THE EFFECTS OF X RAYS ON INSEMINATION AND SPERM
RETENTION IN DROSOPHILA1
ARMON F. YANDERS
Received October 15, 1963
HE rate of radiation induced mutation in Drosophila is most commonly estiTmated by genetic techniques employing mature sperm irradiated either in the
male prior to insemination, or while stored in inseminated females. The mutation
rate is then calculated on the basis of the proportion of sperm with genetic changes
of the specified type which actually fertilize eggs. If the likelihood of containing
a new mutation and the likelihood of success in fertilization are completely independent for each individual in a population of sperm cells, such methods will
yield estimates whose accuracy is limited only by the precision of the genetic
techniques. If, however, there exists an appreciable positive or negative correlation between fertilization success and the presence of a mutation, even the most
sophisticated genetic techniques will yield only spurious estimates of the actual
rate of mutation.
The possibility that there is a relationship between fertilization success and the
presence of a genetic change is rarely taken into consideration in radiation studies,
perhaps because the effect of radiation on the functional capability of sperm is
largely ignored or discounted. For example, it can easily be demonstrated that at
least some Drosophila sperm will survive doses of X rays in excess of 50,000r
(e.g. YANDERS
1959) ; and studies with Habrobracon, where dominant lethality
1938) , show conclucan be distinguished from nonfertilization (e.g. WHITING
sively that the dose required for complete inactivation of sperm is considerably
in excess of that required to produce 100 percent dominant lethality. But it is not
warranted to conclude merely from this evidence that radiation doses within the
range commonly used in genetic studies (below 10,000 r ) produce no detectable
effect on sperm behavior or survival. If it can be shown that some aspect of sperm
behavior is affected by doses of radiation in the range normally employed in mutation rate studies, the problem of whether or not this gross effect is correlated
with the production of mutations is-at least in principle-open to experimental
attack.
Sperm behavior in Drosophila is difficult to evaluate, but one aspect that can be
measured is the relative success in postcopulatory filling of the sperm storage
organs of inseminated females, which, in D. melanogaster, consist of a single
1
Supported by U. S. Atomic Energy Commission Contract AT(11-1)-1033.
Genetics 49: 309-317 February 19F4.
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Department of Zoology, Biology Research Center,
Michigan State Uniuersity, East Lansing
310
A. F. YANDERS
MATERIALS A N D METHODS
Stock: A strain of Oregon-R D. melanogaster was used exclusively. This was obtained from
the University of Chicago (courtesy of W. K. BAKER)in 1959, and has been maintained since by
mass transfers in this laboratory.
Rearing, mating and storing: All flies were reared at 25°C on modified CARPENTER’S
(1950)
medium, and all males were stored on this medium for three to four days after eclosion prior to
treatment. In the experiments not involving storage of inseminated females, virgin females also
were aged for five to seven days after eclosion and mated on this medium. However, the experiments involving sperm retention in inseminated females necessitated that the females be starved
in order to inhibit ovarian development and reduce egg laying. This was achieved by aging the
and SCHMIDT
1936).
females for seven days after eclosion on a minimal medium (OFFERMAN
Mating and further storage of these inseminated females also took place on minimal medium.
Under these conditions the ovaries remain very small, practically no eggs are laid, and the utilization of sperm for fertilization must be nearly absent.
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ventral receptacle and a pair of spermathecae. The number of sperm stored in
these organs is one of the most important factors in the reproductive performance
of the female, for it is unlikely that a significant number of eggs is fertilized by
sperm not first resident in the ventral receptacle or the spermathecae. Because of
their relatively small size in D.melanogaster, the spermathecae probably play a
minor storage role. It is fortunate for the investigator that the storage capacity of
the spermathecae is small, for the structure and color of the walls of the spermathecae make estimates of the relative number of sperm present in them quite
difficult; but the structure of the ventral receptacle, in which the majority of
sperm are stored, is nearly ideal for measurement purposes. The ventral receptacle is quite long (about 2 mm) with thin, transparent walls; this feature, together with the great length of the sperm (about 1.75 mm), makes the presence
of even a single sperm cell readily detectable. Furthermore, the tendency of the
sperm to align themselves longitudinally in the lumen of the ventral receptacle,
especially when large numbers of sperm are present, makes possible quantitative
estimates of the degree of fullness of the organ, enabling comparisons to be made
of the relative insemination success after various treatments.
