] BRAMBELL, F. W. R., The development and morphology of the gonads of
the mouse. I. The morphogenesis of the indifferent gonad and of the
Ovary, Proc. Roy. Soc. (London), (B) 101 (1927) 391.
(11) BORUM, K., Oogenesis in the mouse; a study of the meiotic prophase,
Expti. Cell Res: 24 (1961) 495.
(12) OAKBERG, E. F., The effect ox X-rays on the mouse ovary, Proc. ioth
Int. Cong. Genetics 2 (1958) 207.
(13) AUSTIN, C. R., BISHOP, M. W. H., Fertilization in mammals, Biol. Rev.
32 (1957) 296.
(14] RUSSELL, L. B., "Chromosome aberrations in experimental mammals",
Ch. 7, Progress in Medical Genetics 2 (STEINBERG, A G., BEARN, A. G.,
Eds.), Grune & Stratton, New York (1962).
[15] RUSSELL, L. B., SAYLORS, C. L., "The relative sensitivity of various
germ-cell stages of the mouse to radiation-induced nondisjunction,
chromosome losses and deficiencies", pp. 313-342, Repair from Genetic
Radiation Damage (SOBELS, F. H., Ed.), Pergamon Press, Oxford (1963).
(16) RUSSELL, L. B., "Experimental studies on mammalian chromosome
aberrations", pp. 61-86, Mammalian Cytogenetics and Related Problems
in Radiobiology (PAVAN, C., CHAGAS, C., FROTA-PESOA, 0., CALDAS, L. R.,
Eds.), Pergamon Press, Oxford (1964).
(17) EDWARDS, R. G., SEARLE, A. G., Genetic radiosensitivity of specific
post-dictyate stages in mouse oocytes, Genet. Res. 4 (1963) 389.
LÉONARD, A., Differential radiosensitivity of germ cells of the male
mouse. Canadian J. Genetics and Cytol. 8 (1965) 400.
(19) RUSSELL, W. L., BANGHAM, J. W., GOWER, J. S., Comparison between
mutations induced in spermatogonial and post spermatogonial stages
in the mouse, Proc. 10th Int. Cong. Genetics 2 (1958) 245.
(18)
10
[25]
(20) RUSSELL, W. L., "Evidence from mice concerning the nature of the
mutation process", pp. 257-264, Genetics Today (GEERTS, S. J., Ed.),
Pergamon Press, Oxford (1964).
(21) RUSSELL, W. L., Effect of the interval between irradiation and
conception on mutation frequency in female mice, Proc. Nat. Acad.
Sci. U. S. 54 (1965) 1552.
(22)
RUSSELL, W. L., RUSSELL, L, B., GOWER, J. S., Exceptional inheritance
of a sex-liriked gene in the mouse explained on the basis that the
X/0 sex-chromosome constitution is female, Proc. Nat. Acad. Sci.
U. S. 45 (1959) 554.
[23] WELSHONS, W. J., RUSSELL, L. B., The Y-chromosome as the bearer of
male determining factors in the mouse, Proc. Nat. Acad. Sci. U. S.
45 (1959) 560.
[24] RUSSELL, L. B., CHU, E. H. Y., An XXY male in the mouse, Proc. Nat.
Acad. Sci. U. S. 47 (1961) 571.
RUSSELL, L. .B., SAYLORS, C. L., Factors causing a high frequency of
mice liaving the XO sex-chromosome constitution, Science 131 3409
(1960) 1321.
: [26] RUSSELL, L. B., The genetics of mammalian sex chromosomes, Science
133 (1961) 1795.
[27] RUSSELL, L. B., SAYLORS, C. L., Spontaneous and induced abnormal
sex-chromosome number, in the mouse, Genetics 46 (1961) 894.
(28) RUSSELL, L. B., SAYLORS, C. L., Induction of paternal sex-chromosome
josses by irradiation of mouse spermatozoa, Genetics 47 (1962) 7.
(29] RUSSELL, L. B., MONTGOMERY, C. S., Radiation sensitivity differences
within cell-division cycles during mouse cleavage, Int. J. Rad.
Biol. 10 (1966) 151.
(30) CATTANACH, B. M., Induction of paternal sex-chromosome losses and
deletions and of autosomal gene mutations by the treatment of mouse
post-meiotic germ cells with triethylenemelamine, Mutation Res. 4
(1967) 73.
RUSSELL, L. B., RUSSELL, W. Li, "The sensitivity of different stages
in oogenesis to the radiation induction of dominant lethals and other
changes in the mouse", pp. 187-192, Progress in Radiobiology
(MITCHELL, J. S., HOLMES, P. E., SMITH, C. L., Eds.), Oliver & Boyd',
London (1956).
(32) DEMEREC, M., FARROW, J. G., Relation between X-ray dosage and the
frequency of primary nondisjunction of X-chromosomes in Drosophila
virilis. Proc. Nat. Acaä. Sci. U. S. 16 (1930) 711.
(33) STRANGIO, V. A., Radiosensitive stages in the spermatogenesis of
Drosophila melanogaster, Nature 192 (1961) 781.
(34) SAVHAGEN, R., Relation between X-ray sensitivity and cell stages in
males of Drosophila melanogaster, Nature 188 (1961) 429.
(35) TRAUT, H., The dose-dependence of X-chromosome loss and non-disjunction
induced by X-rays in oocytes of Drosophila i
melanogaster, Mutation
Res. 1 (1964) 157.
(36) FAHMY, O. G., FAHMY, M. J., Radiosensitivity of the stages of spermato-
genesis to different mutagens in Drosophila melanogaster, Mutation
Res. 1 (1964) 247.
RUSSELL, W. L., RUSSELL, L. B. , KIMBALL, A. W., The relative effective-
ness of neutrons from a nuclear detonation and from a cyclotron in
inducing dominant lethals in the mouse, Am. Naturalist 88 (1954) 269.
(31)
(37)
..
.
.
.-
-..
.
...
...
...
(38) RUSSELL, W. L., RUSSELL, L. B., OAKBERG, E. F., "Radi tion genetics
of mammals", Ch. 8, Radiation Biology and Medicine (CLAUS, W. D.,
Ed.), Addison-Wesley, heading (1958).
(39) BATEMAN, A. J., Mutagenic sensitivity of maturing germ cells in the
male mouse, Heredity 12 (1958) 213.
(40) AUERBACH, C., SLIZYNSKI, B. M., Sensitivity of the mouse testis to
the mutagenic action of X-rays, Nature 177 (1956) 376. .
[41) RUSSELL, L. B., Dominant lethals induced at a highly sensitive stage
in mouse oogenesis, Anat. Rec. 125 (1956) 647.
(42) RUSSELL, L. B., SPEAR, R. J., Relation between dominant lethal
incidence and stage in oogenesis irradiated, Rad. Research 3 (1955)
342.
WHITING, A. R., Genetics of Habrobracon, Adv. in Genetics 10 (1961)
295.
1441
PARKER, D. R., "On the nature of sensitivity changes in oocytes of
Drosophila melanogaster", pp. 11-30, Repair from Genetic Radiation
Damage (SOBELS, F. H., Ed.), Pergamon Fress, Oxford (1963).
[45] Work by my student Paul Selvy, unpublished.
6) MAGNI, G. E., "Mutation rates during the meiotic process in yeast",
pp. 77-86, Repair from Genetic Radiation Damage (SOBELS, F. H., Ed.),
Pergamon Press, Oxford (1963).
[47] RUSSELL, L. B. , RUSSELL, W. L., Pathways of radiation effects in the
mother and the embryo, Cold Spring Harbor Symp. Quant. Biol. 19
(1954) 50.
LYON, M. F., SEARLE, A. G., FORD, C. E., OHNO, S., A mouse trans. .
location suppressing sex-linked variegation, Cytogenetics 3 (1964)
306. . '
[49] PETERS, r., Radiation sensitivity of oocytes at different stages of
development in the immature mouse, Rad. Research 15 5 (1961) 582,
...
...
.
.
-
-
-
. .
. .
.
.
.
.

