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Studies on chemically induced dominant lethality. II. Cytogenetic studies of MMS-induced dominant lethality in maturing dictyate mouse oocytes
77
Mutation Research, 37 ( 1 9 7 6 ) 7 7 - - 8 2 © Elsevier S c i e n t i f i c 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
STUDIES ON CHEMICALLY INDUCED DOMINANT LETHALITY. II. CYTOGENETIC STUDIES OF MMS-INDUCED DOMINANT LETHALITY IN M A T U R I N G DICTYATE MOUSE OOCYTES *
J . G . B R E W E N a n d H.S. P A Y N E
Biology Division, Oak Ridge National Laboratory **, Oak Ridge, Tennessee 37830 (U.S.A.) (Received February 25th, 1976) (Accepted June 8th, 1976)
Summary Young adult female mice were injected intravenously with either 50- or 100mg/kg doses of methyl methanesulfonate. The females were superovulated and mated to untreated males at intervals ranging from 0.5 to 14.5 days after treatment. The fertilized ova were collected and cultured to the first cleavage mitosis, at which time the female chromosome complement was analyzed for structural chromosomal damage. Chromatid-type aberrations were observed, b u t at a much lower frequency than previously reported for treatment of post-meiotic male germ cells. The time after treatment at which chromosomal damage was observed and the frequency of affected cells agree, qualitatively, with existing dominant-lethal data derived from treatment of maturing oocytes. Parallel experiments in which metaphase I oocytes were analyzed indicate a lack of MMSinduced chromosomal damage in the meiotic stages. This observation is consistent with the suggestion that an intervening round of DNA synthesis is necessary for MMS-induced lesions to be translated into chromosomal damage. The low yield of chromosomal damage is consistent with the idea that maturing oocytes, unlike late spermatids and spermatozoa, are capable of performing macromolecular repair of premutational lesions.
* Research s u p p o r t e d b y the National Center for Toxicological Research under FDA Interagency Contract No. FDA 224-76-0020. ** Operated by Union Carbide Corporation for the U.S. Energy Research and Development Administrat.lon.
By acceptance of this article, the publisher or recipient a c k n o w l e d g e s the right of the U.S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article.
78
Introduction In a recent article Brewen et al. [1] presented data that correlated the frequency of chromosomal damage observed at the first zygotic cleavage division with the level of dominant lethality after treatment of post-meiotic male germ cells with MMS. The data indicated that the yields of both pre-and lJost-implanration death could be accounted for by simple structural damage to the male chromosome complement. Not only did the frequency of aberrant male chromosomes agree very well with the level of dominant lethality, but there was also an excellent correlation between the two events as a function of the time interval between treatment and mating. Similar results have been reported by Matter and Jaeger [5] using TEM as the mutagen. The induction of dominant lethality in male germ cells has been thoroughly studied for both ionizing radiations and a vast number of chemical agents. The data available on the dominant-lethal effects produced in females by chemicals, however, are much fewer in number. Generoso and co-workers [2--4] have analyzed dominant-lethal induction in maturing oocytes by eight chemicals, including MMS. They found a significant variation in the frequency of dominant lethality as a function of the strain of mice used as well as of the time interval between treatment and mating. The highest yields of dominant lethality were observed to occur during the 0.5- to 4.5-day interval after treatment for both MMS and EMS. There was also a very high level of pre-implantation death. In addition to the data on d o m i n a n t lethal induction in oocytes, there are also several reports on the cytogenetic analysis of metaphase-I and metaphaseII oocytes after treatment of the female with chemical mutagens [7--10], as well as analysis of early cleavage divisions [5,6,15]. The studies on cleavage stages, however, include later stages than the first division and thus make quantitative comparisons to dominant lethal effects difficult