x-ray sterility in the male house mouse

X-RAY STERILITY IN THE MALE HOUSE MOUSE

GEORGE D. SNELL’ University of Texas

INTRODUCTION

During the course of an experiment to determine what genetic changes, if any, are induced by x-rays in the spermatozoa of mice, data have been obtained which contribute to a more precise understanding of the nature of x-ray sterility. I n this paper observations on the x-rayed males and their immediate progeny are described. Results obtained in the later generations will be presented in a subsequent paper.

STOCKS

I n order to determine the optimum dosage for the production of heritable variations, a preliminary experiment was run before the major or final experiment was undertaken. The animals used in this preliminary experiment came from a number of different strains some of which were not inbred. The stocks used in the final experiment will be described in detail when the results obtained in the second and later generations are reported. The male and female parents came from unrelated inbred strains, with the result that the F, individuals, except f o r those whose genetic constitutions had been altered by the x-rays, were uniform and vigorous. In the study of F, embryos from x-rayed males, discard animals of various genotypes were used as parents.

DOSAGE

The male to be x-rayed was placed in a lead chamber large enough to hold him comfortably, but without permitting much

National Research Council Fellow in the Biological Sciences. 421

422 GEORGE D. SNELL

freedom of motion. An aluminum window in the floor of the chamber occupying about two-fifths of its length, permitted the Roentgen rays to reach all parts of the testes, epididymides, and vasa deferentia. In some cases the bones of the leg may have intervened between the genital tract and the source of the rays, but if any shadow was thus cast it does not seem to have affected the results.

The x-ray machine was operated at 110 kilovolts and 5 milli- amperes. The target-skin distance was 14.5 em. During all rayings both copper and aluminum filters were used, the two being placed so that the rays passed through them in the order named. The copper filter was 0.27 mm. thick, the aluminum filter 1 mm. thick. The aluminum window in the floor of the chamber enclosing the mouse was 0.4 mm. thick. A dosimeter placed in the chamber above the window showed that with this set-up the x-ray machine delivered 36.5 Roent- gen-units per minute. All variations in dosage were made by varying the exposure time. During a few of the rayings, owing to some trouble with the x-ray machine, the exact dosage delivered was uncertain. Data obtained from males rayed under these conditions are included only in table 5.

Several of the maIes rayed with the larger doses died during the first week or two after treatment, but the majority of individuals appeared to be uninjured.

METHOD O F MATING

The oestrous cycles of all females used were followed by means of daily vaginal smears. Most smears were observed under the low power of the microscope without staining and without cover-glass, but if the first smear indicated the ap- proach of oestrus a second smear was prepared stained with aceto-carmine. This permitted a more accurate differenti- ation between cornified and epithelial cells. When a female was found in oestrus or approaching oestrus a male was put in the pen with her. If a vaginal plug was present 24 hours later, the occurrence of mating could be inferred. In a few cases litters were obtained from females where no plug had

X-RAY STERILITY I N MALE HOUSE MOUSE 423

been noted. Consequently, a few sterile matings may have gone unrecorded, but the error introduced by this factor is slight.

Pregnant females, with a few exceptions, were isolated in separate pens and examined daily. Stillborn young are not infrequently eaten by the mother shortly after birth, and even with daily examinations some individuals may have gone undetected on this account, but again the error introduced is probably slight.

One fertile mating was obtained from each male before raying to furnish the control. After raying, each male was mated in succession with a number of females. In a few cases a male mated with all of the 2 or 3 females in a pen during one 24-hour period. More commonly there was an interval of one to several days between matings.

HISTOLOGICAL CHANGES I N THE X-RAYED TESTIS

A number of investigators have studied the histological and cytological changes that occur in the testes following exposure to x-rays. Barratt and Arnold ('11) found that the testes of rats killed 14 days after exposure to a heavy dose of x-rays showed marked destructive changes. The seminal cells were destroyed and the Sertoli cells greatly increased in number. The seminiferous tubules were empty of sperm, being filled instead with a shreddy material, made up of ragged strands of lightly staining substance arranged perpendicularly to the basement membrane. The appearance of sections from the fourth day onward appeared to indicate that the tails of spermatozoa took part in the formation of this shreddy material. Even with a moderate dose, division figures in the spermatogonia disappeared almost at once, and after 4 days spermatogonia were no longer recognizable. The primary spermatocytes remained visible for some time after raying but many of them showed multipolar mitoses and other abnormalities. Secondary spermatocytes disappeared within 5 days and spermatids and young spermatozoa began to show necrosis by the fourth day. Unfortunately at the time this

424 GEORGE D. SNELL

study was made, accurate methods of measuring x-ray in- tensity were not available so that the doses used cannot be compared with those employed in the experiment here reported.

