DOMINANT L E T H A L A N D INACTIVATION E F F E C T S OF
NITROGEN MUSTARD O N HABROBRACON S P E R M
A N N A R. WHITING AND ROBERT C. VON BORSTEL
Zoological Laboratory, Uwiversity of Pennsylvania, Philadelphia, Pennsylvania
1
Received September 14, 1953
early as 1911 0. HERTWIC
demonstrated two types of behavior of irradiated Amphibian sperm. One of these, the dominant lethal effect,
resulted in death of embryos due to fusion of injured sperm nucleus with egg
nucleus. The other, inactivation of sperm, stimulated the egg to develop gynogenetically since injured sperm entered it but took no part in development.
PACKARD
(1914) observed, in addition to these phenomena, a second type of
inactivation in which heavily irradiated Nereis sperm failed to penetrate the
egg. With this type likewise, eggs were activated to develop gynogenetically.
MULLER(1927) used the partial sterility of irradiated Drosophila males as
evidence “ f o r the first time, of the occurrence of dominant lethal genetic
changes, both in the X and in the other chromosomes.’’
The parasitic wasp Habrobracon juglandis was used for the present study.
In the cross utilized about 62% of the eggs are fertilized and develop into
diploid females ; 38% are not fertilized and develop parthenogenetically into
haploid males. In this type of reproduction dominant lethal effects can be
distinguished from sperm inactivation without cytological study. Any treatment of sperm which reduces hatchability at the expense of females, leaving
number of males unchanged, has induced dominant lethal effects only. When
hatchability is reduced to 38% and all survivors are males, there has been
induced in each sperm at least one dominant lethal. When, after the attainment of complete dominant lethality, hatchability rises above 38% and all survivors are males, some sperm have been inactivated and are either entering
the eggs without taking part in development or are failing to enter them.
These facts are contrasted with expectation in a form like Drosophila in table
1. In making tests for distinguishing dominant lethal effects from inactivation,
an advantage lies in the fact that polyspermy is rare in Habrobracon. About
1% of fertilized eggs have two sperm in them. Almost every fertilized egg
therefore represents a test of a single sperm so that masking of inactivation by
dominant lethality occurs rarely.
STANCATI
(1932) recognized the possibility of inactivation and the fact
that it could be identified readily in Habrobracon but showed conclusively
that after exposure of males to 2500 r, dominant lethal changes only were
AS
1 This study was supported in part by a research grant to the senior author from the
National Cancer Institute of the National Institutes of Health, Public Health Service.
Part of the work was done while the junior author held an Atomic Energy Commission
Predoctoral Fellowship. The authors express their appreciation to LESLIEE. PECKHAM
and HENRYH. JONES of the Camegie Institution, Cold Spring Harbor, New York, for
preparation of the figures.
GENHICS 391 817 May 1954.
318
ANNA R. WHITING AND ROBERT C. VON BORSTEL
induced. MAXWELL(1938), P. W. WHITING (1938) and HEIDENTHAL
(1945) demonstrated that at X-ray doses above 10,OOO r some sperm are inactivated as demonstrated by the fact that survival ratio increases after having
fallen to 33% at lower doses. Recently (1953) BROWN
and CAVEhave shown
that, due to dominant lethals, after the application of 2OC04000 r to pollen of
Lilium formosarum, few or no seeds are set. In competition with untreated
pollen, treated grains are not handicapped in ability to achieve fertilization.
All investigators agree in finding no inactivation until after the attainment of
complete dominant lethality.
TABLE 1
Theoretical expectations /or hatchability percentages and sex ratios of adult
survivors /or Drosophila and Habrobracon under different conditions of dominant
lethality and sperm inactivation.
Adult survivors
% hatchability
Condi ti ons
% 66
Controls
Drosophila
Habrobracon
%
w
100
100
50
38
50
62
50%dominant lethals
Drosophila
Ha bro bracon
50
50
69
55
50
45
100%dominant lethals
Drosophila
Habrobracon
0
38
0
100
0
0
100% dominant lethals
+ 50%inactivation
Drosophila
Habrobracon
69
0
100
0
0
100%dominant lethals
+ 100%inactivation
Drosophila
Habobracon
0
100
0
100
0
0
0.
Drosophila: lethals are autosomal. Habrobracon: 62%of eggs are fertilized.