H
.
..
.
TA
.
TABLE I ..
Frequency of induced sex chromosome anomalies from irradiation of male gern-cell stages
Adjusted induced
frequency per
--
No. of
Post-irrad. :
days mated
No. with
exceptional
phenotype
Dose
(R)
Cell stage sampled
animals
classified
1
X 10
XMXXY
2.0 A
1-14
(mostly 1-7)
Spermatozoa
(vas & epididymis):
600
.1112
3+5*
0
Control
1285
.
1285
: 2106
* O
8+3* O
15-21
Spermatids
200
4.3
-
Control
1683
..
22-28
Spermatocytes
(postpachytene)
· 200
2118
i
2+1*
0
1.
00
29-35
Spermatocytes
200
2370
3.3
0.4
8+1*
5+1*
1
0
.
36-42
Spermatocytes
200
1726
2.9
0
Control
3423
ed.
Includes: some data from L. B. Russell and Saylors 1962, 1963 (28, 15).
Died before genetic or cytological tests could be comple
Classified on basis of phenotype only.
Irradiated minus contemporary control. Based on 50% of offspring
of singly beterozygous; 100% of offspring of doubly heterozygous mates.
“Induction of XXY not expected since post-divisional stages were irradiated.
--
-
-
-
--
--
-
--
-
-
- -
-
-
-
-
-
.
..
-
-..
.
.
.
-
-
-
-
-
-
-
.
-
-
-
-
:
.
*
-
,
. -
-
-
- -
- -
- -
-
-
-
-
-
-
-
--
-.
.
-
-
..-
.
..
-
-
-
-
-
-
-
.
-
-
-
-
- -
..
.
-
-
.
.
.
-
-
-
.
-
-
-
TABLE II
Frequency of induced sex-chromosome anomalies from irradiation of female germ-cell stages
Age irradiated
Stage sampled
Doses
Individual Weighted
groups
average
No.
animals
classified
_ % loss
XM' pory
Adjusted
induced
frequency
of ox
per r x 10-5
Days 13 1/2-20,1/2, Pre-leptotene
postconception
through diplotene
150,200,250
221
2402
1.4
0.3.0.24-0.3
2.4 0.0 -0.3
Adult
Dictyate
(mature follicles)
100,400
342
331
7.1
Control
0
0
785
'Estimated frequency of occurrence, calculated by taking account of the fact that
only half of all losses of x are detectabl. (0/Y being prenatally lethal).
Groups separated by daily intervals.
The lower number represents only tested or cytologically verified animals.
PRange in other experiments is 0.1-1.1%.
over 90% of classified young were conceived within 2 weeks after irradiation.
TABLE III
Frequency of sex-chromosome less after J.00 R irradiation at
various stages between sperm entry and the first cleavage
Adjusted induced
frequency per
Time irradiated
Stage of development
No.
classified
Series
% loss
r x 10°
xor y M
8:30 a.m.
Completion of second-
polar-body formation.
A few already in pro-
nuclear stage
201
1.0
3.0
4.9
29.8
11:00 a.m.
Most zygotes in early
pronuclear stage .
A
B
422
227
3.3
1.8
1.9
1.8
23.0
12.5
19.0
17.6
3:30 p.m.
All in pronuclear stage.
Nuclear growth and DNA
synthesis probably
completed.
A
0
70
193
1.4
0.5
4.0
0.1
Control
822
196
1.0
0.5
0
0
"All on the day of the vaginal plug. Plug check about 8 a.m.
The two series are separated by 3 years.
Series A: Mothers from 4 different strains, D, T, C3H, (101 X C3H)F, (L. B. Russell and Saylors, 1963).
Series B: Mothers all (101 x C3H)F, (L. B. Russell and Montgomery, 1966).
Estimated frequency of occurrence, calculated by taking account of the fact that only half of
all losses of XM are detectable.
LEGENDS
FIG. 1. Approximate time of occurrence of various gern-cell stages in the
mouse, with wavy arrows indicating those irradiated in experiments
with induced sex-chromosome anomalies. (Modified from [15]).
FIG. 2. Mating scheme for experiments designed to detect sex-chromosome .
anomalies induced by irradiating various germ-cell stages of the
male rouse. Exceptional phenotypes that are phenotypically recogni-
zable are encircled with an unbroken line. A broken circle indicates
those that are only questionably recognizable or detectable by further
test of raro classes.
FIG. 3. Mating scheme for experiments designed to detect sex-chromosome
anomalies induced by irradiating various germ-cell stages of the
female mouse. Circles and broken circles as in Fig. 2.
(Modified from (15]).
FIG. 4.
Theoretical expectations for various abnormal events Layolving sex
chromosomes of zygotes. (Modified from (15)).
11447701