Gatenby and Wigoder ('29) reported that the testes of a guinea-pig 4 days after raying with 1000 r-units showed many apparently normal mitoses of both spermatogonial and spermatocyte stages, though numerous other spermatocytes of both orders were unable to undergo mitosis.

Sections of the testes of some of the males used in the experiment here reported have been prepared and studied by Miss Isabel Thomas, to whom the writer is indebted for the following observations : Males, x-rayed with 800 r-units, were killed at 1, 3, 9, 11, 15, and 27 days after treatment. Mitoses are present in all sections up to and including those prepared at 11 days after raying, though in decreasing numbers. I n the 15-day sections no mitoses are present. The tubules are reduced to about one-half normal size and usually consist of not more than three layers of cells, or sometimes of only a single layer of Sertoli cells. Their lumens often show a considerable amount of debris. The number of spermatozoa is greatly reduced and many tubules lack them entirely. At 27 days the walls of most of the tubules consist of a single layer of cells, and their lumens are filled with a mass of debris. Spermatozoa are practically lacking.

It may be inferred from these observations that the passage of sperm from the testes into the epididymides must be greatly reduced by the second week after raying and cease entirely not later than the end of the fourth week after raying, and the possibility is not precluded that it ceases much earlier than this. If the pressure produced by the formation of new spermatozoa plays an important part in forcing the older spermatozoa out into the epididymides, their passage might be expected to cease within a week of raying, for cell growth largely ceases by this time and the testes lose the firmness normally present due to their internal pressure.


SURVIVAL O F SPERMATOZOA I N X-RAYED MICE

To determine the length of survival of spermatozoa in x-rayed males, a number of males were killed at varying intervals after raying and observations made on the number of motile spermatozoa. Small pieces were cut from the head of the epididymis, the tail of the epididymis, and the vas deferens. These were crushed with forceps in a drop of Ringer’s solution on a slide and examined under the low power of a microscope. The instruments used were thoroughly dried after each observation to prevent the transfer of sperm from one slide to another. The results are summarized in table 1.

It is probable that in most of the males x-rayed with the 200 r-units dose motile sperm were never entirely absent from the reproductive tract. One such male gave a fertile mating at 58 days following raying. The female to which he was mated was killed 10 days after impregnation, eleven embryos of normal size being found in her uterus.

When the dose was 800 or 1600 r-units, motile sperm disappeared sometime between 6 and 7 weeks after raying.

SURVIVAL O F SPERMATOZOA I N MICE WITH BILATERAL VASA EFFERENTIA LIGATION

The presence of motile spermatozoa in the epididymides of x-rayed males for a number of weeks following raying suggests that the x-rays have little o r no effect on the length of life of the spermatozoa. To obtain further evidence on this point, the following experiment was undertaken. An operation was performed on six males which consisted of ligating the vasa efferentia in two places and cutting them between the ligatures. An abdominal incision was used, the testes being withdrawn from the scrota1 sacs to permit access to the vasa efferentia, and replaced in the sacs at the close of the operation. Care was taken to avoid injuring the blood supply of the epididymides. Twenty-four hours after the operation, three of the males were x-rayed with an 800 r-units dose, the other three being kept as controls. Each control male was a

DA

YS

AF

TE

R

RA

YIN

G

L33

0-1 1 5 8

1 i-1

5 22

42

46

46

49

56

65

-66

77

77

88

135

142

+

TA

BL

E 1

Surv

ival

of

sp

erm

atoz

oa i

n x-

raye

d m

ice

m2

L

46

Fd30

DO

SE =

20

0 R

-UN

ITS

++

+ +

Epi

d C

olon

y no

.

L9'

1

L2

I+

gmis

~

Tai

l

+++

+++

+++

0 0 ++

+++

VaS

de

fere

ns

+++

++

+ 0 0 ++

+++

Col

ony

no.

F51

F18'

Fd1

6 R

170

Fd2

L

13

B15

5 G

526

F6

R93

R25

L

7 I3

239

DO

SE =

800

R-U

NIT

S

Epi

didy

mis

Hea

d T

ail

+++

lo O

lo

Vas

de

ferc

n

-t+

++

++

0 + 0 0 0 0 0 0 0 +

+-t

Col

ony

no.