The present study is concerned with the effects of nitrogen mustard, methyl
bis (beta chloroethyl) amine hydrochloride *, on Habrobracon sperm and the
sorting of dominant lethal effects from those due to inactivation. Whether
the sperm inactivation observed is the HERTWIG
type (lack of pronuclear
fusion) or that described by PACKARD
(lack of egg penetration) or a third
kind in which sperm are completely immobile has not been determined. This
is being investigated cytologically at present.
MATERIALS AND METHODS
Mature unmated males or males separated from females for several days
were used. They were exposed to an aerosol of 10% aqueous solution in
2
Provided by Merck & Company.
319
DOMINANT LETHALS IN HABROBRACON
alternating exposures of 50 seconds aerosol and 10 seconds air with time as
the variable. During the 50-second interval 4.7 liters of aerosol were delivered
to the exposure chamber containing the wasps and 4.2 liters of air were delivered during the 10-second interval. The aerosol was produced by a DeVilbiss-40 glass nebulizer. The flow of air served to dilute the atmosphere
and to evaporate coalesced droplets of the mutagenic solution which may have
adhered to the animals or walls of the exposure chamber. The males were
then kept for at least twenty-two hours before mating. This delay in mating
was found to be necessary because of the mutagenic effect on eggs of the
nitrogen mustard carried by freshly exposed males. In most instances matings
TABLE 2
Single matings
Exposure
in
minutes
2.5
5
10
20
30
60
90
Controls
O
01 males after exposure to nitrogen mustard.
% Hatchability
1st day
1st 5 days
120
34 -24.64 -=22.90
138
524
43
317
-=25.15
-=33.12
171
957
29
170
-=50.88
-=38.90
57
43 7
47
239
-=47.47
-=42.60
99
561
34
51
-=48.57
-=44.35
70
115
_.-
49
-=71.m
22
163
0
0
612
1
0
--
377
0
0
-
441
0
7.11
--
329 58.76*2.08
42
0 10.24
119
1 33.81
558
558
gz71.18
2 8 4 ~ 7 1 . 1 8 k 2 . 2 7 113
399
0 53.52
-E
69
22
-=loo
YY
Total
105
225
-=25.99
-=24.25*1.41
404
728
570
-37.52 -887 -35.82f0.96
2476
1519
237
407 -38.43*1.49
-=38.10
622
1057
281
-42.26 520 -42.41
f1.41
1226
665
51
-=44.35*4.63
115
--
%
inactivation
JJ
&Days
-=
32 9
140 57.61 -=58.96
243
Progeny
191
-=97.45
196
139
-=97.89
142
33063
--97.
338
f00.83 101 265
were observed and males were removed after a single mating. Females were
placed individually in small Stender dishes, each of which contained one host
Ephestia larva. After six to eight hours the females were removed and kept
overnight without the host and were given a fresh host larva the following
morning. This was repeated as long as desired. Eggs are deposited on the
surface of the paralyzed host and are easily counted. Records were taken of
number laid at time of removal of females and of number of larvae after a
forty-eight-hour period at 30°C. The six- to eight-hour period of oviposition
has been found best as it allows time for the laying of a number of eggs convenient for observation and counting with no wide duration in time of hatching. Larvae were permitted to mature in order that sex of survivors could
be recorded.
ANNA R. WHITING AND ROBERT C. VON BORSTEL
320
Stocks with different sex alleles were used as sources of males and of
females in order to avoid the production of highly inviable diploid males
characteristic of crosses involving two sex alleles. The males were from virgin
mothers or were carefully checked in order to insure that they were not
sterile diploids. Hatchability of eggs from the type of cross used in this study
is about 98%.
RESULTS
Data from single matings of exfosed males
The data from single matings of exposed males are summarized in table 2.
When males appeared to mate normally but hatchability of eggs was about
lo%,
it was taken for granted that males had no sperm or that mating had
not been completed and data are not included in summaries. It will be seen
TABLE 3
Repeated matings of males e%xpared/or 2.5, 20, and 30 minutes and.
for partponed mating of males expared /or 20 minutes.