due to the loss of "unstable" chromosome aberrations during the intervening mitoses. To better understand the mechanism of dominant lethality in females we undertook the following experiments. Materials and methods Adult female CD1/CR mice, aged 8--12 weeks, were intravenously injected with MMS at doses of 50 or 100 mg/kg body weight. Superovulation was induced by injections of gonadotropin (5 i.u.) from pregnant mare's serum, followed in 48 h by injections of chorionic gonadotropin from human pregnancy urine (5 i.u.). The superovulated females were mated, in single-pair matings, to young male mice at various intervals after treatment with MMS. The mating intervals were so designed that in two instances the MMS was administered between the two hormone treatments used to induce superovulation. 15 to 18 h after the females were caged with the males, they were checked for vaginal plugs: those with plugs were killed and the ova were removed; those without plugs were discarded. The isolated ova were cultured in the presence of 1 × 10 .7 M colchicine for 24 h, at which time slides were made of the first cleavage mitosis. The details
79 of the culture procedure and slide preparation have been published previously [6]. In addition to the study on the first cleavage zygotes, metaphase I oocytes from superovulated females treated with 100 mg/kg b o d y weight of MMS were also analyzed. The intervals between treatment of the females and ovulation were 0.5, 2.5 and 14.5 days. The slide preparation of the metaphase I oocytes is described in the same reference that details the first cleavage mitosis technique [6]. All of the slides were stained with aceto-orcein, and suitable cells were analyzed for chromosomal aberrations at 1360 × magnification using bright-field optics. Female pronuclei were adjudged as such on the basis of the differential contraction of the chromosome complements. In those rare instances in which an aberration occurred in a zygote undergoing karyogamy and the male and female chromosomes were n o t distinguishable, the aberration was assumed to be female in origin. Results
The results of the cytological analyses of the first cleavage divisions and of the metaphase I oocytes are presented in Tables I and II, respectively. The first cleavage division data are presented as the number of female pronuclei with the different numbers of chromatid aberrations. A phenomenon observed in these experiments that was n o t observed in the studies on post-meiotic male germ cells was a shattering effect (Fig. 1B), or almost total fragmentation of the chromosomes. As was the case when post-meiotic male cells were treated, chromatid-type aberrations were observed which were predominantly isochromatid deletions and chromatid deletions (Fig. 1C). The experiments in which metaphase I oocytes were analyzed yielded negative results in that only 1 isochromatid deletion was seen in 300 treated cells (Table II). TABLE I CHROMOSOME ABERRATION YIELDS AT THE FIRST CLEAVAGE MITOSIS AFTER TREATMENT O F D I C T Y A T E O O C Y T E S W I T H MMS Dose (mg/kg)
Control
Interval (days)
--
N o . o f cells scored
100
N o . o f cells w i t h a b e r r a t i o n s
Expected l e t h a l i t y (%)
0
1
2
3
)3
Shattered
100
0
0
0
0
0
0
50 50 50 50
2.5 6.5 10.5 14.5
75 50 50 50
70 a 2 47 3 49 1 50 0
2 0 0 0
0 0 0 0
0 0 0 0
1 0 0 0
8.0 a 6.0 2.0 0
100 100 100 100 100 100
0.5 1.5 2.5 6.5 10.5 14.5
50 50 50 50 50 50
40 4 43 3 40 5 43 a 5 47 3 50 0
0 1 1 0 0 0
0 0 1 0 0 0
0 1 0 1 0 0
6 2 3 1 0 0
20.0 14.0 20.0 16.0 a 6.0 0
a I n c l u d e s a t r i P l o i d z y g o t e ( S e e Fig. 1).
T A B L E II C H R O M O S O M E A B E R R A T I O N Y I E L D S IN M E T A P H A S E I O O C Y T E S F O L L O W I N G A 1 0 0 m g / k g D O S E O F MMS Interval (days)
No. o f cells s c o r e d
No. o f d e l e t i o n s
No. of e x c h a n g e s
Control 0.5 2.5 14.5
200 100 100 100
0 1 0 0
0 0 0 0
l~ - , ~ t ~ t$
,,
B
I ~-d -.-
o~~ "cry"
2',
Fig. 1. E x a m p l e s of first cleavage m i t o s e s in c u l t u r e d m o u s e ova. (A) N o r m a l f e m a l e and m a l e c h r o m o somes~ (B) s h a t t e r e d f e m a l e c h r o m o s o m e c o m p l e m e n t (arrow)~ (C) f e m a l e c h r o m o s o m e s w i t h a c h o m a t i d d e l e t i o n ( a r r o w ) (D) triploid z y g o t e .