F10

R45

A

g49

R99

F

d6

'Rad

iate

d w

ith

86 K

.V.

and

wit

hout

co

pper

fi

lter

ins

tead

of

the

stan

dard

con

diti

ons.

DO

SE =

160

0 R

-UN

ITS

Epi

didy

mis

V

as

defe

rens

++

+++

+++

0 +

In t

his

tabl

e, 0

mea

ns t

hat

no m

otil

e sp

erm

wer

e pr

esen

t, th

ough

it

doe

s no

t im

ply

the

abse

nce

of

imm

otil

e sp

erm

; +

mea

ns t

hat

a fe

w m

otil

e sp

erm

wer

e pr

esen

t; ++

, tha

t a

cons

ider

able

num

ber

wer

e pr

esen

t;

and

+++,

that

man

y w

ere

pres

ent.

X-RAY STERILITY IN MALE HOUSE MOUSE 427

litter mate of one of the x-rayed males and each pair came from a genetically uniform stock. The pairs were killed a t varying intervals after the operation, the vasa efferentia examined under a dissecting microscope to make sure that the operation had been successful, and segments cut from the testis, head and tail of the epididymis, and the vas deferens and observed for motile sperm. The results are summarized in table 2. No difference was found in the condition of the sperm in the x-rayed as compared with the control males, except for the absence of sperm in the left epididymis and vas deferens of control male F31, killed at 36 days after the operation. The absence of sperm in this male probably was

TABLE 2

Survival of spermatozoa in mice with bilateral vasa efferentiu lzgation ~ ~ ~ ~ ___ ~ -

Y-EAYED ( 8 0 0 R U N I T S ) NON-X BAYED DAYS ~

AFPER Epididymis OPhRA- 'IDE

Head Tail wens 1 Head TION 1 no.

36 Right F32 36 Left F32 49 Right F19 49 lLeft ~ 1 9 53 Right D231 53 /Left ~ D231 -

__-- - I-- O I +

~~

++ ++ 0 0 0 0

F31 F12 F12 D224 D224

+ I 0 0 0 ' 0 0

0 0 0 O -

Tail _ _

++ 0 + + 0 0 ~

Vas def-

wens

0 0 0 0 0 0

__

due to the failure properly to replace the left testis and epididymis in the scrota1 sac. In the pair of males killed at 49 days, not more than a dozen motile sperm, and these only feebly motile, were found in the section cut from the tail of each epididymis. No motile sperm were found in the reproductive tracts of the males killed at 53 days, though a considerable number of immotile sperm were present.

Since, with the one exception noted, there was no difference in the survival of spermatozoa in the x-rayed and control males, we may infer that the x-rays have little if any effect on the survival of spermatozoa in the epididymides.

The results obtained check satisfactorily with those reported by Benoit ('26). This investigator found that sper-

THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 65, N O . 3

428 GEORGE D. SNELL

matozoa in the epididymides of mice could be stimulated to motion for approximately 8 weeks following vasa efferentia ligation.

It appears from these data that the passage of sperm from the testes into the epididymides is stopped almost at once by exposure to 800 Roentgen-units. Motile sperm were found in the epididymides of uiioperated males irradiated with this dose up to 46 days following the treatment (table 1). In males with vasa efferentia ligation motile sperm were found up to 49 days after the operation. Since it has been shown that survival of the sperm is not appreciably affected by the x-rays, we may infer from the similar results in the two cases that the x-ray treatment is approximately equivalent to vasa efferentia ligation in its effect on the passage of sperm from the testes into the epididymides. As noted in a previous section, histological observations indicate that the passage of sperm must at least be greatly reduced by 2 weeks after raying, and may perhaps cease earlier than this. The evidence from these two sources is, therefore, in approximate agree- ment.

Strandskov ('32) found motile sperm (as tested by an electrically induced ejaculate) to persist up to 9 weeks following treatment in guinea-pigs rayed with 1296 and 1728 r-units. As in the case of the mice, this is approximately the same length of time that they persist in males with the epididymides separated from the testes by operative procedure. Moore ( '28) found motile sperm in the epididymides of operated guinea-pigs up to 70 days following the operation, though in one individual they disappeared at 37 days. Young ( '29) reported a maximum survival of 50 to 59 days.