Date of
expoewe
Date of
mating
2.5
11./26
11/27
-=
225
928
24.25
f
2.5
11/26
11/28
-=
89
344
25.87
f
20
9/22
9/23
20
9/2 2
9/2 5
20
9/2 2
9/26
20
9/22
10/3
20
9/2 2
10/6
30
5/13
5/14
30
5/13
5/25
20
9/2 2
9/2 5
Expoaure
in
miarctea
X hatchability
Progeny
$8
YY
1.40
163
0
2.36
68
0
-=
356
821
175
-=
395
43.36 f 1.73
305
0
49.37 f 2.52
182
0
-=
185
342
42
-=
102
28
-=
64
54.09
2.67
146
0
41.18 f 4.87
27
0
43.75 f 6.20
16
0
51
e
44.35
30
0
121
6
38
0
115
-54
r
107
f
f
4.63
50.47 f 4.83
that even the lightest treatment, that of two and one-half minutes, induced at
least one dominant lethal in each sperm since no females were produced. Results of this particular experiment are anomalous in one respect and in this are
not comparable with any of those obtained during many years of study of
Habrobracon. Hatchability is significantly lower than expectation, which is
about 38%. One might suggest that matings were made too soon after ex-
321
DOMINANT LETHALS IN HABROBRACON
posure of males to nitrogen mustard so that injury induced in eggs increased
mortality, but these males were handled as were those receiving longer exposure; reference to table 3 discloses that subsequent matings of these males
gave almost identical lowered hatchability. This will be discussed later.
Five- and ten-minute exposures appear to induce dominant lethal effects
only. Hatchability percentages, 35.8 and 38.4, are not significantly different
from expectation, 38%. With the twenty-minute exposure a barely significant
increase in hatchability is apparent. This increase becomes more and more
significant with each extension of length of treatment until at ninety minutes
a hatchability of 71.7% is attained. Theoretically, after a dose of about two
hundred minutes, hatchability should be 98%, but males would probably not
,
loo[
>
-
t
0.
I
0
4;
8;
I
ICjO
too
NITROGEN MUSTARD
-60
*.X-RAYS
60-
,
80
80 -
a
, 7 ,
NITROGEN MUSTARD DOSE (MIN.)
2:
/
;
/
-40:
-20
0
100% DOMINANT LETHALITY
-
#
=!
s4
5
-20
c
1
1
40
I
I
80
I
I
120
I
I
160
I
200
X-RAY DOSE (KILOROENTGENS)
FIGURE
1.-Egg hatchability showing sperm inactivation after exposure of males to
nitrogen mustard or to X-rays; data superimposed. X-ray data from MAXWELL
(1938)
and HEIDENTHAL
(1945). Doses are lethal or higher than lethal.
tolerate an exposure of this length. In the ninety-minute experiment the aerosol and air were delivered at a slightly lower rate of flow than in the other
experiments. A lower rate would consequently decrease the dose, so the true
inactivation at ninety minutes may be somewhat higher than shown in table 2.
Since ninety minutes was very near the lethal dose for the wasps, there was
no repetition at the higher rate of flow used in all other experiments.
Percentages of inactivation can be estimated by subtracting from actual
hatchability that expected if dominant lethals only were induced, that is, 38%,
and dividing the result by the expected percentage of fertilized eggs, in this
case 62. These percentages of inactivation as well as those of hatchability are
plotted in figure 1.
When females are mated they receive only mature sperm. Control females,
322
ANNA R. WHITING AND ROBERT C. VON BORSTEL
after a single mating, may continue to produce daughters for from three to
four weeks. Normal sex ratio is maintained until sperm are exhausted when
there is a sudden cessation of female production. Since mature males, either
unmated or kept from females for several days, were exposed to nitrogen mustard, an ample supply of mature sperm must have been present at time of exposure. The holding of such males for about twenty-two hours before mating
would not be expected to result in the addition of many sperm matured from
earlier stages at time of exposure even should such stages prove resistant to
the treatment.
Tests of hatchability over a period of time after the single mating of treated
males answer several questions if they are carried on for a long enough time.
Should hatchability rise suddently to 98% with all progeny male, all sperm
will have become exhausted. Should hatchability fall to 38% with all progeny
male, sperm will have recovered from inactivation. Should hatchability rise
and females appear or increase among progeny, sperm will have recovered
from injury inducing dominant lethal effects.
Reference to table 2 shows that there has been no significant change in
hatchability within the period covered by the tests which, in some cases, were
continued for twenty-one days after the single mating. In no female among
the one hundred and three used was the sperm supply exhausted. The appearance of two females among the progeny, one in the five-minutes and one in the
sixty-minute experiments, can be explained perhaps by chance escape of an
occasional sperm from injury or the rare maturation of a functional sperm
from a more resistant earlier stage in meiosis during the twenty-two-hour
period between exposure and mating. The fact that the females appeared late
in the experiments, on the 8th and 19th day after the matings, may favor the
idea of some slight recovery.