81 Discussion In their study on the induction of dominant lethals in dictyate oocytes by MMS and EMS, Generoso and co-workers [2--4] reported that there is a restricted time period during which these c o m p o u n d s are effective. The highest frequency of dominant lethality was observed when matings occurred during the interval of 0.5--4.5 days after treatment. The interval of 10.5--14.5 days after treatment resulted in 21.6 and 10% dominant lethality after EMS and MMS, respectively, and the 10% frequency was not significant. The present data agree very well with Generoso's data in that no known cell-lethal chromosomal aberrations were observed after the 10.5-day interval after treatment. Generoso also observed a high frequency of sterile matings and pre-implantation loss in his studies, with a corresponding high level of zygotic arrest at the two-blastomere stage. He postulated that the failure of the zygote to develop b e y o n d the two-blastomere stage was the cause of pre-implantation loss. In fact, the level of pre-implantation loss at the most sensitive time interval was approx. 50%. In his studies, Gene~oso showed that for EMS the reduction in fertile matings could be attributed to death after fertilization rather than to a reduction in fertilization. Presumably this is also the case for MMS, and consequently a toxiCity effect can be ruled out. This interpretation is substantiated by the equal frequency of fertilized ova in the control and treated groups. The present data show that at the time interval corresponding to the peak yield of dominant lethality, the highest frequency of chromosome aberrations was also observed. Furthermore, at the highest dose, i.e. 100 mg/kg, approximately one third of the cells with aberrations had shattered female chromosomes. This shattering phenomenon most probably results in very early embryonic death, and conceivably can account for Generoso's observation of arrest at the two-blastomere stage. The discrepancy in absolute frequency in Generoso's and the present study can most likely be accounted for by the difference in dose (i.e. 150 vs 100 mg/kg, respectively) or by possible strain differences in sensitivity. That Generoso found equal numbers of corpora lutea in his MMS-treated and control females argues against the possibility that the damaged metaphase I oocytes were not ovulated. The lack of a significant level of structural aberrations in the metaphase I oocytes, and a corresponding significant level in the female complement at first cleavage after treatment at the stages of maturation, affirms the premise that an intervening round of DNA synthesis is required for an MMS-induced lesion to be translated into a structural aberration. That the aberrations were at the level of the chromatid confirms the observation made on post-meiotic male germ cells and lends further support to the argument that DNA synthesis is necessary for aberrations to be formed. The data of Jagiello [8,9] are exemplary of the opposite phenomenon, i.e. the non-requirement of an intervening round of DNA synthesis. In this work it is shown that streptonigrin induces classical chromatid aberrations at metaphase I when oocytes are treated. Streptonigrin does n o t require DNA synthesis to produce its clastogenic effect. The time-specificity for the eventual production of chromosome aberrations by MMS, and presumably EMS, in maturing oocytes carries certain implications
82
regarding repair of the pre-mutational lesion. Pederson and Masui [7] have reported that the enzymes necessary for unscheduled D N A synthesis are present in mouse o o c y t e s . In fact, they observed a significant increase in tritiated thymidine incorporation when metaphase I oocytes were irradiated. It has been shown by Regan and Setlow [8] that repair-competent cells do perform unscheduled D N A synthesis after MMS treatment, and Sega [9] has extended these observations to include spermatozoa and spermatid stages in the mouse. It seems reasonable to assume that the efficiency of repair is higher in early oocytes than in more mature oocytes (less than 6.5 days from ovulation), hence the higher aberration frequency could be