It is noteworthy that Strandskov ('32) found motile sperm in the ejaculate of guinea-pigs rayed with 846 r-units up to 12 weeks after the treatment. This is a considerably longer period than they remain following vasa efferentia ligation, and indicates that this dose, contrary to the case in mice, is not sufficient to terminate promptly the passage of sperm from the testes into the epididymides. This is evidence that


Number of matings Number of fertile matings Number of young Mean litter size

the guinea-pig is less susceptible than the mouse to the effect of x-rays.

26 11 12 ' 4 4 1 0 18 9 9 2 1 0 0 49 28 22 3 1 0 0

2.7 3.1 2.4 1.5 1.0

THE LENGTH O F T H E INITIAL FERTILE PERIOD

While motile sperm are present in the epididymides of males rayed with 800 Roentgen-units for 6 o r 7 weeks following raying, the males remain fertile f o r only about 2 weeks following treatment. This is shown by the results summarized in table 3. I n part A of table 3 are given the data ob-

TABLE 3

The length of the initial fertile period of malrs x-rayed with 800 r-units

1 1-3 I 4-6 ~ 7-9 I 10-12 1 13-15 1 16-18 I 19-36 DAYS FROM X-RAYINQ TO MATINQ

A. Data from preliminary and final experiments

B. Data from embryological study. Females killed at G to 14 days after mating

Number of niatings 7 4 2 2 3 5 6 Number of fertilematings I 3 1 4 ! 1 I 1 I 0 1 1 1 0

C. Total data from preliminary and final experiments and from embryological study

Number of matings Number of fertile matings Per cent of niatings fertile

taiiied from the preliminary and the final experiments. In part B are given the data obtained from the embryological study of F, embryos described in more detail in a later section. For many of these data the writer is indebted to Miss Elsie Bodemann. I n the embryological study matings were classified as fertile if any embryos were present regardless of whether or not they were living. In most cases at least some of the embryos were alive, but in the case of the single fertile mating which occurred on the seventeenth day there were only two embryos and both of these were dead.

430 GEORGE D. SNELL

The length of the initial fertile period following x-ray treatment, like the length of survival of spermatozoa, is sug- gestive of the results produced by bilateral ligation of the sperm ducts. Unfortunately, the length of the fertile period in operated mice has not been determined, but Young ('29) found that in guinea-pigs with the epididymides separated from the testes by operative procedure the capacity for fertilization is retained for approximately 25 to 30 days. This checks well with the results of Strandskov ('32 and in correspondence with the writer), who obtained fertile matings from guinea-pigs rayed with 1296 Roentgen-units a t 1, 14, and 25 days after treatment. While these data are not sufficient to be conclusive, they suggest that the duration of fertility in both x-rayed and operated guinea-pigs is approximately the same. The fact that x-rayed mice remain fertile for only about 2 weeks, as compared with 4 weeks for operated guinea-pigs, may perhaps indicate that the x-rays hasten the onset of sterility beyond that to be expected from the mere aging of the spermatozoa, but it seems more likely that a species difference is responsible.

It should be noted that most of the mice which gave sterile matings from the tenth day on had been mated at least four times during the first 9 days. Young found in operated guinea-pigs, however, that the length of the fertile period bore very little relation to the number of matings before the onset of sterility.

X-RAY DOSAGE AND LITTER SIZE

Data showing the relation between x-ray dosage and the size of litters sired during the first 14 days after raying are summarized in table 4. All the data for the 200 r-units dose and the 1600 r-units dose and some of the data f o r the 800 r-units dose were obtained in the preliminary experiment. All other data were obtained in the final experiment. It is to be noted that the litter size falls off as the x-ray dosage increases, the relation between the two being approximately logarithmic. The figures in the last line of table 4 are calculated from the logarithmic equation-


DOSE IN R-UNITS

8 = ke-.oalsr

where r is the dose in r-units, S the mean litter size for the dose T, k the mean litter size of the control, and e the base of the Naperian logarithms. The number .&?I5 is chosen empirically to fit the data.

