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Chromosome aberrations in metaphase II oocytes of Chinese hamster (Cricetulus griseus) I. The sensitivity of the pre-ovulatory phase to triaziquone
Mutation Research, 25 (1974) 347-359 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - P r i n t e d in T h e N e t h e r l a n d s
347
CHROMOSOME A B E R R A T I O N S IN M E T A P H A S E II OOCYTES OF CHINESE HAMSTER
(CRICETULUS GRISEUS)
I. T H E S E N S I T I V I T Y OF T H E P R E - O V U L A T O R Y P H A S E TO T R I A Z I Q U O N E * I. H A N S M A N N * * , J. N E H E R
AND G. R ~ H R B O R N
Institut fiir A nthropologie und Humangenetik der Universitdt Heidelberg (W. Germany) (Received M a y 6th, 1974)
SUMMARY
In previous investigations with triaziquone, cyclophosphamide, amethopterin and X-rays we showed that the pre-ovulatoly stages of mice are sensitive to the induction of numerical and structural chromosome aberrations. In the present investigation, we studied the problem whether or not this sensitivity is restricted to mice. Two doses of triaziquone were injected intraperitoneally to Chinese hamster females at different intervals before ovulation. The ovulated oocytes were collected from the ampulla and analyzed at the metaphase I I stage. Aiter treatment with triaziquone, the frequency of both numerical and structural aberrations was significantly increased over the control values. The proportion of induced structural anomalies was higher than that of the numerical ones. Chinese hamsters were less sensitive to the induction of structural and especially numerical anomalies compared with the findings with one strain of mouse.
INTRODUCTION
The preovulatory phase of mice is very sensitive for the induction of chromosome anomalies by chemical agentsa-6,11, la and by X-rays 9. These induced numerical and structural anomalies could be analyzed immediately after the first meiotic division in ovulated oocytes at the metaphase I I stage4,6,", n. For a better evaluation and extrapolation of germ-cell mutations it is important to confirm the mouse data in other mammals. Because of the very appropriate karyotype we introduced the Chinese hamster (Cricetulus griseus) in these cytogenetic studies of unfertilized oocytes. This species * T h i s w o r k w a s s p o n s o r e d b y t h e D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t a n d w a s p r e s e n t e d b y I. H a n s m a n n in p a r t a t t h e S y m p o s i u m on C h r o m o s o m a l E r r o r s in R e l a t i o n to R e p r o d u c t i v e Failures, i 2 - i 4 S e p t e m b e r 1973, Paris. ** P r e s e n t a d d r e s s : I n s t i t u t f a r H u m a n g e n e t i k , Universitiit G 6 t t i n g e n , N i k o l a u s b e r g e r w e g 5a, 34oo G 6 t t i n g e n , B u n d e s r e p u b l i k D e u t s c h l a n d . A b b r e v i a t i o n s : HCG, h u m a n chorionic g o n a d o t r o p h i n ; PMS, p r e g n a n t m a r e s ' s e r u m ; T, triaziquone.
348
i. HANS.MANN dl O/.
has a low n u m b e r of chromosomes (n -= I I ) which can readily be distinguished fronl each other. This seems to be interesting as, in spontaneous abortions in man, trisomics of the chromosomal groups A and B were less frequently observed than statistically expectedL It has not been decided so far, however, whether these large meta or submetacentric chromosomes are less frequently inwHved in non-disjunction processes, or whether those zygotes are eliminated before an abortion can be recognized. To exclude this possible elinfination (luring early embryonic development we anah'zed the chromosomes already in ovulated oocvtes at the stage of nletaphase I I. During a very short period before ovulation the sensitivity of X-ray-induced d o m i n a n t lethal mutations changed markedly in oogenesis of mice2, .4. A different pattern of sensitivity was observed in oocvtes after treatment with triaziquone at 3 different pre-ovulatory stages n. Based on this cytogenetic study the same experimental set-up was used for Chinese hamster females. A pilot study was done to correlate a different sensitivity with a different chromosomal behavior in oocvtes. MATERIAL AND METHODS
R a n d o m - b r e d virgin Chinese hamster females were used from our own breeding colony. The animals were kept under controlled conditions at 22 ° and at a hmnidity of 5o-7o % . At the time of treatment, tile females were 14--2o weeks old. Fig. i shows the experinaental procedure. 48 11 after the application of 3.o I.U. PMS the females received 2.o I.U. HCG intraperitoneally for stimulated ovulation. Triaziquone was administered at o.25 m g / k g and I.O m g / k g 8 h before, at the same time as (only o.25 mg/kg), and 3 h after application of HCG. The test substance was dissolved in physiological saline solution and injected intraperitoneally. Controls received at the same time an application of physiological saline solution. The total a m o u n t of the liquid was o.5 nd in experiment and control. Matched controls and treated females were dissected 19 h after HCG treatment. The ovulated oocytes were prepared cytologically according to a method published earlier H. Pre-ovulatory oocytes (Figs. 2-9) were prepared by puncturing mature ovarian follicles at various intervals after HCG treatment. The slides with ovulated oocytes were coded and analyzed in a blind study. The Z 2 method was used tor statistical evaluation. RESULTS
(]) Chromosomal behavior in pr~;- and post-ovulatory oocvtes of Chinese hamsters The early stages of meiotic prophase I, leptotene, zygotene, pachytene and onalysis 1.00mg/kg T 1.OOmg/kgT 0.25mg/k9 T O.25rng/kgT /
©
/+,+.,s befo,e aoO 3.0 IU PMS
2.0 IU HCG
after HCG
Fig. i. E x p e r i m e n t a l procedure for t r e a t m e n t of 3 p r o - o v u l a t o r y stages of oogenesis with triaziq u o n e at 0.25 or I.O m g / k g and s u b s e q u e n t c h r o m o s o m e analysis of m e t a p h a s e II oocytes after s t i m u l a t e d ovulation.