The process of insemination and sperm storage has been most recently described by LEFEVRE
and JONSSON
(1962). Although the exact method by which
sperm enter the ventral receptacle is not precisely known, their work indicates
clearly that at least one factor is the activity of the sperm cells themselves. If this
is true, quantitative measurements of the relative success of sperm in reaching
and remaining in the ventral receptacle following irradiation, when compared to
nontreated sperm, will give some indication of the effect of radiation on sperm
activity, and provide a potential technique with which to correlate physiological
with genetic effects of radiation.
The experiments described in this paper were performed to determine whether
or not there is a detectable effect of X rays on sperm behavior as measured by
success in reaching and surviving in the ventral receptacle after irradiation. Evidence is presented which indicates that both insemination success and sperm
retention are reduced by X-radiation delivered to males prior to mating, and that
accelerated sperm loss also occurs after irradiation of inseminated females.
X-RAY EFFECT O N INSEMINATION
311
RESULTS
Insemination success following irradiation of males: The Insemination Indices
and distributions of scores at 13 dose levels are given in Table 1. The Index of
each of the treated groups is lower than that of the controls, but-with the exception of the 500 r level-only doses of 4,000 r or more yield results which differ
significantly from the control results when tested by the chi-square test for independent samples. Each set of scores was tested against the control set. For this
test, it was necessary to combine the numbers of scores in the “0” and “1” categories, so that the expected numbers could be greater than 5 in more than 80
percent of the cases (see SIEGEL1961, p. 178), and the data were cast in a 2 x 4
contingency table.
The Index values (Table 1 ) were ranked and tested against the ranked X-ray
doses by the Spearman rank correlation coefficient test, and a value for r8 of .945
was obtained. This value exceeds the 1 percent level of significance, and supports
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In the experiments in which males were irradiated, they were mated immediately after treatment to virgin females for 24 hr, after which they were removed and discarded. In the experiments in which inseminated females were irradiated, they were mated as virgins to males for
24 hr, after which the males were removed and discarded.
Irradiatiom Flies to be irradiated were placed in plastic tubes in groups of 75 to 1 0 0 and exposed to X rays from a General Electric Maximar-250.111, operating at 250 kv, 15 ma, with no
added filter. The dose rates ranged from 2443 r/min in experiments in which the total was 25,000
r or less, to 900 r/min in experiments involving higher totals.
Scoring for insemination success: Females to be dissected were etherized, placed on a glass
microscope slide, and the storage organs were excised with dissecting needles in a drop of saline
solution. A cover slip was added, and the preparation was observed immediately at 250 x. The
fullness of the ventral receptacle was estimated and given a score of 0 (no sperm), 1 (one sperm
to % full), 2 (% to ‘/z full), 3 (‘/z to % full) or 4 (more than % full). All scoring was done by
the author. To reduce subjective bias, all vials containing groups of females to be dissected were
assigned code numbers by another person, and the scores were recorded using only the code
number as reference.
Znterpreiing the scores: Comparisons of the relative distribution of scores in each of the categories for different treatment groups can be made. Such comparisons, indicating similarities or
differences in the distributions of scores, are especially helpful in demonstrating that, for example,
a low degree of insemination success is due to a n unusually large proportion of “0” scores, or
that the proportion of scores in each category changes with treatment. But in comparing the results from many treatments, as well as in cases where disparities in the distribution patterns are
not particularly striking, the use of a single representative value for each group is helpful. Such
a value, the Insemination Index (referred to as the Index), has been employed. The Index is the
mean proportion of insemination success achieved by all flies within the particular group for
which the value is derived, and permits the relative ranks of several groups to be more easily
compared. The Index is calculated by summing the totals of scores and dividing by the sum
which would have been achieved if all scores had received the maximum value of “4”.
Tests of significance: The usual significance tests cannot be applied to these data, for the
Index values, as well as the scores from which they are derived, are not exact in any numerical
sense. Furthermore, it is not known whether the scores are from a normally distributed population, although it is assumed that there is some sort of underlying continuous distribution. Certain
nonparametric techniques are applicable to data of this type, however, and two of them have
been utilized: the Spearman rank correlation coefficient test, and the chi-square test for independent samples (both given in SIEGEL1956).