The mean litter size in every case is calculated by dividing the total number of young obtained at a given dose by the number of fertile matings obtained at that dose. This method is subject to an error for the higher doses. When the mean litter size is only 2 or 3 young per litter it is to be expected that in some of the matings, even though fertilization occurs,

0

TABLE 4

Results of matings of x-rayed males. Only those mat ings made during t h e first 14 days following raying are included. Data f r o m both the preliminary

experiment and thr final rxperiment are included

Number of matings Number of fertile

Per cent of matings

Number of young Mean litter size Expected litter size

matings

fertile

27

24

89 185

7 . 7 2 2 7.7

6

3

50 4

1.3&.2 1.2

200

11

0

0 0 0

0.7

3

2

67 11

5 . 5 t . 2 5.7

ROO

56

39

70 103

2.62.2 2.3

1200 1 1600

all the zygotes will be genetically inviable and the matings consequently sterile. Properly speaking, the number of these matings should be added to the number of fertile matings in calculating the mean litter size. Of the forty-nine matings by males rayed with 800 r-units made prior to the tenth day after raying, thirteen, or 26.5 per cent, were sterile (table 3A). The first 9 days only are considered because the sterile period begins to appear after this time. In the control only 11 per cent of the matings were sterile (table 4). Hence about 15.5 per cent of the matings at the 800 r-units dose presumably were sterile, not due to failure of fertilization or other causes normally operating, but because all of the zygotes were genetically inviable. When this per cent of all matings

432 GEORGE D. SNELL

is added to the per cent of fertile matings, and the mean litter size calculated using this figure, a value of 2.3 instead of 2.6 is obtained for the 800 r-units dose. The value for the 600 r-units dose, corrected in the same manner, is 2.7.

If some of the stillborn young were eaten by the mother before the daily observation, it is likely that some litters of one in which the single individual was stillborn would have gone unrecorded. This would introduce a second source of error. It is not unlikely that this occurred in a few cases, especially in view of the high rate of stillbirths in small litters (table 5 ) .

TABLE 5

Relation of stillbirths t o litter size

Even with due allowance for these sources of error, it is evident that the observed litter sizes for doses up to 1200 r-units are in good accord with those expected on the basis of the above equation. The litter size of zero obtained with the 1600 r-units dose departs significantly from the expected value of 0.7, the odds being approximately 1000 to 1 that such a departure would not occur due to chance alone. This may indicate that large doses are relatively more effective than small ones in causing dominant lethal changes in the sperm, but more data would be necessary before this conclusion could


be regarded as established. The failure to detect 2 or 3 litters due to eating of the young would account, in part at least, for the results.

Oliver ('32) has shown that a logarithmic relation holds between x-ray dosage and the frequency of induced genetic changes (lethals, inversions, and translocations) in Droso- phila. This logarithmic relation which Oliver has shown to apply in the case of Drosophila and which applies also in the case of mice, at least for doses up to and including 800 r-units, may be interpreted to mean that each added increment of x-rays is exactly as effective as each preceding increment in producing genetic changes. If 500 r-units produce a given change in half the sperm, 500 more r-units will produce the change in half the remaining sperm. There is no threshold value which the x-rays must reach before they begin to be effective.

Strandskov ( '32 and in correspondence with the writer), working with guinea-pigs, obtained litters averaging 1.70 young from males x-rayed with 1296 to 2592 r-units and mated before the onset of sterility. The litter size in the control was 2.77. This is a reduction of 39 per cent. With mice, a reduction of 66 per cent was obtained with a treatment of only 800 r-units. This suggests that the sperm of mice are more susceptible to x-ray treatment than are those of the guinea-pig. However, the difference probably is not as great as the above figures would appear to indicate. Strandskov's figure for the mean litter size is subject to the error already noted as applying to the mean litter size in the case of mice. It is probable that there were a number of matings in which fertilization actually occurred, but in which all the zygotes were inviable due to the genetic changes in the sperm. If these matings were included with the fertile matings in calculating the mean litter size, a smaller value would be obtained.

THE SEX RATIO

With the exception of a few control litters, all F, litters in the final experiment were sexed when first examined. Omit-

434 GEORGE D. SNELL

ting the control litters not sexed when first examined, the number of each sex found is as follows: control, 60 females and 58 males; 400 r-units, 6 females, 11 males, and 4 doubtful; 600 r-units, 17 females, 19 males, and 3 doubtful; 800 r-units, 41 females, 39 males, and 6 doubtful. The doubtful individuals, in most cases, were stillborn young in which the genital region had been eaten by the mother.

