Induction of translocations by cyclophosphamide in different germ cell stages of male mice: Cytological characterization and transmission

Mutation Research, 27 (1975) 375-388 © Elsevier Scientific Publishing Company, A m s t e r d a m - - P r i n t e d in The Netherlands

375

INDUCTION OF TRANSLOCATIONS BY CYCLOPHOSPHAMIDE IN D I F F E R E N T GERM CELL S'I'AGES OF MALE MICE: CYTOLOGICAL CHARACTERIZATION AND TRANSMISSION*

RENI~ E. SOTOMAYOR** AND R O B E R T B. CUMMING***

The University of Tennessee--Oak Ridge Graduate School of Biomedical Sciences and Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. 3783 ° (U.S.A.) (Received September 2nd, 1974)

SUMMARY

Cytological and fertility tests were performed in F1 male mice derived from different germ-cell stages of male parents treated with cyclophosphamide (35o mg/kg body weight). The objectives of the present experiment were: (I) to determine the sensitivity of the male germ-cell stages to the induction of translocations by the compound, and (2) to characterize translocation configurations in F1 and F2 males, in order to obtain information about the pattern of chromosome breakage induced and its transmission to subsequent generations. Of 5o8 F~ males studied, 39 were partially sterile and 9 were fully sterile. The group of males conceived 8-2I days after treatment contained by far the highest proportion of partially sterile animals (3o%). It was also the only group in which totally sterile animals (11%) were found. Of 25 semisterile males from this group, 24 gave evidence of translocations when spermatocytes were scored at diakinesis. The translocation frequencies in F~ derived from treated spermatozoa and spermatocytes were 14 and I ~o, respectively. No translocations were detected cytologically in 6 semisterile males derived from treated spermatogonial stages. These results indicate that spermatid stages are especially sensitive to the mutagenic action of cyclophosphamide. In 21 of the 31 semisterile translocation males (68°/o), the majority of the spermatocytes contained 18 bivalents plus a ring-of-four configuration, indicating that both breakpoints were relatively centrally located; and in several of these males, the frequency of cells with rings was close to lOO%. In another 9 Ft males (29%) the predominant multivalent configuration was a chain-of-four, indicating one of the breakpoints to be relatively more terminally located; and in one male (30), the majority of cells had two unequal bivalents, indicating both breakpoints to be fairly close to * Research sponsored by the U.S. Atomic Energy Commission under contract with the Union Carbide Corporation. ** Fellow from the P a n American H e a l t h Organization, W.H.O. P e r m a n e n t address: Departam e n t o de Ciencias Naturales, Universidad de Chile, Casilla 5539, Santiago (Chile). *** To w h o m reprint requests and correspondence concerning this paper should be addressed. Abbreviations: EMS, ethyl methanesulfonate; TEM, triethylenemelamine; TEPA, tris-(i-aziridinyl)-phosphine oxide.

376

R. E. S O T O M A Y O R , R. B. C U M M I N G

the ends of the chromosomes involved. Determination of centromere positions by the use of C-banding showed that chain-of-four configurations in any one male were predonfinantly of a given type. Associations of more than one translocation were observed in I I of 31 translocation males, but in a very low frequency (about 1% oi the spermatocytes). The results indicate that cyclophosphamide induces a relatively random pattern of chromosome break location. In this respect this mutagen more closely resembles X-rays than it does certain chemicals for which there is evidence that breakpoint position is frequently subterminal. Our data indicate that the pattern of configurations observed in F~ was in general transmitted to F2.

INTRODUCTION

Cyclophosphamide (Cytoxan), an alkylating agent used in human cancer therapy, has been tested for mutagenicity in several different mammalian systems, and various genetic effects have been reported 13. The induction of translocations was detected in liver mitotic cells from F1 mouse embryos by DATTA et al. 6. Their technique provided evidence only for unequal translocations, in which one chromosome is very long and the other very short. Examination of the meiotic chromosomes would undoubtedly have revealed additional translocations. Extensive cytological analysis of meiotic chromosomes from Fl's of treated animals can not only provide the evidence for the presence of translocations but can also yield information on the pattern of induced chromosome breakage. Rings- and chains-of-four chromosomes as well as other configurations can be observed in spermatocytes of translocation heterozygotes. The type of configuration observed during diakinesis will depend on the presence or absence of chiasmata in the pairing arms of the translocation% 1~. In turn, the occurrence of chiasmata is positively related to the length of the pairing arms, which vary according to the position of the breakpoint 1°. Since different types of configurations reflect different positions of induced chromosome breaks, it seems reasonable to use a study of configurations to deternfine whether or not cyclophosphamide induces a random pattern of chromosome breakage, and whether or not the same pattern is transmitted to the progeny. In the present study we have tested for fertility a large number of F1 male mice derived from spermatogonial and postspermatogonial stages of males treated with cyclophosphamide. Those found to be partially or completely sterile were then analyzed cytologically in diakinesis, and most of them were found to carry translocations. The incidence of these depends strongly on the father's germ-cell stage treated. We have also examined meiotic chromosomes of F2 translocation heterozygotes derived from F~ translocation males that showed high frequencies of one or another of various kinds of configurations to determine whether or not the same pattern observed in F~ was transmitted to F~. MATERIALS AND METHODS