EFFECT OF TRIAZIQUONE ON CHINESE HAMSTER OOCYTES
349
Fig. 2. O o c y t e in late d i c t y o t e n e . A p r o m i n e n t nucleolus a n d several c h r o m o c e n t e r s are visible. T h e c h r o m o s o m e fibers progress in coiling. (Figs. 2-9 are direct p r e p a r a t i o n s f r o m t h e ovary.)
Fig. 3. A d v a n c e d late d i c t y o t e n e .
g5 O
I. HANSMANN ~?/
a/.
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Fig. 4. T r a n s i t i o n a l p h a s e b e t w e e n l a t e d i c t v o t e n e a n d d i a k i n c s J s . ( ' h r o m a t i n t i b e r s a r e s u r r o u n d e d b y a thin l a m p - b r u s h - l i k e m ~ t w o r k oil t i l a m e n t s .
Fig. 5. E a r l y d i a k i n e s i s . B i v a l e n t s in a d v a n c e d c o n d e n s a t i o n . H e r e , too, c o n d e n s e d " c o r e c h r o m a t i n " a n d less c o n d e n s e d fine f i l a m e n t s c a n b e seen.
351
EFFECT OF TRIAZIQUONE ON CHINESE HAMSTER OOCYTES
~ttm
Fig. 6. O o c y t e in diakinesis. P a i r i n g of h o m o l o g o u s c h r o m o s o m e s a n d c h i a s m a t a are visible.
05
Fig. 7. M e t a p h a s e I-oocyte. i
I
b i v a l e n t s w i t h c h i a s m a t a m a y be seen.
~5 2
I.
HANS.MANN C/ 0/.
®
Fig. 8. A n a p h a s e i.
Y
Fig. 9. E a r l y m e t a p h a s e I [ o u t of a p r e < ) v u l a t o r y oocyte. In t h i s s t a g e even t h e 3 s ma l l m e t a c m > t r i c c h r o m o s o m e s can be d i s t i n g u i s h e d from each other. This is n o t a l w a y s possible in l a t e r m e t a p h a s e I I stages.
EFFECT OF TRIAZIQUONEON CHINESE HAMSTEROOCYTES
353
Fig. io. The chromosomes from an ovulated metaphase U oocyte (n = II). diplotene occur shortly before or after birth s. Accordingly, homologous chromosomes are paired and chiasmata formed at that time. After females reach sexual maturity, some oocytes leave dictyotene to enter the first meiotic division. Figs. 2-1o show the typical chromosomal behavior in oocytes at various intervals after the stimulation with PMS and HCG.
(2) Chromosomes in ovulated me~aphase I I oocytes We observed numerical and structural chromosome anomalies in oocytes of treated females and less frequently in those of the controls. Some types of aberration are visible in Figs. I I and 12. Each experimental series had its own matched control. As these controls proved to be homogeneous their data are summarized in Tables I and II. The frequencies of structural chromosome abnormalities after triaziquone treatment and from controls can be seen in Table I. Here, the most sensitive stage for the induction of structural anomalies was the stage 8 h before HCG application. At this time, the oocytes are in late dictyotene. This stage was significantly more sensitive (P
1. H A N S M A N N d[ ~l[.
354
Fig. It. Ovulated
o o c y t e w i t h a n e a r d i p l o i d (n
;
N .:
}
2~3 c h r o m o s o m e
set.
@
F i g . ~2. O v u l a t e d m e t a p h a s e l l - o o c y t e w i t h n II chromosomes (the two large metacentric chromosomes are not photographed) and a chromatid-type break on an acrocentric chromosome
4).
E F F E C T OF T R I A Z I Q U O N E ON C H I N E S E H A M S T E R OOCYTES TABLE
355
I
FREQUENCE OF STRUCTURAL CHROMOSOME ABERRATIONS IN THE CONTROL AND AFTER TREATMENT WITH TRIAZIQUONE AT 3 PREOVULATORY STAGES IN RELATION TO H C G
Treatment with T ( m g / h g )
Stage a (h)
N u m b e r of oocytes
Oocytes with structural aberrations Number %
o 0.25 I.OO 0.25 0.25 I.OO
--8 --8 o +3 +3
229 56 19 47 49 27
20 28 16 17 IO io
8. 7 50.0 84.2 36.2 20. 4 37.0
a H o u r s a c c o r d i n g to H C G - a p p l i c a t i o n .
TABLE
II
TYPES AND FREQUENCIES OF CHROMOSOME ANOMALIES OBSERVED IN OVULATED METAPHASE I [ OOCYTES OF CHINESE HAMSTERS
Treatment with 7"(mg/kg)
Stagc (h)
o 0.25 I .oo 0.25 0-25 I .oo
8 -- 8 o + 3 if- 3
N u m b e r of metaphase I I oocytes
N u m b e r of anomalies per ooeyte a B' F' Dt
Et
229 56 19 47 49 27
o.o31 o.196 o. 158 0.03o o.o82 o. I I i
O.Ol 3 o.161 o.21 i o.191 0.020 0.074
0.022 o.125 0.632 o.128 o. 122 0.o74
0.022 0.089 o.316 o.o21 o.o61 o. I I i
a B', C h r o m a t i d t y p e b r e a k s w h e r e t h e f r a g m e n t c a n b e r e l a t e d t o t h e d e l e t e d c h r o m a t i d ; F', c h r o m o s o m e s of t h e o o c y t e a r e all n o r m a l b u t a n a c e n t r i c f r a g m e n t w a s o b s e r v e d ; D ' , e h r o m a t i d t y p e deletion without fragment within the oocyte; E', chromatid type exchange.
stages with T (o.25 mg/kg). When the frequencies of breaks and fragments are compared with those of deletions it becomes obvious that acentric fragments remain about 3 times more often in the metaphase I I oocyte. Chromatid breaks do not seem to be randomly distributed over all the chromosomes. The 2 large metacentric chromosomes (Nos. I and 2) were involved in 75% of all cases, chromosomes Nos. 3, 4 and X in only I6.7%, and the acrocentrics Nos. 5, 6 and 7 in only 8.3% of all cases. No structural abnormality was observed with the small metacentric chromosomes Nos. 8, 9 and IO. No hyperploidy was observed among 22o metaphase I I oocytes ovulated from control females. 5% of these control oocytes, however, proved to be hypoploid (Table III). Hyperploidies could only be induced in the late dictyotene 8 h before HCG with both doses. A dose dependency for numerical anomalies was not seen clearly, although diploid oocytes were observed only in the high dose groups. The frequency of heteroploid oocytes was altogether significantly increased (P
356 TABLH
I. H A N S M A N N
dt a l .
