Tests for heritable translocations in male mammals

Mutation Research, 31 (1975) 291-298 © Elsevier Scientific Publishing Company, Amsterdam--Printed in The Netherlands

291

TESTS FOR H E R I T A B L E TRANSLOCATIONS IN MALE MAMMALS

A. LI~ONARD

Laboratory of Genetics, Department of Radiobiology, C.E.N.-S.C.K., B-24oo Mol (Belgium) (Received December 9th, 1974) (Accepted May i2th, 1975)

INTRODUCTION

The observation of dividing spermatocytes for the presence of reciprocal translocations is, together with the specific locus method and the dominant lethality test, routinely used to estimate genetic hazards in mammalian germ cells. The term of reciprocal translocation designs exchanges of terminal segments between non-homologous chromosomes (Fig. I). At the meiotic synapsis, heterozygosity for a reciprocal translocation results in production of: a quadrivalent configuration in form of a ring (RIV) when the noncentromeric ends of the chromosomes maintain association through terminalyzing chiasmata; a chain of four figure (CIV) by failure of association of chromosomes in one arm; a chain of three plus an univalent figure (C III + I) by failure of association of two adjacent arms; two bivalents, or one bivalent and two univalents or, even, four univalents. More complex configurations such as hexavalents, octovalents or decavalents can be observed if two, three or four translocations occur. Using an appropriate interval between treatment and observation (5O-lOO days) one can estimate the rate at which reciprocal translocations are induced in spermatogonia (the main germ cell type at risk) of the treated animals by scoring multivalent configurations in the derived spermatocytes (spermatocyte test on treated males). The production of reciprocal translocations in the postmeiotic male germ cells is generally ascertained by analysing the dividing spermatocytes of the FI males (FI translocation test). This test can also be used to compare the differential sensitivity of various germ cell stages and to test female germ cells. Refs. 7, 8, 15 discuss the problems related to the induction of heritable reciprocal translocations in mammals by environmental mutagens and their importance for the evaluation of genetic hazards to man.

THE TECHNICAL PROCEDURE Instruments and chemicals required to perform tests for heritable translocations : (z) A centrifuge giving exact speeds from 14 to 88 g and which is equipped with 4-8 carriers for 3-ml centrifuge tubes.

292

A. 1A~;ONARI)

Normal

chromosomes

Exchange

:IIII:-IXI Rearranged chromosomes

:I I:

J

Meiotic

pairing

Oea

Fi~. 1. D i a g r a m o[ a reciprocal t r a n s l o c a t i o n b e t w e e n t w o a c r o c e n t r i c c h r o m o s o m e s .

(:') A microscope. (3) A warming plate. (4) Dissecting instruments (small scissors and small straight and curved forceps).

(5) Pyrex petri dishes (6 cm diameter), conical centrifuge tubes of 3 ml capacity, conventional glass pipettes, Pasteur pipettes drawn out to a fine tip, propipettes of 2 ml, coverglasses, grease-free microslides. (@ Sodium citrate (C~HsNaaO7-2H20), acetic acid (CHaCOOH), ethyl alcohol (CHaCH2OH), chloroform (CHCI:3), toluidine blue_ TEST ANIMALS

The forty chromosomes of the mouse are acrocentric. Therefore the meiotic association configurations are rather simple for these species (Fig. 2) and it is relatively easy to identify the translocation figures in the form of a ring or of a chain in the dividing spermatocytes (Figs 3-6). Due to the presence of metacentric or submetacentric chromosomes the meiotic pairing results in rats and Chinese hamsters in the formation of bivalents of a much more complex nature than the zo bivalents of the mouse. Consequently the mouse is the most convenient animal on which to perform the tests for heritable translocations in male mammals. The frequency of spontaneous translocations among young adult control mice is extremely low. Since a slight increase has been reported in old animals ~, young 3-4-month old adult mice are to be. preferred. EXPERIMENTAL DESIGN

The general outline of the tests for heritable translocations in male mouse is shown in Table I. In general, only very few translocation configurations are recovered in dividing spermatocytes when mouse spermatogonia are treated with mutagenic