Forty ( C 3 H × I o I ) male mice, IO to 12 weeks old, were injected intraperi-

CYCLOPHOSPHAMIDE-INDUCED TRANSLOCATIONS IN MALE MICE

377

toneally with 35o mg/kg body weight of cyclophosphamide (Cytoxan) in isotonic saline solution. Immediately after treatment they were caged individually with 3 virgin females of the T-stock, 5 to 6 months old. Daily vaginal plug checks were made in all females, and those found to have been impregnated were removed to individual cages and replaced with new virgin females. Litters were recorded at birth and at weaning and carefully observed for variations in coat color and other external phenotypic characteristics. Normal daughters were then discarded and sons kept for fertility and cytological studies. Offspring were obtained from both pre- and postmeiotically treated male germ-cell stages. The parental male germ-cell stages exposed to cyclophosphamide were determined b y use of the timetable derived by OAKBERG AND DIMINNO17 and OAKBERG 15. According to these authors, mice conceived o to 7 days after treatment derive from spermatozoa treated in the vas and epididymis; 8 to 14 days, from treated mature spermatids; 15 to 21 days, from treated developing spermatids; 22-36 days, trom treated spermatocytes; and over 36 days, from treated spermatogonia. Each F~ male was mated to 3 or more (C57BLIo × C3H)F1 EBIoC3F~! females, known to be highly fertile, in order to detect any reduction is his fertility. The following c~iteria were established to detect sterility and partial sterility. An F~ male was declared fertile if a litter size ot IO or more was produced in the first litter. He was declared partially sterile if the mean litter size in 3 or more litters was 5 or less. If after at least 3 changes of females no litter was obtained, he was declared sterile. As a general rule, BIoC3F ~ females were replaced with others of the same stock whenever a first litter of io was not obtained. Cytological examinations of meiotic chromosomes at diakinesis were performed in F1 partially o~ completely sterile males. Offspring (F2) of some selected F1 males were also analyzed cytologically to study the transmissibility of certain types of translocation configurations. Preparations were made according to the method of EVANS et al. 8 with two modifications. One of these ~8 consisted in the use of a rubber roller applied over a ground-glass plate to squeeze germ cells out of seminiferous tubules. This permitted us to obtain not only cells at diakinesis and metaphase I, but spermatogonial cells and other cell stages as well. The other modification was in the staining procedure. ~fhe preparation was first stained with Giemsa and scored for translocations, and subsequently destained and reprocessed by Giemsa C-banding techniques to determine the position of the centromeres in the translocation configurations. A minimum of IOO cells 1or each F1 or F2 was scored. RESULTS

G~rm cell sensitivity A total of 5o8 F1 male mice from matings covering pre- and postmeiotic stages of treated male parents were tested for fertility. Table I shows the results obtained in the fertility tests and in the cytological analysis performed on those F1 males that showed less than full fertility. No translocations were detected cytologically in males derived from treated spermatogonial stages. However, 6 ot these males, i.e., about 3%, showed reduced fertility. These six were conceived 72 to 136 days postinjection and thus were derived from treated stem cells 16. The average litter size for the par-

378

R. E. SOTOMAYOR, R. B. CUMMING

TABLE I CYCLOPHOSPHAMIDE-INDUCED

Germ cell stages affected

TRANSLOCATIONS OBSERVED IN V 1 MALES

Days conceived

after

N u m b e r of F 1~ c~ Tested Fertile

treatment

S p e r m a t o z o a in t h e vas and epididymis Mature spermatids Early spermatids Spermatocyte Spermatogonia T o t a l c~c~

1- 7 8-14 15-21 22-35 /> 44

Partially sterile

Sterile

Sterile and carrying trans locations

o o 9 o o 9

o o 5a o o 5

Carrying Cytologitranscally locations normal 35 7 77 187 202 508

29 5 45 185 196 460

5 2 22 2 o 31

i o I o 6 8

a The o t h e r 4 were n o t s u i t a b l e for c y t o l o g i c a l a n a l y s i s .

