- Email: [email protected]
Synaptonemal complexes as indicators of induced structural change in chromosomes after irradiation of spermatogonia
Mutation Research, 122 (1983) 149-154 Elsevier
149
MRLett 0463
Synaptonemal complexes as indicators of induced structural change in chromosomes after irradiation of spermatogonia A . H . C a w o o d and G. Breckon M.R.C. Radiobiology Unit, Harwell, Didcot, Oxon, 0 X l l ORD (Great Britain) (Accepted 13 July 1983)
Summary The incidence of multivalent formation has been compared in primary spermatocytes at pachytene, using synaptonemal complex preparations and at diakinesis/metaphase, using air-dried preparations, from testes of Syrian hamsters several weeks after exposure to an acute dose of X-rays. The pachytene preparations revealed nearly twice the number of multivalents found at diakinesis/metaphase I, and showed the ability to reveal types of structural alteration undetectable in conventional air-dried techniques.
The analysis of chromosome behaviour in primary spermatocytes is used extensively in the estimation of the extent of chromosomal rearrangements induced in spermatogonia after irradiation (for review see Sankaranarayanan, 1982). Particular emphasis has been placed on the induction of translocations since these give rise to multivalent configurations which, given good cytological preparations, are relatively simple to detect at diakinesis/metaphase I (diak/MI). However, for multivalent associations to persist until diak/MI two things must happen; homologous segments must synapse and there must then be chiasma formation in at least one of the translocated segments as well as in the non-translocated segments. As Evans (1979) has pointed out, this requirement for chiasma formation may mean that rearrangements of small segments may be missed unless pachytene cells are analysed. Unfortunately, in air-dried preparations of pachytene cells it is usually possible to analyse only part of the genome, but an alternative method is available by which entire complements of synaptonemal complexes (SC; the structure formed 0165-7992/83/$ 03.00 © 1983 Elsevier Science Publishers B.V.
150 by synapsed chromosomes) can be obtained in large numbers of cells suitable for both light and electron microscopy (see Moses, 1977). In order to determine whether pachytene cells are more sensitive indicators of induced structural chnge than are diak/MI cells we exposed male Syrian hamsters (Mesocricetus auratus) to a single acute dose of X-rays, and compared the frequencies of multivalents at pachytene and diak/MI within each animal and between animals sampled at different times after irradiation.
Materials and methods
Irradiation Karyotypically normal male Syrian hamsters (Mesocricetus auratus, 2n = 44), 25 weeks old, which had been maintained in a 15-h day length at 23°C, were restrained in plastic containers, and the caudal third of the body was exposed to X-rays using a cross-fire technique (dorsal-ventral/ventral-dorsal; 250 KeV X-rays, 1.1 mCu, 1.56 Gy/min) to give a total absorbed dose to the testes of 2.6 Gy. The remaining two thirds of the body were shielded with 5 mm Pb. Control animals were sham irradiated. Testes were examined cytologically from 5 to 15 weeks post-irradiation, and 7 weeks post-sham irradiation in the controls.