HERITABLE TRANSLOCATIONS IN MALE MAMMALS

293

Fig. 2. Mouse d i v i d i n g s p e r m a t o c y t e I w i t h 20 b i v a l e n t s .

compounds. Moreover, the data on ionizing radiations have often revealed great variations among animals given the same treatment. A minimum of IO animals with an analysis of lOO-2OO cells per animal is therefore required for each treatment. To allow the treated spermatogonia to differentiate into dividing spermatocytes, an interval of 5O-lOO days between treatment and examination is suitable. To ascertain that a compound does not produce heritable translocations in postmeiotic germ cells a great number of male offspring must be investigated. In general, spermatids are the most sensitive stage with respect to the induction of chromosome damage by chemical mutagens but, as indicated by the example of mitomycin C which yields higher dominant lethality in spermatocytes 2, the possibility also exists that certain compounds act on spermatocytes but not on other postmeiotic germ cell stages in producing heritable translocations. Depending on the fertility of the strain IO to 25 males are usually used. To avoid fertilisation before the compound could reach the germ cells, the animals are usually treated in the morning and caged with virgin females in the afternoon. The females are exchanged for fresh ones every seven days in order to compare the sensitivity of the different male germ cell stages13,1*: 1- 7 days spermatozoa, 7-14 days spermatids, i 5 - 2 i days early spermatids, 22-35 days spermatocytes. Tile F I females are discarded at weaning and the F I males are examined when mature. Experiments with ionizing radiation 9 and cyclophosphamide TM have shown that in a translocation heterozygote the incidence of multivalent configurations can vary from 12 to 100%. Based on these results it is generally considered that 25 cells are enough to detect heterozygosity for a reciprocal translocation in the F I males. If the 25 analysed cells have been found with 20 bivalents the animal is considered as normal.

294

i . IAIZONARI)

R ;IV

x

f

Fig. 3. Mouse d i v i d i n g s p e r m a t o c y t e [ with IS b i v a l e n t s a n d a ring q u d r i v a l e n t . Fig. 4. Mouse d i v i d i n g s p e r m a t o c y t c I w i t h i8 b i v a l e n t s a n d a ring q u a d r i v a l e n t . Fig. 5. Mouse d i v i d i n g s p e r n l a t o c y t e 1 with 18 b i v a l e n t s a n d a c h a i n q u a d r i v a l e n t . Fig. 6. Mouse dividing s p e r m a t o c y t e I with 18 b i v a l e n t s and u II1 t 1 configuration.

If some n m l t i v a l e n t s have been d e t e c t e d i o o cells are e x a m i n e d for the p r e s u m e d t r a n s l o c a t e d male. DESCRIPTION OF METHODS

Two m a i n techniques h a v e been described for the p r e p a r a t i o n of male m a m m a lian meiotic cllronmsomes (ref. 28 for review). The a i r - d r y i n g m e t h o d d e v e l o p e d b y EVANS et a l . a is more a p p r o p r i a t e for the mouse s p e r m a t o c y t e s . This m e t h o d t a k e s v e r y little t i m e a n d provides a large n u m b e r of scorable cells per animal. Since it involves a t h o r o u g h mixing of t h e testis p o p u l a t i o n t h e cells scored can be con-

HERITABLE TRANSLOCATIONSIN MALE MAMMALS TABLE GENERAL

Test

295

I OUTLINE

OF THE TESTS FOR THE HERITABLE

Number of males to be

Spermatocyte test Examined on the treated males io FI translocation test Mated lO-25

TRANSLOCATIONS

IN MALE MICE

Number of cells to be Interval of time (days) between treatment analyzed per and Treated m a l e

Observation

IOO

.50--1OO

Fz male

Mating Sper- Mature Young Sper- Spermato- sper- s p e r - m a t o - matoFI translocated male zoa matids matids cytes gonia

25

IOO

1-7

8-14

15-21 22-35 35-

sidered as a random sample, representative of the whole spermatocyte population. The method of MEREDITH11 is rather time-consuming and requires the examination of m a n y preparations to avoid sample heterogeneity but is preferable for those F I males which are sterile and have very small testes yielding very few dividing spermatocytes. The two techniques will be described separately. The air-drying method"