III
FREQUENCXlES OF N U M E R I C A L CHROMOSOME A B E R R A T I O N S IN T H E CONTROL AN i) AKTER TRI~;ATMENT \VITII T R I A Z J ~ U O N E AT 3 P R E O V U L A T O R V STAGE=% IN RELATION TO H C G
Treatment with T (m e/kg)
£'ta~c a
0 0.2,5 ~.oo 0.25 0.25 1.00 a In hours
S S o } 3 ] 3 according
Number ~oO,les
o~
°o ,
x:
('i,
1l
'~1,2;~
<'i~ b o l e r o / V o i d
220
.5.0
0.0
0.0
.5,0
37 13 37 -t 6 24
5.4 o.o I6.2 £'7 0,0
,5.4 7-7 o,o o.o 0,0
o.o 7.7 o.o o.o N. 3
I o.S 15,S 162 S. 7 N.[~
to HCG-application.
DISCUSSION
After detailed preparations we obtained well-spread metaphase II chromosomes from ovulated oocytes of mice and Chinese hamsters after spontaneous and induced ovulationV, 10-'~. The use of spontaneously ovulated oocytes is preferable when the frequency of spontaneous abnormalities or mutagenic effects after chronic treatment are to be measured1% x~. However, we used the hormonally stimulated ovulation to get a synchronized timing of the pre-ovulatory stages of female meiosis. The closes of PMS and HCG used in the present study did not influence the frequency of hyperploid metaphase II oocytes in Chinese hamsters. No statement can be made, however, on the influence of these hormones on the frequency of hypoploidies and structural chromosome anomalies because of the low number of comparable oocytes after spontaneous ovulation v. It is noteworthy that no single hyperploid or diploid oocyte was found among 22o control oocytes. The two types of heteroploidies were only observed after treatment with triaziquone. We can conclude, therefore, that the frequency of spontaneous non-disjunction during the first meiotic division is rather low in Chinese hamsters, even if we take hypoploid oocytes into consideration and hypoploidies might be at least partly due to artifacts during preparation. In mice, however, we found a higher frequency of spontaneous non-disjunction when we considered only the frequency of hyperploidies~, 10. But even in mice there exist apparent differences within the strains used", n. The application of triaziquone during 3 pre-ovulatory stages increased the frequency of structural chromosome anomalies in ovulated Chinese hamster oocytes. The frequency of anomalies increased with the (lose injected;however, this increase was only significant in the --8 h series ( P < o . o I ) . We observed a different pattern of sensitivity during the pre-ovulatory course of oogenesis even within the short period from 8 h before to 3 h after treatment with HCG. This change of sensitivity is accompanied by an extreme change of chromosomal behavior in oocytes from the (lespiralized late dictyotene to the diakinesis and metaphase I where the chromosomes are highly condensed (Figs. ~ -7). That stage proved to be the m.ost sensitive [or the induction of structural chromosome anomalies where the chromosomes were less contracted. A similar pattern of sensitivity was found with (IoI × C3H ) F1 hybrid mice after the same kind of treatment 1'. In mice and Chinese hamsters the last stage- the transitional phase between late dictyotene and metaphase I - - w a s the least sensitive. This
EFFECT
OF
TRIAZIQUONE
ON
CHINESE
HAMSTER
357
OOCYTES
90-
0 ~,
i i!i iiilii
70-
.....
60-
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iiili
so
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:,:~:
,0
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lo
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0.25mg/kg T-B
0.25mg/kgTtO 0,25mg/kg T+3
Fig 13. The sensitivity of 3 pre ovulatory phases for the induction of aneuploidies by triaziquone in Chinese hamsters compared with (ioi × C3H ) F 1 hybrid mice (R6HRBORN A N D H A N S m A N N zz)
70
®
60 50o o
;~i~
~= -_= .-.-..° .-.-... .-.-._. .-.-... .-.-... .-.-._. . .-._. .-.-°.. .-.-._. .-.-,.. .-.-... .-.-._. .-.-°.. .-.-. . - . - . .° .-,-..° .-.-._. ,.-.-°.. ,.-.-°°° ,.-.-... .-.-° ° ,.-.-.:. ,,.-.-.:. .-.-,. ,.-.-.:. ,.-.-.:. ,°-.-.:. ,°-°°°:° ,°-.°*:°
40302010
control
".:.2" -._... -..-° ..._.
-.... r.:,." -....
O.25rng/k 9 1 - 8
Fig. 14. The sensitivity of 3 pre-ovulatory malies by triaziquone in Chinese hamsters AND HANSMANNll).
:-:-:. - ....
....