TABLE II A V E R A G E L I T T E R S I Z E -~- S . D . O F V 1 M A L E M I C E F R O M C Y C L O P H O S P H A M I D E - T R E A T E D IN DIFFERENT GERM CELL STAGES

Treated germ cell stage

Fertiles Number

Litter size

Spermatogonia Post-spermatogonia

196 264

9.8 -4- 1.9 io.o :[: 1.9

MALE PARENTS

Partially steriles Translocation heterozygotes N u m b e r Litter size

No translocation detected N u m b e r Litter size

o 31

6 2

4.4 :[: 1.8

3.7 J. 3.o 3.5 :Ix 2.7

C

30-

B

//11

//// zHz

>-

-~ 2 0 E:

//// ////

A

uJ 03

7-

~// ///i

J

IIII ////

//// ////

144;

i--

a. 10

II// //// ....

,,,,

~

////

//// //I/ ////1 F ] ~

1-7 8-14 15-21 2 ~ 3 5 z 4 4 CONCEPTION TIME AFTER TREATMENT (days)

F i g . I . Germ cell s t a g e s e n s i t i v i t y to t h e i n d u c t i o n of t r a n s l o c a t i o n s in F 1 m a l e mi c e d e r i v e d from m a l e p a r e n t s t r e a t e d w i t h c y c l o p h o s p h a m i d e . A, s p e r m ; B, l a t e s p e r m a t i d s ; C, e a r l y s p e r m a t i d s ; D, s p e r m a t o c y t e s ; E, s p e r m a t o g o n i a . The s h a d e d a r e a s r e p r e s e n t c y t o l o g i c a l l y c onfi rme d t r a n s l o c a t i o n h e t e r o z y g o t e s . Sterile a n i m a l s n o t i n c l u d e d in t h i s figure.

C Y C L O P H O S P H A M I D E - I N D U C E D T R A N S L O C A T I O N S IN M A L E MICE

379

tially steriles in this case was 3.7 -+ 3.o compared with 9.8 ~ 1. 9 for the fertiles (see Table II). Only 2 other partially sterile males, one each from treated spermatozoa and treated spermatids, failed to give evidence of translocations at diakinesis. The group of males conceived 8-21 days after treatment contained by far the highest proportion of semisteriles, 3O~o (see Fig. i). It was also the only group in which totally sterile animals (Ii~/o) were found. Almost all of the semisteriles, 23/25, and steriles, 9/9, in this group came from conceptions occurring 17-21 days after treatTABLE

III

TESTIS WEIGHT OF CYCLOPHOSPHAMIDE-INDUCED TRANfiLOCATION HETEROZYGOTES IN F 1 AND F 2 MICE

Weight of one testis (rag) Mean ! S.D. F1 s t e r i l e s a F 1 semisterile translocation heterozygotes F~ f e r t i l e s F 2translocation heterozygotes F2normals

Number ~

49.o ± 6.3

9

i i i .o ± 17.o 122.7 i 16.3

3i 61

i i 2 . 9 ± 17.0 119.3 4- 15.8

52 71

a 5 steriles w e r e cytologically f o u n d to be t r a n s l o c a t i o n h e t e r o z y g o t e s .

ment, and thus presumably from treated early spermatid stages. Of the 9 sterile males, only 5 could be studied cytologically; and these were found to be translocation heterozygotes. Of the other four, two died before the cytological study, and two did not show chromosomes in diakinesis, indicating either spermiogenic arrest prior to diakinesis or failure to find very rare cells. A clear-cut difference between the testis weight of sterile males and that oi semisterile males could be observed (Table III). However, there was no significant difference between semisterile and fertile mice, nor was there a significant difference between F1 and F2 translocation heterozygotes.

Cytological analysis No attempt was made the characterize different translocation configurations in sterile males because of the low yield of cells at diakinesis. Only the 31 partially sterile males carrying translocations were used for extensive cytological analysis of their meiotic chromosomes. A minimum of IOO cells at diakinesis or first metaphase was analyzed per translocation male. The results of this cytological analysis are shown in Table IV. Some of the types of configurations are shown in Figs. 2 and 3All 31 translocation males showed two types of spermatocytes: cells with 18 bivalents plus a ring oi four chromosomes (I8II + RIV) or cells with 18 bivalents plus a chain of four chromosomes (I8II + CIV). Both configurations were present in all cases, but their frequencies were different in different males. Ring-of-four configurations varied from 12 to 97%, while chain-of-four figures varied from 3 to 75~o. No correlation was found between the predominant type of configuration exhibited by a male and his litter size. Other types of configurations with 18 bivalents were also found in some of the