( I ) The mice are sacrificed by cervical dislocation and the testes removed immediately. In order to wash away fat they are quickly placed in 2.2% (w/v) trisodium citrate (CeHsNa307 •2H20) solution in a 6-cm diameter Petri dish maintained at room temperature. It is important to use fresh citrate solutions prepared once a week with water double-distilled in a quartz apparatus and stored in a refrigerator. (2) The washed testes are then transferred into 2.5 ml of a 2.2% citrate solution in fresh petri dishes and the tunica is removed. The contents of the tubules are gently teased out with curved forceps. In the F I translocation test, the contents of the two testes are generally mixed together whereas in the spermatocyte test the two testes are prepared separately in order to detect any differences between testes in the frequency of induced reciprocal translocations. (3) The cell suspension produced is transferred into a 3-ml conical tube with a Pasteur pipette and centrifuged at 22 g for 5 rain. (4) The supernatant is discarded, and the pellet is resuspended in 2 ml of a 1% (w/v) citrate solution added drop by drop with a Pasteur pipette while flicking the tube with a finger to mix well. (5) After IO rain the suspension is centrifuged for 5 min at 22 g, and as much if the supernatant as possible is removed. 2 ml of fixative (3 parts ethyl alcohol to I part glacial acetic acid) is added dropwise flicking the tube vigorously after each drop. (6) The fixative is changed after 5 min and again after IO rain. (7) Three to four droplets of the final suspension are allowed to fall from a fine tipped Pasteur pipette on to a grease-free slide and blown upon to dry. According to the cell concentration the process is repeated 5 to IO times to cover the whole slide with a homogeneous layer of cells. Meredith's method (z) The first step is the same as in the air-drying method. (2) After removing the testis tunica the tubules are gently washed in i % citrate

solution, the total duration of the hypotonic treatment being 12 min.

296

a. LiX~
(3) The tubules would then be transferred to fixative (3 parts ethyl alcohol to I part acetic acid) for a least 15 rain. The tubules can be stored in a stoppered glass tube for several days in refrigerator if necessary. (4) Three or four pieces of the tubules (about 1 cm long) are transferred to a glass tube containing I ml of 6o% acetic acid, and the tubes are gently flicked until the tubules become transparent. (5) With a fine tipped Pasteur pipette, one drop is put on a grease-free slide kept at 6o ° on a warming plate. The drop is drawn into and expelled from the pipette several times until the liquid has disappeared. This procedure is repeated with three or four drops. MICROSCOPIC EXAMINATION

For immediate examination, the preparations are normally stained with toluidine blue but a lactic-acetic-orcein stain m a y be used for finer examination and for slides. The preparations are scanned at low power magnification (IO × 15), and the dividing spermatocytes are examined for quality of spreading. The selected cells are analysed at higher magnification (lO × 4 ° or Io × IOO). DRAWBACKS

It must be pointed out that selective elimination m a y occur before the sperrnatogonia bearing reciprocal translocations reach the spermatocyte stage thus decreasing the frequency of detectable multivalents. Failure of certain translocations to form multivalent configurations also reduced the frequency observed. A review of the literature shows that, for compounds giving positive results in the spermatocyte test on treated males, the frequency of reciprocal translocations is generally very low ( < i % ) when compared with the rate of reciprocal translocations induced in premeiotic male germ cells b y ionizing radiation 7. It is noteworthy that most of the chemicals that fail to induce reciprocal translocations in spermatogonia have been shown to be potent mutagens in other organisms or other test systems and are even capable of causing chromosome breakage in the post-meitoic male germ cells of the mouse as demonstrated by the dominant lethality test. Furthermore, some compounds (e.g. mitomycin C) which fail to produce chromosome rearrangements in premeiotic male germ cells~, 6 are able to produce chromosome breaks resulting in the killing of spermatogonia as shown by the induction of a temporary sterile period ~°. ACKNOWLEDGEMENT