O.25mg/k 9 T+_O O.25mg/k9 T+3
phases for the induction of structural compared with (ioi × C3H) F1 hybrid
chromosome anomice (R6HRBORN
comparison of sensitivity from two species (Figs 13 and 14) shows that the Chinese hamster was less sensitive than this hybrid m o u s e The difference in sensitivity for the induction of structural chromosome anomalies is significant in the o h series ( P < o.o2) and in the + 3 h series ( P % I o - 5 ) This becomes even more apparent when only the frequency of induced aneuploidies are considered Not only is the frequency of spontaneous non-disjunction lower in Chinese hamsters but also the possibility of enhancing this process of non-disjunction by applying the same chemical mutagen as used in m i c e
35 ~
I. HANSMANN C[ a[.
The decreasing sensitivity in Chinese h a m s t e r p r e - o v u l a t o r y oogenesis is in contrast with studies on the d o m i n a n t lethal assay in mice2, t4. The s e n s i t i v i t y for Xr a y - i n d u c e d d o m i n a n t lethal m u t a t i o n s increased during the course of w e - o v u l a t o r y oogenesis. The late p r e - o v u l a t o r y stages were the most sensitive. This a p p a r e n t difference m i g h t be due to a different action of chenficals and X-rays, to different sensitivities for the induction of d o m i n a n t lethal n m t a t i o n s and structural chrom~some anomalies and even to a different e l i m i n a t i o n - r e p a i r process. The ch r o m o s o m e s of the Chinese h a m s t e r oocyte can be distinguished nmch b e t t e r from each o th e r than can those of the mouse. The former species is, therefore, v e r y suitable for answering the question which chromosonles are preferentially inwflved in meiotic c h r o m o s o m e abnormalities, i n the present s t u d y the large m e t a c e n t r i c chronlosomes Nos. I and 2 were nlore often i n v o l v e d in s t r u c t u r a l anomalies than the group 3, 4 and X. A n d the m e t a c e n t r i c chromosomes 3, 4 and X were twice as freq u e n t l y i n v o l v e d as the acrocentric chromosomes Nos. 5, 6 and 7. No s t r u c t u r a l chrom o s o m e a n o m a l y was observed with th e small m e t a c e n t r i c s Nos. 8, 9 and Io. These chromosomes, however, were more often inwflved in hypoploidies (in 6o°o of all cases) t h a n the others. As the total f r e q u e n c y of aneuploidies, and especially ot hyperploidies, was too low, no s t a t e m e n t can be m a d e ab o u t which chromosonles are freq u e n t l y missegregated. Our results show t h a t there exist q u a l i t a t i v e and q u a n t i t a t i v e differences with respect to the induction of s t r u c t u r a l and n u m e r ical chromosome anomalies in oogenesis of m a m m a l s and th a t , on the o th e r hand, the sen si t i v i t y of the p r e - o v u l a t o r y phase is not r e s t r i c t e d to mice. ACKNOWLEDGEMENTS
The a u t h o r s are v e r y grateful to Miss 1. BECK, Mrs. M. FRANKE, Mrs. L. RUBES, Mrs. V. SEZER and Mrs. R. WEBER for skillfull technical assistance and for the photographic reproductions.
REFERENCES I Bout, J. G., AND A. Bou£, Les ab6rrations chromosonliques dans les abortements spontands humains, Presse Med., 14 (197o) 635 641. 2 EDWARDS, R. G., AND A. G. SEARLE, Genetic radiosensitivity of specific post-dictyate stages ill mouse oocytes, Genet. Res., 4 (1963) 389-398. 3 HANSMANN, I., Induced chromosomal aberrations in pronuclei, 2-cell stages and in morulae of mice, Mutation Res., 2o (1973) 353-367 • 4 HANSMANN, I., Chromosome aberrations in metaphase lI oocytes. Stage sensitivity in the mouse oogenesis to amethopterin and cyclophosphamide, 3Iulation Res., 22 (1974) I75 191. 5 HANSMANN, i., AND G. R()HRBORN, Chromosome aberrations in preinlplantation stages of mice after treatment with triazoquinone, Humangenetik, 18 (I973) IOI-IO9. 6 JAGIELLO,G., Action of phleomycin on the meiosis of the mouse ovum, Mutation Res., 6 (1968) 289-295. 7 NEHER, J., Experimentelle zytogenetische Untersuchungen zum Ablaut der pr~- und periovulatorischen Phase der Oogenese und der priiimplantativen Embryogenese des chinesischen Hamsters (Cricetulus griseus), P h . D . thesis, UniversitXt Heidelberg, 1974. 8 NEHER, J., Zytogenetische Untersuchungen zunl Verlauf der Oogenese und der frflhen Elnbryogenese beim ehinesischen Hamster (Cricetulus griseus), Diplomarbeit, Universit~t Heidelberg, 1973. 9 REICHERT, W., I. HANSMANN AND G. ROHRBORN, Chromosome anomalies in mouse oocytes after irradiation, Humangenetik, (T974) in press.
EFFECT OF TRIAZlQUONE ON CHINESE HAMSTER OOCYTES
359
IO ROHRBORN, G., F r e q u e n c i e s of s p o n t a n e o u s n o n - d i s j u n c t i o n in m e t a p h a s e II o o c y t e s of mice,
Humangenetik, 16 (1972) 123-125. I I R6HRBORN, G., AND I. HANSMANN, I n d u c e d c h r o m o s o m e a b e r r a t i o n s in unfertilized o o c y t e s of mice, Humangenetik, 13 (1971) 184-198. 12 R6HRBORN, G., AND I. HANSMANN, Oral c o n t r a c e p t i o n a n d c h r o m o s o m e s e g r e g a t i o n in oocytes of mice, Mutation Res., 26 (1974) 535-54413 ROHRBORN, G., O. Ki)HN, I. HANSMANN AND K. THON, I n d u c e d c h r o m o s o m e a b e r r a t i o n s in e a r l y e m b r y o g e n e s i s of mice, Humangenetik, II (1971) 316-322. 14 RUSSELL, L. B., AND L. L. SAYLORS, T h e relative s e n s i t i v i t y of v a r i o u s g e r m cell s t a g e s of t h e m o u s e to r a d i a t i o n i n d u c e d n o n - d i s j u n c t i o n , c h r o m o s o m e losses a n d deficiencies, in F. H. SOBELS (Ed.), Repair from Genetic Damage and Differential Radiosensitivity in Germ Cells, P e r g a m o n , Oxford, 1963 , pp. 313-34 ° .