FROM

3I F 1 TRANSLOCATION HETEROZYGOTES

I 4 5 5 7

12 14 17 17 17

17 17 17 18 18

18 18 18 18 19

19 19 19 20 2o

20 20 20 21 23 29

CT-43 CT-44 CT-53 CT-55 CT-6I

CT-62 CT-65 CT-68 CT-72 CT-74

CT-8o C T 82 C T 83 C T 84 C T IOI

CT-Io2 CT-Io4 CT-Io5 CT-93 CT-III

CT-II2 CT-II 3 CT-I2O CT-I26 CT-I45 CT-312

1.9 1.7 1.4 1.4

• ::E ~± i

± ± ~ :J_ :k

1-5 1.6 1.4 2.0 17

1.6 2. 3 i.o I.I 2.2

-4- 1.8 ::k 1.2 ~: 1.3 ± 2.0 :J:: 1.6

-4± ± ::k

5.o 4.6 3.0 4.1 3.7 3.2

:J: -k j_ ± 4:~

2.1 2,5 1. 3 2.1 1.6 1.8

3-7 :~: i . i 4.0 ::k 1.8 5.5 -L 1.3 5.1 ± 1.5 3.6 :j_ 1. 3

4-4 3.2 3.8 4.8 4-2

4.1 4.0 3,1 5.0 4.6

3-4 1.9 5.1 4.6 5.2

5'4 4-4 4.8 4.9

4.8 ~2 2. 5

57.0 58.0 9o.o 46.6 64,0 84.0

53.0 38.0 72.0 92.0 91.o

91.° 15.o 89.0 88.0 72.o

97.0 90.0 14.6 12,o 35'.0

25.0 88.o 55.7 76,0 93.o

15.O 31.O 92.0 69.6 45.o

2.o 4.0 2.0 41.7 3.0 3.o

o,o 13.o 12.o o.o 7.0

o.o o.o 3.0 5.0 o o

3.o 2.o lO. 7 6.0 52.o

51.o 4.0 37.4 o.o o.o

36.0 15.o 3.0 4.9 7.o

35.0 27.0 2.0 3.9 23.0 II.O

41.o 34.0 5.0 8.0 o.o

9.0 75.0 4.o 4.0 23.o

o.o 6.o 62.1 22.0 8.0

7.o 8.o 1.1 17.o 5.0

19.O 47.0 o.o 23.5 44.0

37.0 31.o 4.0 45.6 26.0 14.o

41.o 47.0 17.o 8.0 7.0

9.0 75.o 7.0 9.0 230

3.0 8.o 72.8 28.0 6o.0

58.0 12.o 38.5 17.o 5.o

55.0 62.0 3.0 28.4 51.o

o.o IO.O 3.o 3.9 9.0 o.o

3.0 5.0 6.0 o.o I.O

o.o 2.0 4.o 2.0 IO

o.o o.o o.9 58.0 I.O

I.O o.o 3.9 4.0 o.o

20.0 3.0 5.0 o.o i.o

6.0 I.O I.O 1-9 I.O 2.0

3.0 io.o 5.0 o.o o.o

o.o 7.o o.o I.O i.o

o.o 2.o 3-9 o.o 4.0

16.o o.o o.8 i.o 2.0

9.0 4.0 o.o 1.9 I.O

Days Litter size Translocation configurations recorded in percent conceived produced z8 bivalents plus after Mean ± S,D. R I V CI VA C I VB Total II + II III+ I treatment CI V