312
A. F. YANDERS
TABLE 1
Insemination success following irradiation of males at 12 X-ray doses
Pelrentage of scores in category
N
0
1
2
3
4
Or
500r
1,000r
2,000 r
2,500r
4,000r
5,000r
7,500r
10,000r
20,000r
40,000 r
60,000r
75,000r
926
275
145
192
1%
190
266
155
223
267
195
99
49
1.4
2.2
3.4
2.1
2.1
3.2
1.9
2.6
1.8
0.4
1.5
23.2
100.0
2.1
1.5
2.1
2.1
2.6
3.7
4.9
3.9
7.6
4.9
19.5
57.6
0.0
3.1
6.5
1.4
6.3
4.1
10.5
6.4
5.2
9.9
20.6
23.6
18.2
0.0
24.2
29.5
24.1
24.0
23.7
27.9
25.2
32.9
32.3
45.7
33.8
1.0
0.0
69.2
60.4
69.0
65.6
67.5
54.7
61.7
55.5
48.4
28.5
21.5
0.0'
0.0
N=Number of ventral receptacles scored; I n d e x = I n s t " a t i n n
are samplec from the sanie population.
P
Index
.89
..
.86 .02>P>.Ol
.88 .5>P>.3
.87 .2>P>.l
.88
.9>P>.8
.82
P<.OOl
.85 .01 >P>.oO~l
.84 .01 >P>.OO1
.80
P<.OOl
.75
P<.oOl
.64
P<.OOI
.24
P<.OOl
.oo P<.OOl
Index; P=probahility that control and treated group
the notion that the reduction in the Index is roughly proportional to the total dose.
These results show quite clearly that the success in insemination is significantly
reduced when the males are irradiated prior to mating at doses of 4,000 r o r more.
It is possible that lower doses could also be found to produce significant effects,
a
0.80
L,' ,I,
:::::/-,[[
0.20
0.00
1
2
3
4
0
1
2
3
4
CATEGORY
FIGURE1.-Distribution
60,000r
(1.1..
.24)
40,000 r
(1.1. = . 6 4 1
20.000 r
(1.1. = . 7 5 )
I-
10,000 r
(1.1. = .eo)
5,000 r
(1.1. = . 8 5 )
Control
(1.1..
.89)
W
-
0
1
2
3
4
of fullness categories among Drosophila females inseminated by
X-irradiated males. 1.1.= Insemination Index.
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X-ray dose
313
X-RAY EFFECT O N INSEMINATION
1.00r
‘
0.00
0
I
5
I
IO
I
I
20
40
DOSE IN kr
-
FIGURE
2.--Reduction in insemination success following X-irradiation of males at doses to
4Qkr.The weighted linear regression line calculated on these data is indicated.
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but the use of much larger numbers, or some refinement in scoring technique,
would be necessary to demonstrate this conclusively.
The reduction in the Index with dose is due to shifts toward lower scores, not
to the presence of more scores in the “0” category except at the two highest dose
levels. The distributions at six representative doses are shown in Figure 1. Inspection of the percentages of scores in each category shows that the fraction of “0”
scores remains at a low level at all doses below 60,000 r. This is an important feature of the scoring pattern evaluation, for scores in the “0” category may represent females who never mated, as well as females who were inseminated but
stored no sperm, yet the data indicate that mating success was not influenced at
doses of 4.0,OOO r and below. This may not be true at 60,000 r and above, however.
Although the survival and behavior of the males receiving 60,000 r seemed normal, males receiving 75,000 r exhibited a marked depression in activity immediately after treatment, and only about 25 percent of them survived the 24-hour
mating period. This suggests that the absence of stored sperm noted at this dose
was due to lack of mating. For this reason, the data at 60kr and 75kr were omitted
from the regression calculations. A weighted linear regression analysis calculated
on the data from 0 to 40kr yields a slope value for which the drop in the Index
is equal to 0.0066 times the dose in kiloroentgens (see Figure 2). The more extreme reductions at 60kr and 75kr suggest that two factors may contribute to the
reduction in the Index with dose: at the lower doses (500 to 40,000 r ) the drop
appears to be linear, but there is a more abrupt drop at the two higher doses. Such
a distribution could be achieved if low doses reduced insemination success by
nonlethal means, while higher doses reduced the likelihood of mating or caused
sperm death. A more extensive study of the dose range between 40,000 r and
314
A. F. YANDERS
TABLE 2
Retention of sperm in females mated to males irradiated prior to insemination
Days after irradiation
1
14
7
N
Index
N
0
4,000 r
25,000 r
50,000 r
68
45
23
20
375
789
.5uo
.loo
65
49
18
18
Index
.738
.679
.222
,000
N
Index
65
50
12
17
.638
.615
.042
,000
N = Number of ventral receptacles scored; Index = Insemination Index.