347
CHROMOSOME A B E R R A T I O N S IN M E T A P H A S E II OOCYTES OF CHINESE HAMSTER
(CRICETULUS GRISEUS)
I. T H E S E N S I T I V I T Y OF T H E P R E - O V U L A T O R Y P H A S E TO T R I A Z I Q U O N E * I. H A N S M A N N * * , J. N E H E R
AND G. R ~ H R B O R N
Institut fiir A nthropologie und Humangenetik der Universitdt Heidelberg (W. Germany) (Received M a y 6th, 1974)
SUMMARY
In previous investigations with triaziquone, cyclophosphamide, amethopterin and X-rays we showed that the pre-ovulatoly stages of mice are sensitive to the induction of numerical and structural chromosome aberrations. In the present investigation, we studied the problem whether or not this sensitivity is restricted to mice. Two doses of triaziquone were injected intraperitoneally to Chinese hamster females at different intervals before ovulation. The ovulated oocytes were collected from the ampulla and analyzed at the metaphase I I stage. Aiter treatment with triaziquone, the frequency of both numerical and structural aberrations was significantly increased over the control values. The proportion of induced structural anomalies was higher than that of the numerical ones. Chinese hamsters were less sensitive to the induction of structural and especially numerical anomalies compared with the findings with one strain of mouse.
INTRODUCTION
The preovulatory phase of mice is very sensitive for the induction of chromosome anomalies by chemical agentsa-6,11, la and by X-rays 9. These induced numerical and structural anomalies could be analyzed immediately after the first meiotic division in ovulated oocytes at the metaphase I I stage4,6,", n. For a better evaluation and extrapolation of germ-cell mutations it is important to confirm the mouse data in other mammals. Because of the very appropriate karyotype we introduced the Chinese hamster (Cricetulus griseus) in these cytogenetic studies of unfertilized oocytes. This species * T h i s w o r k w a s s p o n s o r e d b y t h e D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t a n d w a s p r e s e n t e d b y I. H a n s m a n n in p a r t a t t h e S y m p o s i u m on C h r o m o s o m a l E r r o r s in R e l a t i o n to R e p r o d u c t i v e Failures, i 2 - i 4 S e p t e m b e r 1973, Paris. ** P r e s e n t a d d r e s s : I n s t i t u t f a r H u m a n g e n e t i k , Universitiit G 6 t t i n g e n , N i k o l a u s b e r g e r w e g 5a, 34oo G 6 t t i n g e n , B u n d e s r e p u b l i k D e u t s c h l a n d . A b b r e v i a t i o n s : HCG, h u m a n chorionic g o n a d o t r o p h i n ; PMS, p r e g n a n t m a r e s ' s e r u m ; T, triaziquone.
348
i. HANS.MANN dl O/.
has a low n u m b e r of chromosomes (n -= I I ) which can readily be distinguished fronl each other. This seems to be interesting as, in spontaneous abortions in man, trisomics of the chromosomal groups A and B were less frequently observed than statistically expectedL It has not been decided so far, however, whether these large meta or submetacentric chromosomes are less frequently inwHved in non-disjunction processes, or whether those zygotes are eliminated before an abortion can be recognized. To exclude this possible elinfination (luring early embryonic development we anah'zed the chromosomes already in ovulated oocvtes at the stage of nletaphase I I. During a very short period before ovulation the sensitivity of X-ray-induced d o m i n a n t lethal mutations changed markedly in oogenesis of mice2, .4. A different pattern of sensitivity was observed in oocvtes after treatment with triaziquone at 3 different pre-ovulatory stages n. Based on this cytogenetic study the same experimental set-up was used for Chinese hamster females. A pilot study was done to correlate a different sensitivity with a different chromosomal behavior in oocvtes. MATERIAL AND METHODS
R a n d o m - b r e d virgin Chinese hamster females were used from our own breeding colony. The animals were kept under controlled conditions at 22 ° and at a hmnidity of 5o-7o % . At the time of treatment, tile females were 14--2o weeks old. Fig. i shows the experinaental procedure. 48 11 after the application of 3.o I.U. PMS the females received 2.o I.U. HCG intraperitoneally for stimulated ovulation. Triaziquone was administered at o.25 m g / k g and I.O m g / k g 8 h before, at the same time as (only o.25 mg/kg), and 3 h after application of HCG. The test substance was dissolved in physiological saline solution and injected intraperitoneally. Controls received at the same time an application of physiological saline solution. The total a m o u n t of the liquid was o.5 nd in experiment and control. Matched controls and treated females were dissected 19 h after HCG treatment. The ovulated oocytes were prepared cytologically according to a method published earlier H. Pre-ovulatory oocytes (Figs. 2-9) were prepared by puncturing mature ovarian follicles at various intervals after HCG treatment. The slides with ovulated oocytes were coded and analyzed in a blind study. The Z 2 method was used tor statistical evaluation. RESULTS
(]) Chromosomal behavior in pr~;- and post-ovulatory oocvtes of Chinese hamsters The early stages of meiotic prophase I, leptotene, zygotene, pachytene and onalysis 1.00mg/kg T 1.OOmg/kgT 0.25mg/k9 T O.25rng/kgT /
©
/+,+.,s befo,e aoO 3.0 IU PMS
2.0 IU HCG
after HCG
Fig. i. E x p e r i m e n t a l procedure for t r e a t m e n t of 3 p r o - o v u l a t o r y stages of oogenesis with triaziq u o n e at 0.25 or I.O m g / k g and s u b s e q u e n t c h r o m o s o m e analysis of m e t a p h a s e II oocytes after s t i m u l a t e d ovulation.
EFFECT OF TRIAZIQUONE ON CHINESE HAMSTER OOCYTES
349
Fig. 2. O o c y t e in late d i c t y o t e n e . A p r o m i n e n t nucleolus a n d several c h r o m o c e n t e r s are visible. T h e c h r o m o s o m e fibers progress in coiling. (Figs. 2-9 are direct p r e p a r a t i o n s f r o m t h e ovary.)