THE EFFECT O F X RAYS ON THE INCIDENCE O F STILLBORN YOUNG

Of 187 progeny of x-rayed fathers, twenty-five were stillborn (or dead when first examined) ; whereas, of 185 progeny of untreated fathers, onIy twelve were stillborn. The prob- ability of this difference in incidence of stillbirths occurring due to chance alone, as calculated by the chi-square method, is 0.03, and the difference, therefore, is probably significant. It does not necessarily follow, however, that any of the stillbirths are a direct consequence of the x-rays or, in other words, that the young are stillborn because of changes in their genetic constitution induced by the x-rays.

In table 5 the test and control litters are arranged accord- ing to litter size. It will be noted that, whereas 59 per cent of the test young born in litters of 1 are stillborn, none of the young born in litters of 5 and 6 are stillborn. I n litters of intermediate size the per cent of stillbirths decreases as the litter size increases. In the controls 8 of the 12 stillbirths occurred in one litter of 8 in which all the young were dead. There is obviously, therefore, a marked tendency for stillbirths to occur in the small litters. This is not because more of the small litters came from fathers treated with the larger doses; where one dose alone is considered, the same relation still holds.

The most probable explanation of this relation appears to be that the mothers experienced difficulties in parturition when litters numbered only 1 or 2 young. The young in these small litters were, on the average, conspicuously bigger at birth than those in litters of normal size. This, together perhaps with other unknown factors operating when the litters were small,


might be expected to lead to a difficult labor. As evidence for this explanation may be cited the fact that a number both of the liveborn and the stillborn young showed bruises about the head and shoulders when they were first found. If this explanation is correct, the stillbirths are only very indirectly a result of the x-rays.

One stillborn individual entirely lacking hind legs appeared in a litter of 2 in the x-rayed group. It was carefully examined under a dissecting microscope and the skin found to be everywhere intact, showing that the absence of legs was not due to their having been eaten. Death in this case perhaps is attributable directly to the effect of the treatment.

Strandskov ( '32), using guinea-pigs, found a slightly but not significantly lower incidence of stillbirths among the progeny of x-rayed males mated before the onset of x-ray sterility than among the progeny of the control males. Since a de- crease in litter size presumably would not increase mortality at birth in the guinea-pig, whose litters are normally rather small, this result is in accord with the result here reported. I n the litters which the x-rayed males sired after the return of fertility Strandskov found a considerable and significant increase in stillbirths as compared with the controls. These data are not comparable, however, with any obtained in the writer's experiments.

F, EMBRYOS FROM X-RAYED MALES

An embryological study has shown that the cause of the small size of the litters sired by x-rayed males is the degeneration of a part of the embryos, usually at or shortly after implantation. Discard females of mixed stocks were used in this study. The females were mated in the usual fashion except that observations for vaginal plugs were made twice daily. They were killed at varying intervals following impregnation and the embryos classified as normal or degenerating, with the results shown in table 6. Each line in the table contains the embryos from a single female. The classification of the embryos up to and including 7 days of

436 GEORGE D. SNELL

X-RAYED (800 %UNITS) EMBRYO AGE

age is based on sections prepared and studied by Miss Elsie Bodemann, to whom the writer is indebted for the data here presented. The embryos from 10 to 22 hours of age were in the fallopian tube and had not yet begun to cleave. Three, unfortunately, were lost and hence are classified as doubtful. All of the remaining ones showed both male and female pronuclei, proving that fertilization had occurred, and hence are classified as normal. Of the 5- to 7-day embryos in the x-ray group, thirteen out of twenty-four showed signs of degenera-

CONTROL

TABLE 6 F , embryos from x-rayed males

10-20 hours 11-22 hours

5-7 days

9-11 days

Total 5-11 days

7 0 3 7 0

1 5 1 10 0 4 5 5 0 5 3

2 3 6 1 1 2 5 8 0 5 4 9 0 0 1 6 1 2 5 8 0

8 0 ) 1

2 1 31 1 65 3 1

tion and one was doubtful. All the 5- to 7-day embryos in the control group were normal. The 9- to 11-day embryos were studied by dissecting out the uterus, pinning it in a dish filled with Locke’s solution, slitting it open, and observing the embryos under a dissecting microscope. I f the heart was beat- ing, the embryo was classified as normal. In many cases the inviable embryos had died so soon after implantation that the embryos themselves could not be found, but the swellings in the uterus where they had implanted were still quite large and easily recognized as late as 11 days after mating.