OF SPERMATOCYTES

IV

CT-9 CT-I5 CT-I7 CT-25 CT-35

F 1 translocation male

ANALYSIS

TABLE

O.O

I.O

o,o 0.0 0.0

I.O

o.o 0.0 0.0

o.o 0.0 0.0

0.0

0.0

0,0

0.0

0.0 0.0

0.0

0,0

O.O

0.0

O.O O.O

O.O O.O

O.O

O.O

O.O

O.O

O.O

O.O

O,O

O.O

O.O

O.O

O.O

O.O

O.O

O,O

0.0

O.O

0.0

0.0

0.0

O.O

O.O

0.0

O,O

O.O O.O

2.0 O.O 0.0

o.9

o.o

0.0

0.0

0.0

0.0

2.0

o.o

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0. 5

0.0

0.0

0.0

0.0

0.0

0.0

o.o 0.0 0.0

O.O

0.0

0.0

1.0

O.O

O.O

O.O

O.O

O.O

O.O

O.O

I.O

0.0

O.O

O.O

1.9

0.0

0.0

0.0

0.0

0. 3

0.0

0.0

2.0

0.0

0.0

0.0

0.0

1. 9 0.0 0.0

O.O

0.0

0.0

0.0

O.O

O.O

O.O

O.O

3.0

O.O

O.O

0.0

0.0

O.O

O.O

4.9

0.0

0.0

0.0

0.0

0. 3

0.0

0.0

0.0

0.0

0.0

0.0

1.0

z6 bivalents plus RIV CIV CIV + + + RIV CIV RIV

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

z 7 bivalents plus CVI RVI

1. 9 0.0 0.0

2.0

0.0

0.0

1.0

O.O

O.O

O.O

O.O

3.O

O.O

O.O

1.0

0.0

O.O

2.0

7-7

0.0

0.0

0.0

2.0

I.I

0.0

0.0

2.0

0.0

0.0

0.0

1.0

Total multiple translocations

ioo IOO ioo lO 3 ioo IOO

lOO ioo IOO ioo IOO

ioo ioo IOO ioo IOO

lO 3 ioo IOO

IO0

IOO

I oo

I oo

361

IOO

IOO

IOO

I02

IOO

IOO

IOO

Number of cells

o

o

o

O0 ©

CYCLOPHOSPHAMIDE-INDUCED TRANSLOCATIONS IN MALE MICE

381

b

0 O

Q

|

4

i~i!~i~~iI~ C

d

0

Fig. 2. Configurations observed in s p e r m a t o c y t e s from F 1 translocated male mice: a and b, two e x a m p l e s of i 8 I I + R I V ; c and d, two examples of 1811 + CIV t y p e A (chiasmata fully terminalized in c and n o t fully terminalized in d; e and f, two examples of 181I + CIV t y p e B (chiasmata fully terminalized in e and n o t fully terminalized in f. Arrows indicate q u a d r i v a l e n t configurations.

382

R. E. SOTOMAYOR, R. B. CUMMING

Fig. 3. a t h r o u g h d, localization of centromeres after C-band staining: a, 1811 + R I V ; b, 1811 + CIV t y p e A; c, i811 + CIV t y p e B; d, 18II + C I I I + I. Arrows indicate multivalent or u n i v a l e n t configurations, e and f, spermatogonial m e t a p h a s e s from two different translocations; one of the translocation p r o d u c t s is a p p a r e n t in f.

383

C Y C L O P H O S P t I A M I D E - I N D U C E D TRANSLOCATIONS -IN MALE MICE

animals, as follows: two presumably unequal bivalents (I8II + II + If) which cytologically were indistinguishable from 20 normal bivalents; and one trivalent plus one univalent (18II + C l I I + I). In general, these configurations occurred in appreciable proportions only in those males with a relatively low RIV frequency. To assess the possibility that more than one type of chain could occur in any one translocation male, slides were stained with the Giemsa C-banding technique, and the position of the centromeres was determined. Two types of chain quadrivalents were detected: in Type A (18II 4- CIVA) all centroineres are at intermediate positions in the chain; in Type B (I8II + ClVB) two pairs are at the ends of the chain (see Figs. 2 and 3). Most males showed preponderantly or exclusively one type or the other. The average frequencies for all the configurations observed in ceils having at least 18 bivalents are shown in Table V. The average frequency of ring-of-four configurations for the 31 translocation males was 62.6 ~ 28.1 compared with 29.1 ± 22. 4 for chain-of-four configurations. There was no significant difference between the overall frequencies of Type A and B chains. In addition to configurations involving two translocation breaks, we found, in I I of 31 translocations studied, a few cells with either 16 bivalents plus two quadrivalents (either rings, chains, or both) or 17 bivalents plus a chain of six or a ring of six (see Fig. 4). In some cases these configurations were difficult to interpret, even with Giemsa C-banding, because of the possibility that a chain quadrivalent could be contiguous to a bivalent, or two contiguous bivalents could simulate a chain quadrivalent. In several cases, locating the position of the centromeres by Giemsa C-banding helped us to discard or confirm our first diagnosis. In spite of the tact that some of the presumed multiple translocation figures are subject to other interpretations, there is little doubt that many of them are real.

Analysis ofF2 translocation stocks To determine whether or not the pattern of chromosome configurations is transmitted from one generation to the next, we studied 46 sons of 8 F1 translocation males. Four of these Fl'S were chosen because over 80% of their cells contained rings of four chromosomes; three, because the majority of their cells showed the chain-oftour configuration; and one because over 50% of its cells had the II + II configuration. Of the total oi 46 sons 22 carried translocations (Table VI), and 18 of these were used for cytological analysis of configurations. As expected, the pattern of TABLE

V

MEAN FREQUENCIES CHROMOSOME

OF SPERMATOCYTES

FROM

F 1 PARTIALLY

CONFIGURATIONS

Configurations

Frequency ( % ) Mean ± S.D.

Number of cells

I 8 I I -- R I V I8II / CIVA I8II + CIVB i8II + II + II i8II + III + I More than one translocation T o t a l n u m b e r cells

62.6 -c 28.1 lO.6 -t- 15.5 18. 5 ± 19.2 4.8 ± lO.7 2. 7 ± 3.6

2088 427 580 158 88

0.8 ± 1.5

28 3369

STERILE

MALES SHOWING

DIFFERENT

384

R. E. SOTOMAYOR, R. B. CUMMING

O

.