Fig. 3. A d v a n c e d late d i c t y o t e n e .
g5 O
I. HANSMANN ~?/
a/.
®
Fig. 4. T r a n s i t i o n a l p h a s e b e t w e e n l a t e d i c t v o t e n e a n d d i a k i n c s J s . ( ' h r o m a t i n t i b e r s a r e s u r r o u n d e d b y a thin l a m p - b r u s h - l i k e m ~ t w o r k oil t i l a m e n t s .
Fig. 5. E a r l y d i a k i n e s i s . B i v a l e n t s in a d v a n c e d c o n d e n s a t i o n . H e r e , too, c o n d e n s e d " c o r e c h r o m a t i n " a n d less c o n d e n s e d fine f i l a m e n t s c a n b e seen.
351
EFFECT OF TRIAZIQUONE ON CHINESE HAMSTER OOCYTES
~ttm
Fig. 6. O o c y t e in diakinesis. P a i r i n g of h o m o l o g o u s c h r o m o s o m e s a n d c h i a s m a t a are visible.
05
Fig. 7. M e t a p h a s e I-oocyte. i
I
b i v a l e n t s w i t h c h i a s m a t a m a y be seen.
~5 2
I.
HANS.MANN C/ 0/.
®
Fig. 8. A n a p h a s e i.
Y
Fig. 9. E a r l y m e t a p h a s e I [ o u t of a p r e < ) v u l a t o r y oocyte. In t h i s s t a g e even t h e 3 s ma l l m e t a c m > t r i c c h r o m o s o m e s can be d i s t i n g u i s h e d from each other. This is n o t a l w a y s possible in l a t e r m e t a p h a s e I I stages.
EFFECT OF TRIAZIQUONEON CHINESE HAMSTEROOCYTES
353
Fig. io. The chromosomes from an ovulated metaphase U oocyte (n = II). diplotene occur shortly before or after birth s. Accordingly, homologous chromosomes are paired and chiasmata formed at that time. After females reach sexual maturity, some oocytes leave dictyotene to enter the first meiotic division. Figs. 2-1o show the typical chromosomal behavior in oocytes at various intervals after the stimulation with PMS and HCG.
(2) Chromosomes in ovulated me~aphase I I oocytes We observed numerical and structural chromosome anomalies in oocytes of treated females and less frequently in those of the controls. Some types of aberration are visible in Figs. I I and 12. Each experimental series had its own matched control. As these controls proved to be homogeneous their data are summarized in Tables I and II. The frequencies of structural chromosome abnormalities after triaziquone treatment and from controls can be seen in Table I. Here, the most sensitive stage for the induction of structural anomalies was the stage 8 h before HCG application. At this time, the oocytes are in late dictyotene. This stage was significantly more sensitive (P
1. H A N S M A N N d[ ~l[.
354
Fig. It. Ovulated
o o c y t e w i t h a n e a r d i p l o i d (n
;
N .:
}
2~3 c h r o m o s o m e
set.
@
F i g . ~2. O v u l a t e d m e t a p h a s e l l - o o c y t e w i t h n II chromosomes (the two large metacentric chromosomes are not photographed) and a chromatid-type break on an acrocentric chromosome
4).
E F F E C T OF T R I A Z I Q U O N E ON C H I N E S E H A M S T E R OOCYTES TABLE
355
I
FREQUENCE OF STRUCTURAL CHROMOSOME ABERRATIONS IN THE CONTROL AND AFTER TREATMENT WITH TRIAZIQUONE AT 3 PREOVULATORY STAGES IN RELATION TO H C G
Treatment with T ( m g / h g )
Stage a (h)
N u m b e r of oocytes
Oocytes with structural aberrations Number %
o 0.25 I.OO 0.25 0.25 I.OO
--8 --8 o +3 +3
229 56 19 47 49 27
20 28 16 17 IO io
8. 7 50.0 84.2 36.2 20. 4 37.0
a H o u r s a c c o r d i n g to H C G - a p p l i c a t i o n .
TABLE
II
TYPES AND FREQUENCIES OF CHROMOSOME ANOMALIES OBSERVED IN OVULATED METAPHASE I [ OOCYTES OF CHINESE HAMSTERS
Treatment with 7"(mg/kg)
Stagc (h)
o 0.25 I .oo 0.25 0-25 I .oo
8 -- 8 o + 3 if- 3
N u m b e r of metaphase I I oocytes
N u m b e r of anomalies per ooeyte a B' F' Dt
Et
229 56 19 47 49 27
o.o31 o.196 o. 158 0.03o o.o82 o. I I i
O.Ol 3 o.161 o.21 i o.191 0.020 0.074
0.022 o.125 0.632 o.128 o. 122 0.o74
0.022 0.089 o.316 o.o21 o.o61 o. I I i
a B', C h r o m a t i d t y p e b r e a k s w h e r e t h e f r a g m e n t c a n b e r e l a t e d t o t h e d e l e t e d c h r o m a t i d ; F', c h r o m o s o m e s of t h e o o c y t e a r e all n o r m a l b u t a n a c e n t r i c f r a g m e n t w a s o b s e r v e d ; D ' , e h r o m a t i d t y p e deletion without fragment within the oocyte; E', chromatid type exchange.
stages with T (o.25 mg/kg). When the frequencies of breaks and fragments are compared with those of deletions it becomes obvious that acentric fragments remain about 3 times more often in the metaphase I I oocyte. Chromatid breaks do not seem to be randomly distributed over all the chromosomes. The 2 large metacentric chromosomes (Nos. I and 2) were involved in 75% of all cases, chromosomes Nos. 3, 4 and X in only I6.7%, and the acrocentrics Nos. 5, 6 and 7 in only 8.3% of all cases. No structural abnormality was observed with the small metacentric chromosomes Nos. 8, 9 and IO. No hyperploidy was observed among 22o metaphase I I oocytes ovulated from control females. 5% of these control oocytes, however, proved to be hypoploid (Table III). Hyperploidies could only be induced in the late dictyotene 8 h before HCG with both doses. A dose dependency for numerical anomalies was not seen clearly, although diploid oocytes were observed only in the high dose groups. The frequency of heteroploid oocytes was altogether significantly increased (P
356 TABLH
I. H A N S M A N N
dt a l .