O GERMLINE
LATE FETUS,
NEONATAL
$ GERMLINE
MEIOTIC PROPHASE
(through diplotene)
m
POSTNATAL
and ADULT
...
..
(all stages present at
DICTYATE
all times)
beginning follicles
mi to
SPERMATOGONIA A
most mature follicles SPERMATOGONIA B
DIAKINESIS
MEIOTIC PROPHASE
(hours preovulation)
MEIOTIC METAPHASE 1,1
MEIOTIC METAPHASE I
SPERMATIDS
SPERMATOZOA
MEIOTIC METAPHASE I
SPERM PENETRATION OF VITELLUS
W PRONUCLEAR STAGES
FIRST CLEAVAGE
..
..
..-37
*
*
S*
POST-
COPULATION
10336-) :
TO +
x
+ Blo
NORMAL PROGENY
NON-C.O : C.O.
EXCEPTIONAL PROGENY
NON-C.o.
C.O.
Ta +
+ +
TO Blo
+ +
m
To
+
Ta Blo
oyen :
TO
+
+
+
*
*
+ Blo
+
t.
+
+
+ Blo
+
+
DIO
+ Blo

Fig 2.
.:'.
. per
. .
.
.
EL
T
..
.
I
17550
PRENATAL (VARIOUS STAGES)
NEONATAL, OR ADULT

NORMAL PROGENY
EXCEPTIONAL PROGENY
NON-C.O.
CO.
"Ta t
cele
Tat
+ Blo
Fig. 3

10339-1
ZYGOTE
CHROMOSOME!
LOSS
CHROMATID
LOSS .
NONDISJUNCTION
CHROMOSOME
LOSS
CHROMATID
LOSS
NONDISJUNCTION

dies

-
-
'12.
2
95

1
75W
"I
"",
ideia
B
*
.
RI
END
DATE FILMED
8 / 25 /67







..:.
.
.
.
.
G
NIE!
at
7
,
..
.