'l C

b

|

P ¢

T

m d

Fig. 4. Double t r a n s l o c a t i o n configurations w h i c h are p r e s e n t in low f r e q u e n c y in a b o u t half of t h e t r a n s l o c a t i o n s studied, a a n d b, two e x a m p l e s of 1611 + R I V + CIV t y p e B; c, ITII + R I V after C - b a n d s t a i n i n g ; d, 17II + CVI. Arrows indicate m u l t i v a l e n t configurations.

chromosome configurations observed in F1 translocation heterozygotes was, in general, transmitted to the F~ (Table VII), and in some cases there is remarkable similarity in the relative frequency of the various configurations. Normal and balanced translocation F2 sons occurred in about a I :I ratio, indicating no detectable departure from theoretical expectations (Table VI). DISCUSSION

Translocation yield and germ cell sensitivity BRITTINGER1 reported that cyclophosphamide induced dominant lethal mutations in all stages of mouse spermatogenesis, but that postmeiotic stages were most sensitive. We tested 202 F1 males derived from treated spermatogonial stages (conceived 44 days or longer after treatment) and found 6 of these to be partially sterile. How-

385

C Y C L O P H O S P H A M I D E - I N D U C E D T R A N S L O C A T I O N S I N MALE MICE TABLE

VI

TRANSMISSION OF THE

TRANSLOCATIONS

SHOWN IN TABLE

VII

F 1 translocation P r e d o m i n a n t heterozygotes configurations

N u m b e r of F23(~ analyzed Normal Translocation

Total

CT-6I 83 84 93

1811 181I I8II I8II

RIV RIV RIV RIV

5 5 2 4

3a 3 2 2

8 8 4 6

CT-68 74 82

1811 + C I V I8II + CIV 1811 + C I V

o 3 3

4b I 5e

4 4 8

CT-72 Total

I8II + II + II

+ + + +

2 24

2 22

4 46

SI s t e r i l e c~ s h o w i n g f e w cells a t d i a k i n e s i s .

bI (J t e s t e d t h r o u g h o f f s p r i n g (F3). c2 c~3 t e s t e d t h r o u g h o f f s p r i n g (F3).

TABLE ANALYSIS LOCATION

VII F R O M F 2 T R A N S L O C A T I O N HETEROZYGOTES DERIVED FROM F 1 T R A N S STOCKS SELECTED FOR HIGH FREQUENCIES OF DIFFERENT TYPES OF CONFIGURATIONS

OF S P B R M A T O C Y T E S

Translocation male

Translocation configurations recorded 18 bivalents plus RIV CIVA CIVB Total II + II III + I CI V

Multiple translocations

Total cells

F 1 CT-61 /6I-9 F2 /61-66

93 87 77

o I 3

5 7 II

5 8 14

o 4 7

2 o 2

o I o

lOO IOO IOO

F1 C T - 8 3 {83-17 F~ 183-I8 ~83-I 9

89 82 83 78

3 I I I

4 12 12 16

7 13 13 17

4 3 3 4

o o I I

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I~. E. SOTOMAYOR, R. B. CUMMING

ever, extensive cytological analysis revealed only spermatocytes with 2o normal bivalents. The average size of litters sired by these males was not significantly different from that of litters sired by postspermatogonially induced translocation heterozygotes. I t was not determined whether the low litter size was the result of the death of embryos or had some other cause. As pointed out by LYON AND MEREDITH 14, the absence of cytological abnormality can indicate either failure of quadrivalent formation in a translocation heterozygote or a cause of partial sterility other than reciprocal translocation. We have reported preliminary results on the induction of specific-locus mutations by cyclophospbamide 5, and although the number of animals analyzed was low for this type of experiment, 3 mutants were found in 511o mice scored. In that study all mutants were conceived 19 or 20 days after treatment with 350 mg/kg body weight of cyclophosphamide and were thus presumably derived from treated early spermatids ~5. In tile present study, the yield of translocation heterozygotes was highest (at least 35~o) in matings occurring between 15 and 21 days after treatment of the father. These findings appear to indicate that early spermatids are especially sensitive to the mutagenic action of cyclophosphamide. The results of DATTA et al. 6 also point to sensitivity of the spermatid stage. These authors used a dose oi 21o mg/kg body weight of cyclophosphamide and scored chromosomes from embryonic liver cells. Only unequal translocations were detectable with their method. Of 34 embryos analyzed, they found 2 carrying translocations. Both of these derived from treated spermatids. However, they did not analyze spermatogonial stages. A comparison of cyclophosphamide with some other chemical mutagens indicates two areas of difference. First, the relative sensitivity of the various male germcell stages is different. While T E P A and EMS induce most translocations in late spermatids and early spermatozoa4,7,~L cyclophosphamide induces the highest frequency ot translocations in early spermatid stages. Second, there is a difference in the relative frequencies of sterile and partially sterile sons. TEPA, TEM, and EMS induce about the same frequencies of sterile and partially sterile mice3,~,7; with cyclophosphamide, however, the overall frequency of sterility is only about one-fourth that of partial sterility. In this respect cyclophosphamide is more similar to X-rays~2, ~°. In summary, our results show that cyclophosphamide induces translocations in postspermatogonial stages and that of these, early spermatids are the most sensitive. Cytology ofF1 and F~ translocation stocks Reciprocal translocations can form several types of chromosome configurations at diakinesis. Rings of four chromosomes are formed when chiasmata were present in all four pairing arms of the translocation. Failure to form a chiasma in one arm produces a chain-of-four configuration; failure in two pairing arms produces a chainof-three plus a univalent, or, alternatively, two unequal bivalentsl°, 1~. On the assumption that the location of breakpoints, by determining the length of the pairing arms of a translocation, influences the probability that chiasmata will occur in these arms, a study of types of configurations should shed light on breakpoint location. This would permit th e general pattern of chromosome breakage induced by a given mutagen to be analyzed. Reports on extensive cytological analysis of chemically-induced translocation