This work has been performed with a grant from the Fonds National de la Recherche Fondamentale Collective. REFERENCES I ADLER, 1. D., C o m p a r a t i v e cytogenetic s t u d y after t r e a t m e n t of mouse s p e r m a t o g o n i a with m i t o m y c i n C, Mutation Res., 23 (1974) 369-379. 2 EHL1NG, U. H., Comparison of radiation- and chemically-induced d o m i n a n t lethal m u t a t i o n s in male mice, Mutation Res., i i (1971) 35-44-

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2} EVANS, E. P., G. BRECKON AND C. E. FORD, A n a i r - d r y i n g m e t h o d for meiotic p r e p a r a t i o n s f r o m m a m m a l i a n testes, Cytogenetics, 3 (1964) 289-294. 4 FORD, C. E., A. G. SEARLE, E. P. EVANS AND B. J. WEST, Differential t r a n s m i s s i o n of t r a n s locations i n d u c e d in s p e r m a t o g o n i a of mice b y irradiation, Cytogenetics, 8 (1969) 447-47 o. 5 GERBER, G. B., AND A. L~ONARD, Influence of selection, n o n - u n i f o r m cell p o p u l a t i o n a n d repair on dose-effect c u r v e s of genetic effects, Mutation Res., 12 (1971) 175-182. 6 GILLIAVOD, N., AND A. Lt~ONARD, T e s t s for m u t a g e n i c effects of chemicals in mice, I. Effects of m i t o m y c i n C on s p e r m a t o g o n i a , Mutation Res., 13 (1971) 274-275. 7 L~ONARD, A., R a d i a t i o n i n d u c e d t r a n s l o c a t i o n s in s p e r m a t o g o n i a of mice, Mutation Res., I I (1971 ) 71-88. 8 LI~ONARD,A., O b s e r v a t i o n s on meiotic c h r o m o s o m e s of t h e male m o u s e as a t e s t of t h e p o t e n t i a l m u t a g e n i c i t y of chemicals in m a m m a l s , in A. HOLLAENDER (Ed.), Chemical Mutagens: Principles and Methods for their Detection, Vol. 3, P l e n u m , New York, 1973, pp. 21-56. 9 LI~ONARD,A., AND GH. DEKNUDT, T h e s e n s i t i v i t y of v a r i o u s germ-cell stages of t h e male m o u s e to r a d i a t i o n i n d u c e d t r a n s l o c a t i o n s , Can. J. Genet. Cytol., IO (1968) 496-5o7 . IO LI~ONARD, A., AND N. GILLIAVOD, T e s t i c u l a r c h a n g e s in mice after t r e a t m e n t w i t h m i t o m y c i n C, Toxicology, i (1973) 217-223. i I MEREDITH, R., A simple m e t h o d for p r e p a r i n g meiotic c h r o m o s o m e s from m a m m a l i a n testis, Chromosoma, 26 (1969) 254-258. 12 MURAMATSU,S., F r e q u e n c y of s p o n t a n e o u s t r a n s l o c a t i o n s in m o u s e s p e r m a t o g o n i a , Mutation Res., 24 (1974) 81 82. 13 OAKBERG, E. V., D u r a t i o n of s p e r m a t o g e n e s i s in t h e m o u s e a n d t i m i n g of stages of t h e cycle of t h e seminiferous epithelium, Am. ]. Anat., 99, (1956) 5o7-516. 14 OAKBERG,E. F., AND R. L. DIMINNO, X - r a y s e n s i t i v i t y of p r i m a r y s p e r m a t o c y t e s of t h e mouse, Int. J. Radiat. Biol., 2 (196o) 196-2o9. 15 SEARLE, A. G., C h r o m o s o m e d a m a g e a n d risk a s s e s s m e n t , in J. DE GROUCHY, F. J. G. EBLING AND I. W. HENDENSON (Eds.), Proc. 4th Int. Congr. Hum. Genet., z 9 7 z , pp. 58-66. 16 SOTOMAYOR, R. E., AND R. B. CUMMING, I n d u c t i o n of t r a n s l o c a t i o n s b y c y c l o p h o s p h a m i d e in different g e r m cell stages of male mice: cytological c h a r a c t e r i z a t i o n a n d t r a n s m i s s i o n , Mutation Res., 27 (1975) 375-388.