Frequently females of these experiments were set in vials after hatchabilityrecord-taking was discontinued and allowed to reproduce as long as they survived. The two F1 females mentioned above were the only ones produced in
a total of 3798 progeny. Females mated to males exposed to the lowest doses,
two and one-half and five minutes, produced 764 males, 0 females, and 728
males and 1 female respectively.
Data from repeated matings of exposed males
Another question which arises concerns the nature of the effects of nitrogen mustard on cells in earlier stages of meiosis at time of exposure. In table
3 are recorded some data from successive matings of exposed males and from
one experiment in which two males were mated for the first time three days
after exposure. Data are not as extensive as would be desirable. In the series
exposed for twenty minutes and then mated one, three, four, eleven and fourteen days after exposure, hatchability remains constant with the exception of
the four-day test where the fact that one female exhausted her supply of
sperm increased hatchability.
In the two tests of the two and one-half minute exposure there is, again,
323
DOMINANT LETHALS IN HABROBRACON
no evidence of any change in sperm behavior. The low hatchability of both
groups for this dose is to be noted. Data for the thirty-minute experiments are
given, although hatchability records were taken in one only, because of the
appearance of six females from the mating made twelve days after the exposure. The consistently late appearance of all females is to be noted. The supply of mature sperm in males which have not mated must be large since control
males may mate as many as fourteen times in rapid succession and produce
daughters in every mating. All tests herein described may, therefore, deal with
sperm mature at time of exposure.
All data obtained agree in giving no convincing evidence for recovery of
exposed sperm from either dominant lethal or inactivation effects. Neither is
there evidence for augmentation of either effect in the exposed sperm whether
stored in the male or the female. The data suggest, in addition, that sperm
remain “ functional’’ as long as do those in control females after a single
mating.
DISCUSSION
The dose of X-rays inducing at least one dominant lethal in every sperm is
approximately the same for Habrobracon (P. W . WHITING1938; HEIDENT H A L 1945) and for the frog (RUGH,1939). HEIDENTHAL
obtained one female among 1106 progeny after exposure of males to 10,000 r and RUGH
fmnd that only 1.6% of eggs hatched after exposure of frog sperm to this
dose. Both reported that as dose was increased above this, hatchability increased. Inactivation of sperm, then, followed after the attainment of 100%
dominant lethality .
In figure 1, X-ray data on inactivation are plotted for comparison with
nitrogen mustard effects. In order to obtain a dominant lethal curve for
Habrobracon sperm after exposure to nitrogen mustard for comparison with
that induced by X-rays, much shorter exposures, or probably better, much
more dilute solutions, should be used.
The anomalous hatchability ratios of eggs from females mated to males
exposed to nitrogen mustard for two and one-half minutes are difficult to
explain. Actually, from first matings of such males, progenies from eleven
females were tested. Of these, four gave hatchabilities not significantly different from expectation, 32.4, 33.3, 34.0 and 38.0%. The remaining seven
gave percentages ranging from 8.3 to 25. Subsequent tests of four of these
males selected at random gave 19.3, 21.6, 28.7 and 29.3% respectively. These
differ significantly from expectation of 38%. No convincing explanation of
this comes to mind, but it appears that the treated sperm have been stimulated” to fertilize more eggs than do the untreated sperm. The effect has
never been observed after X-ray treatment.
The nature of sperm inactivation in Habrobracon is still unknown. Theoretically, sperm could enter the egg but take no part in development, or they
could be active but not enter the egg or, in a form with natural parthenogenesis
such as this, they could be inactive literally, moving little or not at all.
“
A N N A R. WHITING A N D ROBERT C. VON BORSTEL
324
MACBRIDE(1946) found that although sperm of sterile diploid Habrobracon males are motile, penetration of the eggs does not occur. A similar
study is now being conducted on sperm of heavily irradiated males.
AUERBACH
and ROBSON(1947) mentioned that no histological study had
been made of the effects of nitrogen mustard on the testes of Drosophila. Mature sperm remained motile and induced dominant lethal effects after exposure.
Extensive breeding tests indicated an inhibition of spermatogenesis and
absence of mature sperm after exhaustion of supply present at time of exposure. Tests herein reported are not extensive enough to permit the formation of conclusions in respect to inhibition of meiosis.