C Y C L O P H O S P H A M I D E - I N D U C E D T R A N S L O C A T I O N S I N M A L E MICE

387

configurations in partially sterile progeny are very limited, so only a few comparisons can be made with other chemicals at present. On the other hand, several reports have provided abundant information about the types of configurations induced by X-rays in spermatogonial la and postspermatogoniaP2O ~ stages of male mice. In the case of translocations induced by X-rays in spermatogonia, analysis of configurations in diakinesis or metaphase I of the treated animals yielded an average of 7o% ring and 24% chain quadrivalents ~3. In the case of irradiated postgonial stages, rings also outnumber chains in partially sterile sons. Thus, LYoN AND MEREDITH14 found 63% of the cells of 25 such animals to contain the ring-of-four configuration; and LgONARD AND DEKNUDT1L in a more extensive cytological study involving 32 partially sterile sons, found the frequencies of cells with ring and chain quadrivalents to be 46 and 32%, respectively. In our experiments, 31 partially sterile sons derived from treated postspermatogonial stages showed an average of 62% rings and 3o% chains. The ring-of-four figure was found in the majority of cells in 21 of the 31 males, and with very high frequencies in some of these: 8 had 9 ° to 97% ; 4 had 84 to 89% ; 4 had 7 ° to 76% ; and 5 had 54 to 64O/o. Our results resemble those from X-ray-induced translocations more than they do results from chemically induced ones. Thus, TEPA, one of the few chemical mutagens studied in this respect, induces an average of 7o% chains and only 2o% rings in partially sterile males derived from treated postspermatogonial stages 7. This means that for TEPA-induced translocations, breaks frequently occur close to one of the ends of a chromosome. This pattern contrasts with that induced by cyclophosphamide, which shows a more random pattern of chromosome breakage. High frequencies of chain-of-four configurations have been found in totally sterile sons derived from X-ray or EMS treatment2,1~. The authors of the more recent paper 2 relate F1 sterility (as opposed to partial sterility) to breakpoint localization near one end of a chromosome and suggest that position effects from translocation of centromeric heterochromatin m a y cause the male sterility. As noted, when chemical mutagens such as T E P A and EMS are used, the frequency of sterile F~ males is almost as great as that of partially sterile ones4, 7. In our experiments, the overall yield of steriles was only about one-fourth that of partially steriles. If induced F1 sterility is indeed positively related to such configurations as chains-of-four or chains-of-three plus univalent, then one would expect that those chemicals that induce high frequencies of rings-of-four would be less effective in inducing sterility. This is, in fact, the case with cyclophosphamide. On the other hand, in the case of partial sterility there is no correlation between the type of configuration present and the litter size produced. The finding of a low frequency of multiple translocation configurations in a number of the translocation heterozygotes studied is unexpected and at the present time not fully explained. It differs from results reported by other authors who have dealt primarily with radiation-induced translocations~O 3. We think it unlikely that the phenomenon is due to multiple translocation heterozygosity with low expression of all but a single translocation. We also think it unlikely that this finding is artifactual in nature. The most likely interpretation at present is that tor some reason these animals have a high "spontaneous" frequency of translocation heterozygosity at diakinesis which interacts with translocations already present to produce the effect, though the strain used, when untreated, shows a frequency of spontaneous trans-