75,000 r would be necessary to reveal whether there was indeed an abrupt drop,
and--if so-whether it was due to nonmating.
Retention of irradiated sperm: (a) Sperm irradiated in male prior to mating.
Males were treated at doses of 0, 4,000 r, 25,000 r, and 50,000 r, mated for 24
hours to starved females, and discarded. The females were stored on minimal
medium and dissected 1, 7, or 14 days following irradiation. The results given
in Table 2 suggest that the rate of loss of irradiated sperm is greater than that of
the unirradiated controls. The data in Table 2 also indicate that premating starvation of the females reduces the initial insemination success. In this experiment,
the Indices at Day 1 are lower than those obtained with nonstarved females at
similar dose levels (Table 1) . It is hardly surprising that the physiological state
of the female is a factor in sperm success, but how and when the influence of the
female occurs is not known.
(b) Sperm irradiated in female after insemination. Starved females inseminated prior to treatment were exposed to X rays to determine whether there
occurred reductions in the number of sperm stored when the sperm were already
present in the storage organs of the female. The data in Table 3 are the results of
TABLE 3
Retention of sperm irradiated in females after insemination
Dags after irradiation
1
0
X-ray dose
N Index
N Index
N
0
5,000 r
10,000 r
12,500 r
15,OOO r
20,000 r
25,000 r
37,500 r
50,000 r
25 .820
79
24
23
.
27
24
24
70
22
50
44 312
..
. .
30 .750
26 .846
23 .620
48 ,708
332
333
.598
,898
,656
.438
.643
,488
.420
Index
N
Index
23 ,804
26 ,692
26 .529
25 .810
45 ,728
28 .429
43 ,267
5
3
2
24 .573
22 ,455
25 5 8 0
7
9
17
Index
N Index
N
51 ,833
22 .739
24 .698
27 .731
25 ,560
24 .490
52 .452
25.240
41 ,030
25 ,730
27 .648
20 .613
22 .784
26 .657
25 .520
16 .359
23 .250
22 250
25 .460
22 .307
25 ,290
25 .490
23 ,228
24 ,208
22 .227
18 .I11
22 ,136
N
N=Number of ventral receptacles scored; Index= Insemination Index.
Index
N
Index
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X-ray dube
X-RAY EFFECT O N INSEMINATION
315
DISCUSSION
These data are consistent with the hypothesis that Drosophila sperm which
receive doses of X rays within the range customarily employed in genetic studies
(10,000 r and below) suffer physiological damage as well as genetic damage. At
the highest dose levels used (25,000 r and above) some sperm are either killed or
inactivated, and the average length of time that a sperm cell is retained in the
ventral receptacle is reduced.
Physiological damage resulting in a reduction in motility can be postulated as
and
the primary effect of treatment. According to the hypothesis of LEFEVRE
JONSSON
(1962), sperm not only migrate from the spermatophore to the storage
organs after insemination, but remain in continual motion in and out of these
organs during the storage period. One suspects that a less active sperm cell would
be both less likely to successfully migrate to a storage organ, and more likely to
be lost during one of its periodic trips to the genital chamber, if such trips do indeed take place.
Whether sperm disappearance is due to the death of the cells is not revealed by
these experiments. M y observations suggest that, if the sperm cells do die in the
ventral receptacle, they are expelled prior to cytolysis. Even after the highest
doses were given to inseminated females, in no case were any sperm fragments
seen in the ventral receptacle, yet some debris would be expected if the sperm
actually died and disintegrated in this organ. This observation prompts the sugand JONSSON
gestion that, even if the circulation of sperm postulated by LEFEVRE
does not occur, sperm must remain active merely to be retained in the ventral
receptacle; inactive sperm will be expelled as a result of pressure exerted on the
organ by muscular contractions of the female’s abdomen, or by the activity of
more vigorous sperm. The latter notion implies that there is a competition among
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dissections of females inseminated I to 24 hours prior to irradiation and dissected
at a time between 2 hours (Day 0) and 17 days after irradiation. Although the
numbers in each group are small and considerable variation within groups appears, two conclusions seem warranted.
First, the number of sperm stored by untreated females, in the absence of egg
laying, remains relatively high for at least seven days after mating; the first significant reduction is noted only after 17 days. Other tests, not included in the data
of Table 3, revealed that some motile sperm are retained in nonlaying females
held on minimal medium for as long as six weeks after insemination.