Cytology For each animal, one testis was injected with 0.05 ml colchicine (at an equivalent dose of 8 mg/kg body weight) 2 h before the animal was killed with ether. Both testes were removed and their weights recorded. Sperm was collected from the epididymis of the uninjected testis for counting. The testis exposed to colchicine was used for air-dried preparations of diak/MI and metaphase II by the method of Breckon (1982). The other testis was used for SC preparations by a method developed by E.P. Evans (personal communication). The tunica was removed and the tubulues macerated in Hanks' basic salt solution. The tubule fragments were aspirated 30-40 times with a Pasteur pipette then gently rolled with a 25 mm x 5 mm plastic-covered steel roller until they appeared translucent. The resulting cell suspension was centrifuged at 800 r.p.m, for 5 min and the pellet resuspended in 1-2 ml Hanks' solution to which an equal amount of 0.3°7o v/v aqueous Nonidet P40 (B.D.H. Ltd.), pH 8.5-9, was added. After 5 min the tube was filled with fixative (407o w/v aqueous paraformaldehyde in 0.1 M sucrose) and centrifuged at 500 r.p.m, for 5 min. The pellet was resuspended in just enough fixative to give a slightly cloudy suspension. Up to this point the cell suspension and all solutions should be kept on ice. 0.1 M sucrose may be used in place of Nonidet. A thin layer o f the suspension was put on a slide and dried on a warm hotplate. When dry the slide was flooded with distilled water for 20-30 sec and gently blown
151
dry. To stain the SCs for light microscopy 3 drops of 50°70 w/v AgNO3 solution were placed on the cells, a coverslip added and the slide put into a closed, moist glass petri dish and illuminated with a 60 W tungsten filament light bulb 30-50 mm above the lid of the dish for about 45 min. The coverslip was rinsed o f f with running distilled water, and the slide was dried, cleared in xylene, and mounted in D.P.X. For electron microscopy the slides were covered with a plastic film (after Felluga and Martinucci, 1976: a 0.5°70 w/v solution o f a Falcon petri dish in chloroform was used; this gives a film showing silver-gold in reflected light). Staining was carried out as above, but for only 30 min. The positions of suitable cells were marked on the filmed slide using a marker objective fitted with an inked rubber ring, the film was floated o f f on distilled water, grids were placed at the marked positions, and the film and grids picked up from the surface of the water using Parafilm.
Results and discussion
Normal cells at pachytene and diak/MI contain 21 autosomal bivalents and an X-Y bivalent (pachytene, Fig. 1; diak/MI, Fig. 2). 100 cells were examined at both pachytene and diak/MI in all animals. The numbers of cells in these samples with structural changes are shown in Table 1. Despite the changes in sperm counts and relative testis weights there is good homogeneity among the irradiated samples, both at pachytene 0(6=8.13, 2 P = 0.23) and at diak/MI 0(~ = 2.69, P = 0.85). All irradiated samples show a higher incidence of structural abnormalities than the corresponding control samples at both meiotic stages, and in 1-sided Fisher's exact tests all but one of these differences (diak/MI in animal 1581) are statistically significant ( P ¢ 0 . 0 1 2 ) . For all animals except 1563 more structural abnormalities are seen at pachytene than at diak/MI, and although in Fisher's exact test the individual differences are not significant, the overall difference is highly significant ( U = 2.98, P = 0.0028). Of the 60 abnormal pachytene cells 52 contained a single quadrivalent (Fig. 3), 1 two quadrivalents, 3 a hexavalent, 2 a trivalent plus a univalent, 1 a ring X-Y bivalent, and 1 a small, interstitial loop having a morphology which suggests that it is the result o f a deletion or insertion/duplication rather than of an inversion (Fig. 5). This loop shows the sensitivity of the SC technique for the analysis of induced structural change; such a small change would be completely undetectable at diak/MI. Of the 32 abnormal diak/MI cells 31 contained a single quadrivalent, and 1 a hexavalent (Fig. 4). In approximately 10°70 of pachytene cells some o f the SCs were associated in such a way that more than one interpretation was possible using the light microscope. All such ambiguous associations were counted as overlapped bivalents rather than as multivalents. This interpretation was checked by the examination of 20 ambiguous associations with the electron microscope. Of these only one proved to involve a
152
I
.
.
2
d
A
~*~'~-
-
~
'
~; t
w
. . . . . . .
Fig. 1. Normal complement of SCs at pachytene, X-Y bivalent arrowed. Fig. 2. Normal diak/MI cell, X-Y bivalent arrowed. Fig. 3. Pachytene cell with radiation-induced quadrivalent (arrowed). Fig. 4. Diak/MI cell with radiation-induced hexavalent (arrowed). Fig. 5. Pachytene bivalent with interstitial deletion insertion loop (arrowed). Fig. 6. Electron micrograph of ambiguous association of SCs showing a quadrivalent (1V) and a bivalent (II). Bars equal 5/zm.