In the x-rayed group the average litter size for the 5- to 11- day embryos, normal, degenerate, and doubtful embryos being included, was 6.6. I f we omit the one litter which contained a single embryo, as being probably abnormal, the value is 7.4. In the control the average size was 7.7. The close agree- ment between these two figures indicates that the x-rays do not cause the degeneration of any embryos, or certainly not of more than a very few embryos, prior to implantation. An attempt was made to get further evidence on this point through corpora lutea counts, but it was found that at the stage at which the females were killed the corpora lutea of the last ovulation could not always be distinguished from those of earlier ovulations, thus making accurate counts impossible.

The results reported above confirm and extend the observation of Regaud and Dubreuil published in 1908. These investigators found numerous degenerating embryos in the uteri of female rabbits mated to x-rayed males.

Any attempt to name the cause of the degeneration of embryos in the litters sired by x-rayed males must be regarded as somewhat speculative. In, view, however, of the proved potency of x-rays in causing deletions, translocations, and other chromosome changes, and the disturbances in develop- ment that are known to be produced by changes in chromosome balance, it may be inferred with some degree of prob- ability that many and perhaps all of the inviable embryos are the result of chromosome abnormalities. As three of many investigations showing the production by x-rays of chromosome changes, we may cite the work of Muller and Altenburg ( '30), of Stadler ( '31), and of Patterson ( '33). Some evidence that many translocations are produced in mice by means of x-rays has already been presented by the author ('33) in a preliminary communication. Two types of changes are probably responsible for most of the dominant lethals with which we are here concerned. These are, first, deletions and other deficiencies, and, second, translocations in which one of the resulting chromosomes receives two spindle fiber at-

438 GEORGE D. SNELL

tachments and the other none. I t is well established that in Drosophila most changes of chromosome balance involving the autosomes and some of those involving the x-chromosome, are lethal, and it is reasonable to suppose that the same would be true in mice. It is therefore postulated that the inviability of embryos sired by x-rayed males is caused in most and perhaps in all cases by hypoploidy resulting from chromosomal abnormalities.

THE RETURN O F FERTILITY

The investigation here reported has been chiefly concerned with the changes that appear in xrayed males before the onset of x-ray sterility. A few data have been gathered, however, bearing on the return of fertility. Table 1 shows that motile sperm had reappeared in the epididymides of two males x-rayed with 800 r-units by 20 weeks after the treatment. One male x-rayed with 800 r-units was left with two females, each of which gave birth to a litter, one litter number- ing 8 young, the other 7. The litters were born at 16 weeks after the treatment and must have been sired about 3 weeks earlier. A male x-rayed with 600 r-units was similarly mated. Litters of 6 and 11 young resulted at 26 and 28 weeks after treatment. Three other x-rayed males under similar conditions failed to produce young. The number of young in the above four litters would suggest that the x-ray treatments have no effect on the size of litters sired after the return of fertility. The more extensive data of Strandskov ('32), however, show a reduction in the size of litters sired by x-rayed guinea-pigs following the return of fertility. The reduction is undoubtedly significant, though not as great as that found before the onset of sterility.

ACKNOWLEDGMENTS

The author is indebted to Professor Muller and Professor Patterson for valuable suggestions and assistance during the course of the work. A mouse colony intended for a study of the effect of x-rays on mammals had been developed at the


University of Texas by Professor Muller and some of his students before the writer arrived. This colony was very kindly turned over to the writer by Professor Muller, the start of the experiment being thereby greatly facilitated. The author wishes to thank Miss Isabel Thomas and Miss Elsie Bodemann for some of the data herein reported.

SUMMARY

1. Mitoses disappear from the testes of males x-rayed with 800 r-units at about 11 days after treatment. At 15 days the tubules are shrunken and the number of spermatozoa is greatly reduced. At 27 days most of the tubules consist of a single layer of Sertoli cells, spermatozoa being practically absent. It may be inferred from these histological observations that the passage of Spermatozoa from the testes into the epididymides must cease not much later than 2 weeks after raying, and the possibility is not precluded that it ceases earlier than this.

2. The x-rays have little or no effect on the survival of spermatozoa in the epididymides. This was shown by per- forming a bilateral vasa efferentia ligation on six males and raying three of them with 800 r-units, the other three being kept as controls. One x-rayed and one control male were killed at 36 days after the operation, a considerable number of motile spermatozoa being present in each. A very few motile sperm were present in each member of the pair killed at 49 days, none in the pair killed at 53 days.