-
.-
--
X-RAY DOSE (KILOROENTGENS)
FIGURE
2.-Egg hatchability showing sperm inactivation after exposure of males to
nitrogen mustard or to X-rays ; relationship of sensitivities arranged on basis of
comparison with egg sensitivities. Sperm five times as resistant to X-rays as to nitrogen
mustard. X-ray data from MAXWELL
(1938) and HEIDENTHAL
(1945). Doses are lethal
or higher than lethal.
A N N A R. WHITING (1945) has demonstrated that approximate lethal
X-ray doses are 2000 r for eggs in first meiotic metaphase and 50,000 r for
those in first meiotic prophase. The approximate lethal dose for sperm, as
pointed out above, is 10,000 r. Lethal dose of nitrogen mustard for eggs in
first meiotic metaphase is about 8-10 minutes. Eggs treated in the first meiotic
metaphase and first meiotic prophase show the same relative sensitivity
whether treated with nitrogen mustard or X-rays (WHITINGand VON BORSTEL 1952). O n this basis the lethal dose for sperm after nitrogen mustard
treatment should be 40-50 minutes. Actually, the lethal dose for sperm is two
and one-half minutes or less. Figure 2 compares sperm inactivation induced
by nitrogen mustard with that induced by X-rays when egg metaphase and
DOMINANT LETHALS IN HABROBRACON
325
prophase sensitivity are used as criteria. The idea presents itself that small
size of sperm cells and their position in the body permit greater accessibility
of chromosomes to the mutagen than is true of the relatively large yolk-laden
eggs. Relative sensitivities derived from X-ray studies would be expected to
be more accurate than those made with nitrogen mustard.
SUMMARY
Data are presented which show that nitrogen mustard, methyl bis (beta
chloroethyl) amine hydrochloride, produces a dominant lethal effect in every
sperm in the shortest time during which mature males of the parasitic wasp
Habrobracon were exposed. As length of exposures was increased beyond
lethal dose, increased sperm inactivation occurred. This is expressed as an
increase in parthenogenetic development and haploid male production due
either to failure of sperm to penetrate eggs or to undergo syngamy if they
enter. There is no evidence for recovery of sperm from either of these effects.
LITERATURE CITED
AUERBACH,
C., and J. M. ROBSON,1947 The production of mutations by chemical substances. Proc. Roy. Soc. Edin. B. 62: 271-283.
BROWN,S. W., and M. S. CAVE,1953 Induced dominant lethality in Lilium. Proc. Nat.
Acad. Sci. 39: 97-102.
HEIDENTHAL,
G., 1945 The occurrence of X-ray induced dominant lethal mutations in
Habrobracon. Genetics 30: 197-205.
HERTWIG,
O., 1911 Die Radiumkrankheit tierischer Keimzellen. Ein Beitrag zur experimentellen Zeugungs- und Vererbungslehre. Arch. f. mikr. Anat. 77: 1-164.
MACBRIDE,
DOROTHY
H., 1946 Failure of sperm of Habrobracon diploid males to penetrate the eggs. (Abstr.) Genetics 31: 224.
MAXWELL,
J., 1938 Inactivation of sperm by X-radiation in Habrobracon. Biol. Bull.
74: 253-255.
MULLER,H. J., 1927 Artificial transmutation of the gene. Science 66: 84-87.
PACKARD,
C., 1914 Effect of radium radiations on fertilization of Nereis. J. Exp. Zool.
16: 85-129.
RUGH,R., 1939 Developmental effects resulting from exposure to X-rays. I. Effect on
the embryo of irradiation of frog sperm. Proc. Am. Phil. Soc. 81: 447-471.
STANCATI,
M. F., 1932 Production of dominant lethal genetic effects by X-radiation of
sperm in Habrobracon. Science 76: 197-198.
WHITING,ANNAR., 1945 Effects of X-rays on hatchability and on chromosomes of
Habrobracon eggs treated in first meiotic prophase and metaphase. Amer. Nat. 79:
193-227.
WHITING,ANNAR., and R. C. VON BORSTEL,
1952 Comparison of sensitivity to nitrogen
mustard and X-rays of Habrobracon eggs and sperm. (Abstr.) Genetics 37: 635.
WHITING,P. W., 1938 The induction of dominant and recessive lethals by radiation in
Habrobracon. Genetics 23: 562-572.