153
TABLE 1 I N C I D E N C E OF STRUCTURAL C H A N G E IN PRIMARY SPERMATOCYTES Animal
Weeks Wt. both testes Sperm count post-irradiatiWt. whole x 100% (103/ml) on
1512 1560 1513 1581 1514 1563 1515
5 6 7 8 11 13 15
Pachytene
Diak/MI
6_+ 2.37 14_+3.47 9_+2.86 7 + 2.55 10_+3 4_+ 1.96 10-+ 3
4 + 1.96 6_+2.37 6_+2.37 2 + 1.40 5_+2.18 4 + 1.96 5 _+2.18
body 0.84 1.17 1.71 2.21 2.52 3.11 3.24
19.2 32.15 88.5 95.2 104_+2 112.8 68.2
Overall 1477 1478
Number abnormal cells (-+ S.E.)
8.57+ 1.06 Control Control
3.53 3.73
107.2 84.4
0 0
4.57_+0.79 0 0
multivalent (shown in Fig. 6 - a quadrivalent plus an overlapping bivalent), which suggests that counting only unequivocal multivalents using light microscopy does not lead to a serious underestimate o f the extent of structural change. The proportion of cells with ambiguous associations depends upon the quality o f the preparations; the less well spread the cells the more necessary it is to use the electron microscope to validate the light microscopy. Segregation at anaphase I, as judged by counts at metaphase II, appeared unaffected by the dose of irradiation used here. All ceils examined had 22 chromosomes, and there was no significant deviation from a 1:1 ratio in the segregation of the sex chromosomes. The greater incidence of scorable multivalents at pachytene compared with diak/MI reflects, at least in part, the requirement in the former for synapsis alone in contrast to the need in the latter for both synapsis and chiasma formation. However, it is possible that some o f the pachytene cells containing multivalents make no further progress through meiosis and thus make no contribution to the genetic burden carried by the gametes. This possibility is extremely difficult to test although a comparison o f the dose-response curves based on pachytene and diak/MI cells may give some indication. This work is under way. There is no indication of preferential selection against multivalents arising from exchanges between the sex chromosomes and autosomes since the ratio of their numbers in pachytene and diak/MI cells (6:3) matches that o f the total numbers of multivalents (60:32). Moses (1977) argued that the analysis of SCs could be o f great benefit in the analysis of a variety o f experimental treatments o f the germ ceils. The results reported here support this view, and show that the analysis of diak/MI cells alone
154 m a y l e a d to a serious u n d e r e s t i m a t e o f the a m o u n t o f s t r u c t u r a l c h a n g e i n d u c e d in s p e m a t o g o n i a l cells a f t e r i r r a d i a t i o n . T h e SC t e c h n i q u e is s i m p l e a n d q u i c k , a n d can p r o v i d e i n f o r m a t i o n n o t o b t a i n a b l e f r o m a n y o t h e r m e t h o d . It is o f p o t e n t i a l v a l u e in a n y s t u d y i n v o l v i n g e x p e r i m e n t a l t r e a t m e n t o f p r e m e i o t i c a n d m e i o t i c g e r m cells.
Acknowlegements W e w o u l d like to t h a n k M r . D . G . P a p w o r t h for p e r f o r m i n g t h e statistical analyses, M r . M . C o r p for p r o v i s i o n o f r a d i a t i o n facilities, a n d M r . J. Bowler for a n i m a l husbandry.
References Breckon, G. (1982) A modified hypotonic treatment for increasing the frequency and quality of meiotic metaphases from spermatocytes of the Syrian hamster, Stain Technol., 57, 349-353. Evans, E.P. (1979) Cytological methods for the study of meiotic properties in mice, Genetics, 92, s97-s103. Felluga, B., and G.B. Martinucci (1976) A simple method for karyotyping by transmission electron microscopy, J. Submicr. Cytol., 8, 347-352. Moses, M.J. (1977) Microspreading and the synaptonemal complex in cytogenetic studies, in: A. de la Chapelle and M. Sorsa (Eds.), Chromosomes Today, Vol. 6, Elsevier, Amsterdam, pp. 71-82. Sankaranarayanan, K. (1982) Genetic Effects of Ionizing Radiation in Multicellular Eukaryotes and the Assessment of Genetic Radiation Hazards in Man, Elsevier, Amsterdam.