Second, the effect of irradiation on sperm present in treated females, even at
the highest doses, does not begin to show itself immediately. Only after 24 hours
of storage do the Index values for the irradiated groups show a clear difference
from the controls, and then only at the two highest doses. Differences between
control and treated groups at Days 3 and 5 appear at doses as low as 15,000 r. The
reduction in the 50,000 r group is especially striking at Day 5.
These results also agree with those of the preceding experiment id that a lower
initial insemination success is noted in females starved prior to mating.
316
A. F. YANDERS
SUMMARY
The effect of X rays on insemination success and retention of sperm during
storage in inseminated females was studied by direct microscopic estimates of the
relative fullness of excised ventral receptacles in female Drosophila melanogaster
dissected at intervals following mating.
Males were treated at 12 X-ray dose levels ranging from 500 to 75,000 r and
mated for 24 hours to untreated, virgin females. The insemination success in
groups of females dissected immediately after the mating period was significantly
lower in treated groups than in the controls at all doses of 4,000 r or more. The
reduction in insemination was proportional to -0.0066 times the dose in kiloroentgens at doses to 40kr, and was even more extreme at doses of 60kr and 75kr.
Males were treated with 4,000,25,000, or 50,000 r of X rays and mated for 24
hours to virgin untreated females. The insemination success in females dissected
immediately after the mating period was lower in each treated group, and the
retention of sperm after 7 and 14 days of storage in the females was drastically
reduced in the 25,000 and 50,000 r groups.
Inseminated females were treated at eight X-ray dose levels ranging from
5,000 r to 50,000 r, and dissected at times ranging from 2 hours to 17 days after
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sperm for storage space, and this is supported by the genetic evidence of LEFEVRE
and JONSSON that partial displacement of nonirradiated sperm by irradiated
sperm does occur.
The problem of whether physiological damage and genetic damage are in any
way correlated remains to be solved. In any population of irradiated sperm,
some will exhibit severe genetic damage and some will not, and the same is undoubtedly true for physiological damage as well. Experimental demonstration of a
relationship between the two will be difficult, for, although a sperm with severe
genetic damage and little physiological damage can fertilize an egg, the reverse
might not be true. However, a positive correlation between genetic damage and
physiological damage would lead to underestimates of the genetic effects of radiation, especially in studies employing dominant lethality as the experimental test.
Furthermore, since the measurement of dominant lethality in Drosophila
customarily uses egg hatch as the sole criterion, and since failure to hatch can
result from simple failure of fertilization, as well as genetic damage, the potential
importance of physiological damage leading to sperm loss should not be disregarded. It is hoped that experiments now in progress will enable the relative importance of these factors to be assessed.
The technique employed in these experiments has definite limitations, but it
is the best available method with which to approach directly and quantitatively
the problem of sperm survival in Drosophila. Refinements in scoring and experimental design which are being employed in current studies may permit more
sensitive estimates of the effect of irradiation and other environmental variables
on sperm behavior to be made, but the existence of a measurable effect is demonstrated by the data presented here.
X-RAY E F F E C T O N I N S E M I N A T I O N
317
irradiation. Reductions in the success of sperm retention were first noted 24 hours
after irradiation at the highest dose levels, and became evident even at lower
doses within the 17-day storage period.
It is concluded that some aspect of sperm behavior, possibly motility, is affected
by X rays at doses within the range commonly employed in genetic studies. This
finding is significant for the analysis of induced mutation rates in Drosophila.
CARPENTER,
J. M., 1950 A new semi-synthetic food medium for Drosophila. Drosophila Inform.
Serv. 24: 96.
LEFEVRE,
G., JR., and U. B. JONSSON,
1962 Sperm transfer, storage, displacement, and utilization in Drosophila. Genetics 47 : 1719-1 736.
OFFERMAN,
C., and I. SCHMIDT,
1936 Culture media for Drosophila. Drosophila Inform. Serv. 6:
64.
SIEGEL,S., 1956 Non-parametric Statistics for the Behavioral Sciences. McGraw-Hill, New
York.
WHITING,
P. W., 1938 The induction of dominant and recessive lethals by radiation in Habrobracon. Genetics 23: 562-572.
A. F., 1959 The effects of X rays on sperm activity in Drosophila. Genetics 44: 545YANDERS,
546.
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LITERATURE CITED