III
FREQUENCXlES OF N U M E R I C A L CHROMOSOME A B E R R A T I O N S IN T H E CONTROL AN i) AKTER TRI~;ATMENT \VITII T R I A Z J ~ U O N E AT 3 P R E O V U L A T O R V STAGE=% IN RELATION TO H C G
Treatment with T (m e/kg)
£'ta~c a
0 0.2,5 ~.oo 0.25 0.25 1.00 a In hours
S S o } 3 ] 3 according
Number ~oO,les
o~
°o ,
x:
('i,
1l
'~1,2;~
<'i~ b o l e r o / V o i d
220
.5.0
0.0
0.0
.5,0
37 13 37 -t 6 24
5.4 o.o I6.2 £'7 0,0
,5.4 7-7 o,o o.o 0,0
o.o 7.7 o.o o.o N. 3
I o.S 15,S 162 S. 7 N.[~
to HCG-application.
DISCUSSION
After detailed preparations we obtained well-spread metaphase II chromosomes from ovulated oocytes of mice and Chinese hamsters after spontaneous and induced ovulationV, 10-'~. The use of spontaneously ovulated oocytes is preferable when the frequency of spontaneous abnormalities or mutagenic effects after chronic treatment are to be measured1% x~. However, we used the hormonally stimulated ovulation to get a synchronized timing of the pre-ovulatory stages of female meiosis. The closes of PMS and HCG used in the present study did not influence the frequency of hyperploid metaphase II oocytes in Chinese hamsters. No statement can be made, however, on the influence of these hormones on the frequency of hypoploidies and structural chromosome anomalies because of the low number of comparable oocytes after spontaneous ovulation v. It is noteworthy that no single hyperploid or diploid oocyte was found among 22o control oocytes. The two types of heteroploidies were only observed after treatment with triaziquone. We can conclude, therefore, that the frequency of spontaneous non-disjunction during the first meiotic division is rather low in Chinese hamsters, even if we take hypoploid oocytes into consideration and hypoploidies might be at least partly due to artifacts during preparation. In mice, however, we found a higher frequency of spontaneous non-disjunction when we considered only the frequency of hyperploidies~, 10. But even in mice there exist apparent differences within the strains used", n. The application of triaziquone during 3 pre-ovulatory stages increased the frequency of structural chromosome anomalies in ovulated Chinese hamster oocytes. The frequency of anomalies increased with the (lose injected;however, this increase was only significant in the --8 h series ( P < o . o I ) . We observed a different pattern of sensitivity during the pre-ovulatory course of oogenesis even within the short period from 8 h before to 3 h after treatment with HCG. This change of sensitivity is accompanied by an extreme change of chromosomal behavior in oocytes from the (lespiralized late dictyotene to the diakinesis and metaphase I where the chromosomes are highly condensed (Figs. ~ -7). That stage proved to be the m.ost sensitive [or the induction of structural chromosome anomalies where the chromosomes were less contracted. A similar pattern of sensitivity was found with (IoI × C3H ) F1 hybrid mice after the same kind of treatment 1'. In mice and Chinese hamsters the last stage- the transitional phase between late dictyotene and metaphase I - - w a s the least sensitive. This
EFFECT
OF
TRIAZIQUONE
ON
CHINESE
HAMSTER
357
OOCYTES
90-
0 ~,
i i!i iiilii
70-
.....
60-
!!!!!
iiili
so
ii~ii
:,:~:
,0
iiiil
iliii!
:::::
:!:i: :::~:
:.:.:. ..
iiili
i!!iii
30.
lo
i!!ii
:::::
control
~iiii!
!!!!i!
0.25mg/kg T-B
0.25mg/kgTtO 0,25mg/kg T+3
Fig 13. The sensitivity of 3 pre ovulatory phases for the induction of aneuploidies by triaziquone in Chinese hamsters compared with (ioi × C3H ) F 1 hybrid mice (R6HRBORN A N D H A N S m A N N zz)
70
®
60 50o o
;~i~
~= -_= .-.-..° .-.-... .-.-._. .-.-... .-.-... .-.-._. . .-._. .-.-°.. .-.-._. .-.-,.. .-.-... .-.-._. .-.-°.. .-.-. . - . - . .° .-,-..° .-.-._. ,.-.-°.. ,.-.-°°° ,.-.-... .-.-° ° ,.-.-.:. ,,.-.-.:. .-.-,. ,.-.-.:. ,.-.-.:. ,°-.-.:. ,°-°°°:° ,°-.°*:°
40302010
control
".:.2" -._... -..-° ..._.
-.... r.:,." -....
O.25rng/k 9 1 - 8
Fig. 14. The sensitivity of 3 pre-ovulatory malies by triaziquone in Chinese hamsters AND HANSMANNll).
:-:-:. - ....
....