149
MRLett 0463
Synaptonemal complexes as indicators of induced structural change in chromosomes after irradiation of spermatogonia A . H . C a w o o d and G. Breckon M.R.C. Radiobiology Unit, Harwell, Didcot, Oxon, 0 X l l ORD (Great Britain) (Accepted 13 July 1983)
Summary The incidence of multivalent formation has been compared in primary spermatocytes at pachytene, using synaptonemal complex preparations and at diakinesis/metaphase, using air-dried preparations, from testes of Syrian hamsters several weeks after exposure to an acute dose of X-rays. The pachytene preparations revealed nearly twice the number of multivalents found at diakinesis/metaphase I, and showed the ability to reveal types of structural alteration undetectable in conventional air-dried techniques.
The analysis of chromosome behaviour in primary spermatocytes is used extensively in the estimation of the extent of chromosomal rearrangements induced in spermatogonia after irradiation (for review see Sankaranarayanan, 1982). Particular emphasis has been placed on the induction of translocations since these give rise to multivalent configurations which, given good cytological preparations, are relatively simple to detect at diakinesis/metaphase I (diak/MI). However, for multivalent associations to persist until diak/MI two things must happen; homologous segments must synapse and there must then be chiasma formation in at least one of the translocated segments as well as in the non-translocated segments. As Evans (1979) has pointed out, this requirement for chiasma formation may mean that rearrangements of small segments may be missed unless pachytene cells are analysed. Unfortunately, in air-dried preparations of pachytene cells it is usually possible to analyse only part of the genome, but an alternative method is available by which entire complements of synaptonemal complexes (SC; the structure formed 0165-7992/83/$ 03.00 © 1983 Elsevier Science Publishers B.V.
150 by synapsed chromosomes) can be obtained in large numbers of cells suitable for both light and electron microscopy (see Moses, 1977). In order to determine whether pachytene cells are more sensitive indicators of induced structural chnge than are diak/MI cells we exposed male Syrian hamsters (Mesocricetus auratus) to a single acute dose of X-rays, and compared the frequencies of multivalents at pachytene and diak/MI within each animal and between animals sampled at different times after irradiation.
Materials and methods
Irradiation Karyotypically normal male Syrian hamsters (Mesocricetus auratus, 2n = 44), 25 weeks old, which had been maintained in a 15-h day length at 23°C, were restrained in plastic containers, and the caudal third of the body was exposed to X-rays using a cross-fire technique (dorsal-ventral/ventral-dorsal; 250 KeV X-rays, 1.1 mCu, 1.56 Gy/min) to give a total absorbed dose to the testes of 2.6 Gy. The remaining two thirds of the body were shielded with 5 mm Pb. Control animals were sham irradiated. Testes were examined cytologically from 5 to 15 weeks post-irradiation, and 7 weeks post-sham irradiation in the controls.