3. Motile spermatozoa remain in the epididymides of males x-rayed with 800 o r 1600 r-units for about 6 or 7 weeks following treatment. This is approximately the same length of time that they remain following vasa efferentia ligation. I t may be inferred that with these doses the passage of viable spermatozoa from the testes into the epididymides ceases with a few days or a week of raying. While this inference could not be drawn from the histological observations, it does not necessarily conflict with these observations. Motile sperm reappear in the epididymides of males rayed with 800 r-units

440 GEORGE D. SNELL

at about 13 to 19 weeks following treatment. Motile sperm probably never disappear entirely from the epididymides of most males treated with 200 r-units or less. 4. Mice x-rayed with 800 r-units remain fertile for approxi-

mately 10 to 14 days after treatment. 5. It is suggested that the duration of the initial fertile

period following treatment with the larger doses of x-rays, like the length of survival of spermatozoa in the epididymides, is to be explained by the fact that the passage of viable spermatozoa from the testes into the epididymides ceases within a few days after the application of such doses.

6. The size of the litters sired by x-rayed males during the initial fertile period is markedly reduced, the relation between x-ray dosage and litter size being logarithmic for doses up to and including 800 r-units, and perhaps also for larger doses. When the treatment is 800 r-units, the mean litter size is 2.6.

7. The sex-ratio among the progeny of x-rayed males is unaltered.

8. There is a significant excess of stillbirths in the x-rayed group as compared with the controls, most of these stillbirths occurring in the very small litters. A number of liveborn young in the very small litters showed bruises about the head and shoulders. These facts indicate that it is difficulties in parturition due to the abnormally small size of many of the litters rather than genetic changes produced in the stillborn individuals by the treatment which are usually the immediate cause of their death. One stillborn individual in the x-rayed group lacked hind legs. Death in this case may have been a direct consequence of the treatment.

9. An embryological study has shown that the small size of the litters sired by x-rayed males is due to the death of many of the F, embryos, usually at or shortly after implantation. The presence of both male and female pronuclei in all of fourteen eggs fixed and sectioned at about 16 hours after impregnation shows that failure of fertilization is not in- volved.


10. The death of these embryos is most plausibly explained as the result of hypoploidy caused by x-ray induced chromosome abnormalities.

LITERATURE CITED

BARRAW, J. 0. W., AND G. ARNOLD 1911 Cell changes in the testis due to

BENOIT, J. 1926 Recherches anatomiques, cytologiques et histophysiologiques Arch. Anat.

The effect of x-radiation on the Proc. Roy. SOC. B., vol. 104, pp.

MOORE, C. R. 1928 On the properties of the gonads as controllers of somatic and psychical characteristics. J. Exp. Zool., vol. 50, pp. 455-494.

MULLER, H. J., AND E. ALTENBURG 1930 The frequency of translocations produced by x-rays in Drosophila. Genetics, vol. 15, pp. 283-311.

OLIVER, C. P. 1932 An analysis of the effect of varying duration of x-ray treatment upon the frequency of mutations. Zeits. f . ind. Abst. u. Vererb., Bd. 61, S. 447-488.

PATTERSON, J. T. 1933 The mechanism of mosaic formation in Drosophila. Genetics, vol. 18, pp. 32-52.

REQAUD, C., AND G. DUBREUIL Perturbations dans le d6veloppement des oeufs f6condi.s par des spermatozoYdes roentgenids chez le lapin. C. R. SOC. Biol., T. 64, pp. 1014-1016.

SNELL, G. D. 1933 Genetic changes in mice induced by x-rays. Am. Nat., vol. 67, p. 24.

STADLER, L. J. 1931 The experimental modification of heredity in crop plants. I. Induced chromosomal irregularities. Scientific Agri., vol. 11, pp.

STRANDSKOV, H. H. Effect of x-rays on an inbred strain of guinea-pigs.

YOUNO, W. C. 1929 A study of the function of the epididymis. 11. The im- portance of an ageing process in sperm for the length of the period during which fertilizing capacity is retained by sperm isolated in the epididymis of the guinea-pig. J. Morph. and Phys., vol. 48,

x-rays.

sur les voies excrbtrices du testicule, chez les mammifhres. Hist. et Emb., T. 5, pp. 173-412.

1926 spermatogenesis of the guinea-pig.

Archiv f . Zellforschung, Bd. 7, S. 242-276.

GATENBY, J. B., AND SYLVIA WIQODER

351-370.

1908

557-572. 1932

J. EXP. ZoOl., V O ~ . 63, pp. 175-202.

pp. 475-491.

x-ray sterility in the male house mouse

Documents