388

R. E. SOTOMAYOR, R. B. CUMMINS

l o c a t i o n h e t e r o z y g o s i t y w i t h i n n o r m a l l i m i t s . T h i s p h e n o m e n o n is c u r r e n t l y u n d e r a c t i v e i n v e s t i g a t i o n in o u r l a b o r a t o r y . I t is c l e a r f r o m o u r r e s u l t s t h a t t h e p a t t e r n o b s e r v e d in FI w a s in g e n e r a l t r a n s m i t t e d t o F~. T h i s m a y b e a n t i c i p a t e d o n a p r i o r i g r o u n d s , b u t it h a s n o t b e e n d e m o n s t r a t e d b e f o r e . A s e x p e c t e d , a n o v e r a l l r a t i o of I : I n o r m a l t o t r a n s l o c a t i o n h e t e r o z y g o t e m a l e s w a s f o u n d in F2. ACKNOWLEDGEMENTS W e t h a n k Dr. LIANE B. RUSSELL for m a n y v a l u a b l e s u g g e s t i o n s d u r i n g t h e c o u r s e of t h i s s t u d y . W e also t h a n k Mrs. MARVA 1v. WALTON for u s e f u l d i s c u s s i o n s in t h e p l a n n i n g a n d e x e c u t i o n of t h i s e x p e r i m e n t a n d b o t h h e r a n d Mrs. KARIN SCHREIBER for v a l u a b l e t e c h n i c a l a s s i s t a n c e . REFERENCES I BRITTINGER, D., Die mutagene Wirkung von Endoxan bei der Maus, Humangenetik, 3 (1966) 156-165 . 2 CACHEIRO, N. L. A., L. B. RUSSELL AND M. S. SWARTOUT, Translocations, the predominant cause of total sterility in sons of mice treated with mutagens, Genetics, 76 (1974) 73-913 CATTANACH,B. M., The sensitivity of the mouse testis to the mutagenic action of triethylenemelamine, Z. Vererbungslehre, 90 (I959) i-6. 4 CATTANACH, B. M., C. E. POLLARD AND J. H. ISAACSON, Ethyl methanesulfonate-induced chromosome-breakage in the mouse, Mutation Res., 6 (1968) 297-307 . 5 CUMMINS, R. B., AND M. WALTON, Genetic effects of cyclophosphamide in the germ cells of male mice, Genetics, 68 (1971) SI 4. 6 DATTA, P. K., H. FRIGGER AND E. SCHLEIERMACHER, The effect of chemical mutagens on the mitotic chromosomes of the mouse in vivo, in F. VOGEL AND G. ROHRBORN (Eds.), Chemical Mutagenesis in Mammals and Man, Springer, Heidelberg, 197 o, pp. 194-213. 7 EPSTEIN, S. S., W. BASS, E. ARNOLD, Y. BISHOP, S. JOSHI AND I. D. ADLER, Sterility and semisterility in male progeny of male mice treated with the chemical mutagen TEPA, Toxieol. Appl. Pharmacol., 19 (1971) 131-146. 8 EVANS, E. P., G. BRECKON AND C. E. FORD, An air-drying method for meiotic preparations from mammalian testes, Cytogenetics, 3 (1964) 289-292. 9 FORD, C. E., AND H. M. CLEGG, Reciprocal translocations, Brit. Med. Bull., 25 (1969) 11o-114. IO FORD, C. E., A. G. SEARLE, E. P. EVANS AND B. J. WEST, Differential transmission of translocations induced in spermatogonia of mice by irradiation, Cytogenetics, 8 (1969) 447-47 o. I I GENEROSO, W. M., W. L. RUSSELL, S. W. HUFF, S. K. STOUT AND D. G. GOSSLEE, Effects of dose on the induction of dominant-lethal mutations and heritable translocations with ethyl methanesulfonate in male mice, Genetics, 77 (1974) 741-752. i2 LI~ONARD,A., AND GH. DEKNUDT, The sensitivity of various germ-cell stages of the male mouse to radiation induced translocations, Can. J. Genet. Cytol., io (1968) 495-507. 13 LI~ONARD, A., Observations on meiotic chromosomes of the male mouse as a test of the potential mutagenicity of chemicals in mammals, in A. HOLLAENDER (Ed.), Chemical Mutagens: Principles and Methods for Their Detection, Vol. 3, Plenum, New York, 1973, pp. 21-56. 14 LYON, M. F., AND R. MEREDITH, Autosomal translocations causing male sterility and viable aneuploidy in the mouse, Cytogenetics, 5 (1966) 335-354. 15 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) 5o7-5 I6. 16 OAKBERG,E. F., Spermatogonial stem-cell renewal in the mouse, Anat. Rec., 169 (1971) 515-532. 17 OAKBERG,E. F., AND R. L. DIMINNO, X-ray sensitivity of primary spermatocytes of the mouse, Intern. J. Radiation Biol., 2 (196o) 196-2o9. 18 PRESTON, R. J., AND J. G. BREWEN, X-ray induced translocations in spermatogonia, I. Dose and fractionation responses in mice, Mutation Res., 19 (1973) 215-223. 19 RUSSELL, L. B., Chromosome aberrations in experimental mammals, in A. G. STEINBERG AND A. G. BEARN (Eds.), Progress in Medical Genetics, Vol. 2, Grune and Stratton, New York, 1962, pp. 230-294. 20 RUSSELL, W. L., Genetic effects of radiation in mammals, in A. HOLLAENDER (Ed.), Radiation Biology, Vol. i, McGraw-Hill, New York, 1954, pp. 825-859.