Cytology For each animal, one testis was injected with 0.05 ml colchicine (at an equivalent dose of 8 mg/kg body weight) 2 h before the animal was killed with ether. Both testes were removed and their weights recorded. Sperm was collected from the epididymis of the uninjected testis for counting. The testis exposed to colchicine was used for air-dried preparations of diak/MI and metaphase II by the method of Breckon (1982). The other testis was used for SC preparations by a method developed by E.P. Evans (personal communication). The tunica was removed and the tubulues macerated in Hanks' basic salt solution. The tubule fragments were aspirated 30-40 times with a Pasteur pipette then gently rolled with a 25 mm x 5 mm plastic-covered steel roller until they appeared translucent. The resulting cell suspension was centrifuged at 800 r.p.m, for 5 min and the pellet resuspended in 1-2 ml Hanks' solution to which an equal amount of 0.3°7o v/v aqueous Nonidet P40 (B.D.H. Ltd.), pH 8.5-9, was added. After 5 min the tube was filled with fixative (407o w/v aqueous paraformaldehyde in 0.1 M sucrose) and centrifuged at 500 r.p.m, for 5 min. The pellet was resuspended in just enough fixative to give a slightly cloudy suspension. Up to this point the cell suspension and all solutions should be kept on ice. 0.1 M sucrose may be used in place of Nonidet. A thin layer o f the suspension was put on a slide and dried on a warm hotplate. When dry the slide was flooded with distilled water for 20-30 sec and gently blown
151
dry. To stain the SCs for light microscopy 3 drops of 50°70 w/v AgNO3 solution were placed on the cells, a coverslip added and the slide put into a closed, moist glass petri dish and illuminated with a 60 W tungsten filament light bulb 30-50 mm above the lid of the dish for about 45 min. The coverslip was rinsed o f f with running distilled water, and the slide was dried, cleared in xylene, and mounted in D.P.X. For electron microscopy the slides were covered with a plastic film (after Felluga and Martinucci, 1976: a 0.5°70 w/v solution o f a Falcon petri dish in chloroform was used; this gives a film showing silver-gold in reflected light). Staining was carried out as above, but for only 30 min. The positions of suitable cells were marked on the filmed slide using a marker objective fitted with an inked rubber ring, the film was floated o f f on distilled water, grids were placed at the marked positions, and the film and grids picked up from the surface of the water using Parafilm.
Results and discussion
Normal cells at pachytene and diak/MI contain 21 autosomal bivalents and an X-Y bivalent (pachytene, Fig. 1; diak/MI, Fig. 2). 100 cells were examined at both pachytene and diak/MI in all animals. The numbers of cells in these samples with structural changes are shown in Table 1. Despite the changes in sperm counts and relative testis weights there is good homogeneity among the irradiated samples, both at pachytene 0(6=8.13, 2 P = 0.23) and at diak/MI 0(~ = 2.69, P = 0.85). All irradiated samples show a higher incidence of structural abnormalities than the corresponding control samples at both meiotic stages, and in 1-sided Fisher's exact tests all but one of these differences (diak/MI in animal 1581) are statistically significant ( P ¢ 0 . 0 1 2 ) . For all animals except 1563 more structural abnormalities are seen at pachytene than at diak/MI, and although in Fisher's exact test the individual differences are not significant, the overall difference is highly significant ( U = 2.98, P = 0.0028). Of the 60 abnormal pachytene cells 52 contained a single quadrivalent (Fig. 3), 1 two quadrivalents, 3 a hexavalent, 2 a trivalent plus a univalent, 1 a ring X-Y bivalent, and 1 a small, interstitial loop having a morphology which suggests that it is the result o f a deletion or insertion/duplication rather than of an inversion (Fig. 5). This loop shows the sensitivity of the SC technique for the analysis of induced structural change; such a small change would be completely undetectable at diak/MI. Of the 32 abnormal diak/MI cells 31 contained a single quadrivalent, and 1 a hexavalent (Fig. 4). In approximately 10°70 of pachytene cells some o f the SCs were associated in such a way that more than one interpretation was possible using the light microscope. All such ambiguous associations were counted as overlapped bivalents rather than as multivalents. This interpretation was checked by the examination of 20 ambiguous associations with the electron microscope. Of these only one proved to involve a
152
I
.
.
2
d
A
~*~'~-
-
~
'
~; t
w
. . . . . . .
Fig. 1. Normal complement of SCs at pachytene, X-Y bivalent arrowed. Fig. 2. Normal diak/MI cell, X-Y bivalent arrowed. Fig. 3. Pachytene cell with radiation-induced quadrivalent (arrowed). Fig. 4. Diak/MI cell with radiation-induced hexavalent (arrowed). Fig. 5. Pachytene bivalent with interstitial deletion insertion loop (arrowed). Fig. 6. Electron micrograph of ambiguous association of SCs showing a quadrivalent (1V) and a bivalent (II). Bars equal 5/zm.