O.25mg/k 9 T+_O O.25mg/k9 T+3
phases for the induction of structural compared with (ioi × C3H) F1 hybrid
chromosome anomice (R6HRBORN
comparison of sensitivity from two species (Figs 13 and 14) shows that the Chinese hamster was less sensitive than this hybrid m o u s e The difference in sensitivity for the induction of structural chromosome anomalies is significant in the o h series ( P < o.o2) and in the + 3 h series ( P % I o - 5 ) This becomes even more apparent when only the frequency of induced aneuploidies are considered Not only is the frequency of spontaneous non-disjunction lower in Chinese hamsters but also the possibility of enhancing this process of non-disjunction by applying the same chemical mutagen as used in m i c e
35 ~
I. HANSMANN C[ a[.
The decreasing sensitivity in Chinese h a m s t e r p r e - o v u l a t o r y oogenesis is in contrast with studies on the d o m i n a n t lethal assay in mice2, t4. The s e n s i t i v i t y for Xr a y - i n d u c e d d o m i n a n t lethal m u t a t i o n s increased during the course of w e - o v u l a t o r y oogenesis. The late p r e - o v u l a t o r y stages were the most sensitive. This a p p a r e n t difference m i g h t be due to a different action of chenficals and X-rays, to different sensitivities for the induction of d o m i n a n t lethal n m t a t i o n s and structural chrom~some anomalies and even to a different e l i m i n a t i o n - r e p a i r process. The ch r o m o s o m e s of the Chinese h a m s t e r oocyte can be distinguished nmch b e t t e r from each o th e r than can those of the mouse. The former species is, therefore, v e r y suitable for answering the question which chromosonles are preferentially inwflved in meiotic c h r o m o s o m e abnormalities, i n the present s t u d y the large m e t a c e n t r i c chronlosomes Nos. I and 2 were nlore often i n v o l v e d in s t r u c t u r a l anomalies than the group 3, 4 and X. A n d the m e t a c e n t r i c chromosomes 3, 4 and X were twice as freq u e n t l y i n v o l v e d as the acrocentric chromosomes Nos. 5, 6 and 7. No s t r u c t u r a l chrom o s o m e a n o m a l y was observed with th e small m e t a c e n t r i c s Nos. 8, 9 and Io. These chromosomes, however, were more often inwflved in hypoploidies (in 6o°o of all cases) t h a n the others. As the total f r e q u e n c y of aneuploidies, and especially ot hyperploidies, was too low, no s t a t e m e n t can be m a d e ab o u t which chromosonles are freq u e n t l y missegregated. Our results show t h a t there exist q u a l i t a t i v e and q u a n t i t a t i v e differences with respect to the induction of s t r u c t u r a l and n u m e r ical chromosome anomalies in oogenesis of m a m m a l s and th a t , on the o th e r hand, the sen si t i v i t y of the p r e - o v u l a t o r y phase is not r e s t r i c t e d to mice. ACKNOWLEDGEMENTS
The a u t h o r s are v e r y grateful to Miss 1. BECK, Mrs. M. FRANKE, Mrs. L. RUBES, Mrs. V. SEZER and Mrs. R. WEBER for skillfull technical assistance and for the photographic reproductions.
REFERENCES I Bout, J. G., AND A. Bou£, Les ab6rrations chromosonliques dans les abortements spontands humains, Presse Med., 14 (197o) 635 641. 2 EDWARDS, R. G., AND A. G. SEARLE, Genetic radiosensitivity of specific post-dictyate stages ill mouse oocytes, Genet. Res., 4 (1963) 389-398. 3 HANSMANN, I., Induced chromosomal aberrations in pronuclei, 2-cell stages and in morulae of mice, Mutation Res., 2o (1973) 353-367 • 4 HANSMANN, I., Chromosome aberrations in metaphase lI oocytes. Stage sensitivity in the mouse oogenesis to amethopterin and cyclophosphamide, 3Iulation Res., 22 (1974) I75 191. 5 HANSMANN, i., AND G. R()HRBORN, Chromosome aberrations in preinlplantation stages of mice after treatment with triazoquinone, Humangenetik, 18 (I973) IOI-IO9. 6 JAGIELLO,G., Action of phleomycin on the meiosis of the mouse ovum, Mutation Res., 6 (1968) 289-295. 7 NEHER, J., Experimentelle zytogenetische Untersuchungen zum Ablaut der pr~- und periovulatorischen Phase der Oogenese und der priiimplantativen Embryogenese des chinesischen Hamsters (Cricetulus griseus), P h . D . thesis, UniversitXt Heidelberg, 1974. 8 NEHER, J., Zytogenetische Untersuchungen zunl Verlauf der Oogenese und der frflhen Elnbryogenese beim ehinesischen Hamster (Cricetulus griseus), Diplomarbeit, Universit~t Heidelberg, 1973. 9 REICHERT, W., I. HANSMANN AND G. ROHRBORN, Chromosome anomalies in mouse oocytes after irradiation, Humangenetik, (T974) in press.
EFFECT OF TRIAZlQUONE ON CHINESE HAMSTER OOCYTES
359
IO ROHRBORN, G., F r e q u e n c i e s of s p o n t a n e o u s n o n - d i s j u n c t i o n in m e t a p h a s e II o o c y t e s of mice,
Humangenetik, 16 (1972) 123-125. I I R6HRBORN, G., AND I. HANSMANN, I n d u c e d c h r o m o s o m e a b e r r a t i o n s in unfertilized o o c y t e s of mice, Humangenetik, 13 (1971) 184-198. 12 R6HRBORN, G., AND I. HANSMANN, Oral c o n t r a c e p t i o n a n d c h r o m o s o m e s e g r e g a t i o n in oocytes of mice, Mutation Res., 26 (1974) 535-54413 ROHRBORN, G., O. Ki)HN, I. HANSMANN AND K. THON, I n d u c e d c h r o m o s o m e a b e r r a t i o n s in e a r l y e m b r y o g e n e s i s of mice, Humangenetik, II (1971) 316-322. 14 RUSSELL, L. B., AND L. L. SAYLORS, T h e relative s e n s i t i v i t y of v a r i o u s g e r m cell s t a g e s of t h e m o u s e to r a d i a t i o n i n d u c e d n o n - d i s j u n c t i o n , c h r o m o s o m e losses a n d deficiencies, in F. H. SOBELS (Ed.), Repair from Genetic Damage and Differential Radiosensitivity in Germ Cells, P e r g a m o n , Oxford, 1963 , pp. 313-34 ° .