153
TABLE 1 I N C I D E N C E OF STRUCTURAL C H A N G E IN PRIMARY SPERMATOCYTES Animal
Weeks Wt. both testes Sperm count post-irradiatiWt. whole x 100% (103/ml) on
1512 1560 1513 1581 1514 1563 1515
5 6 7 8 11 13 15
Pachytene
Diak/MI
6_+ 2.37 14_+3.47 9_+2.86 7 + 2.55 10_+3 4_+ 1.96 10-+ 3
4 + 1.96 6_+2.37 6_+2.37 2 + 1.40 5_+2.18 4 + 1.96 5 _+2.18
body 0.84 1.17 1.71 2.21 2.52 3.11 3.24
19.2 32.15 88.5 95.2 104_+2 112.8 68.2
Overall 1477 1478
Number abnormal cells (-+ S.E.)
8.57+ 1.06 Control Control
3.53 3.73
107.2 84.4
0 0
4.57_+0.79 0 0
multivalent (shown in Fig. 6 - a quadrivalent plus an overlapping bivalent), which suggests that counting only unequivocal multivalents using light microscopy does not lead to a serious underestimate o f the extent of structural change. The proportion of cells with ambiguous associations depends upon the quality o f the preparations; the less well spread the cells the more necessary it is to use the electron microscope to validate the light microscopy. Segregation at anaphase I, as judged by counts at metaphase II, appeared unaffected by the dose of irradiation used here. All ceils examined had 22 chromosomes, and there was no significant deviation from a 1:1 ratio in the segregation of the sex chromosomes. The greater incidence of scorable multivalents at pachytene compared with diak/MI reflects, at least in part, the requirement in the former for synapsis alone in contrast to the need in the latter for both synapsis and chiasma formation. However, it is possible that some o f the pachytene cells containing multivalents make no further progress through meiosis and thus make no contribution to the genetic burden carried by the gametes. This possibility is extremely difficult to test although a comparison o f the dose-response curves based on pachytene and diak/MI cells may give some indication. This work is under way. There is no indication of preferential selection against multivalents arising from exchanges between the sex chromosomes and autosomes since the ratio of their numbers in pachytene and diak/MI cells (6:3) matches that o f the total numbers of multivalents (60:32). Moses (1977) argued that the analysis of SCs could be o f great benefit in the analysis of a variety o f experimental treatments o f the germ ceils. The results reported here support this view, and show that the analysis of diak/MI cells alone
154 m a y l e a d to a serious u n d e r e s t i m a t e o f the a m o u n t o f s t r u c t u r a l c h a n g e i n d u c e d in s p e m a t o g o n i a l cells a f t e r i r r a d i a t i o n . T h e SC t e c h n i q u e is s i m p l e a n d q u i c k , a n d can p r o v i d e i n f o r m a t i o n n o t o b t a i n a b l e f r o m a n y o t h e r m e t h o d . It is o f p o t e n t i a l v a l u e in a n y s t u d y i n v o l v i n g e x p e r i m e n t a l t r e a t m e n t o f p r e m e i o t i c a n d m e i o t i c g e r m cells.
Acknowlegements W e w o u l d like to t h a n k M r . D . G . P a p w o r t h for p e r f o r m i n g t h e statistical analyses, M r . M . C o r p for p r o v i s i o n o f r a d i a t i o n facilities, a n d M r . J. Bowler for a n i m a l husbandry.
References Breckon, G. (1982) A modified hypotonic treatment for increasing the frequency and quality of meiotic metaphases from spermatocytes of the Syrian hamster, Stain Technol., 57, 349-353. Evans, E.P. (1979) Cytological methods for the study of meiotic properties in mice, Genetics, 92, s97-s103. Felluga, B., and G.B. Martinucci (1976) A simple method for karyotyping by transmission electron microscopy, J. Submicr. Cytol., 8, 347-352. Moses, M.J. (1977) Microspreading and the synaptonemal complex in cytogenetic studies, in: A. de la Chapelle and M. Sorsa (Eds.), Chromosomes Today, Vol. 6, Elsevier, Amsterdam, pp. 71-82. Sankaranarayanan, K. (1982) Genetic Effects of Ionizing Radiation in Multicellular Eukaryotes and the Assessment of Genetic Radiation Hazards in Man, Elsevier, Amsterdam.