due to more residual unrepaired lesions. The reason for this may be the more highly condensed nature of the chromosomes as the oocyte approaches the ovulation stage and the consequent inaccessibility of the lesion to attack by the repair enzymes. No attempt was made to analyze the first cleavage divisions for aneuploidy. The reason for this is that the preparation techniques employed lead to a significant level of chromosome loss through cell rupture, which it is felt would mask any increase in non-disjunction. Two triploid zygotes were seen, both of which {Fig. 1D) appeared to contain a diploid female complement as adjudged by the differential degree of chromosome condensation. References 1 B r e w e n , J . G . , H.S. P a y n e , K.P. J o n e s a n d R.J. P r e s t o n , S t u d i e s on c h e m i c a l l y - i n d u c e d d o m i n a n t let h a l i t y . I. T h e c y t o g e n e t i c basis o f M M S - i n d u c e d d o m i n a n t l e t h a l i t y in p o s t - m e i o t i c m a l e g e r m cells, M u t a t i o n Res., 33 ( 1 9 7 5 ) 2 3 9 - - 2 5 0 . 2 G e n e r o s o , W.M.° C h e m i c a l i n d u c t i o n of d o m i n a n t lethals in f e m a l e m i c e , G e n e t i c s , 61 ( 1 9 6 9 ) 4 6 1 - 470. 3 G e n e r o s o , W.M. a n d W.L. Russell, Strain a n d sex v a r i a t i o n s in t h e sensitivity of m i c e to d o m i n a n t lethal i n d u c t i o n w i t h e t h y l m e t h a n e s u l f o n a t e , M u t a t i o n Res., 8 ( 1 9 6 9 ) 5 8 9 - - 5 9 8 . 4 G e n e r o s o , W.M., S.W. H u f f a n d S.K. S t o u t , C h e m i c a l l y i n d u c e d d o m i n a n t l e t h a l m u t a t i o n s and cell killing in m o u s e o o c y t e s in a d v a n c e d stages o f follicular d e v e l o p m e n t , M u t a t i o n Res., 11 ( 1 9 7 1 ) 4 1 1 - 420. 5 H a n s m a n n , I. a n d G. R S h r b o r n , C h r o m o s o m e a b e r r a t i o n s in p r c i m p l a n t a t i o n stages of m i c e a f t e r t r e a t m e n t w i t h t r i a z o q u i n o n e , H u m a n g e n e t i k , 18 ( 1 9 7 3 ) 1 0 1 - - 1 0 9 . 6 H a n s m a n n , I., I n d u c e d c h r o m o s o m a l a b e r r a t i o n s in p r o n u c l e i c , 2-cell stages, and m o r u l a e of m i c e , M u t a t i o n Res., 20 ( 1 9 7 3 ) 3 5 3 - - 3 6 7 . 7 H a n s m a n n , I., C h r o m o s o m e a b e r r a t i o n s in m e t a p h a s e I I - o o c y t e s stage s e n s t i t i v i t y in the m o u s e oogenesis to a m e t h o p t c r i n , M u t a t i o n Res., 22 ( 1 9 7 4 ) 1 7 5 - - 1 9 1 . 8 Jagiello, G., S t r e p t o n i g r i n : e f f e c t on t h e first m e i o t i c m e t a p h a s e of t h e m o u s e egg, Science 152 (1967) 453--454. 9 Jagiello, G., A c t i o n o f p h l e o m y c i n on t h e m e i o s i s of the m o u s e o v u m , M u t a t i o n Res., 6 ( 1 9 6 8 ) 2 8 9 - 295. 10 Jagiello, G. a n d P.E. Polani, M o u s e g e r m cells a n d L S D - 2 5 , C y t o g e n e t i c s ° 8 ( 1 9 6 9 ) 1 3 6 - - 1 4 7 . 11 M a t t e r , B.E. a n d I. J a e g e r , P r e m a t u r e c h r o m o s o m e c o n d e n s a t i o n , s t r u c t u r a l c h r o m o s o m e a b e r r a t i o n s a n d m i c r o n u c l e i in early m o u s e e m b r y o s o f f e r t r e a t m e n t of p a t e r n a l g e r m cells w i t h t r i e t h y l e n e m e l a m i n e : a possible m e c h a n i s m f o r c h e m i c a l l y - i n d u c e d d o m i n a n t l e t h a l m u t a t i o n s , M u t a t i o n Res., 33 (1975) 251--260. 12 P a y n e , H.S. a n d K.P. J o n e s , T e c h n i q u e for m a s s isolation a n d c u l t u r e of m o u s e ova f o r c y t o g e n e t i c analysis of the first cleavage m i t o s i s , M u t a t i o n Res., 33 ( 1 9 7 5 ) 2 3 9 - - 2 5 0 . 13 P e d e r s e n , R . A . a n d Y. Masui, U n s c h e d u l e d D N A s y n t h e s i s a f t e r U V i r r a d i a t i o n of m o u s e o o c y t e s , A b s t r a c t s of P a p e r s for the 2 3 r d Mtg. of the R a d i a t i o n R e s e a r c h S o c i e t y , May 1 1 - - 1 5 , 1 9 7 5 , A b s t r . Be-2. 14 R e g a n , J . D . a n d R.B. S e t l o w , T w o f o r m s of r e p a i r in the D N A o f h u m a n cells d a m a g e d b y c h e m i c a l caxcinogens a n d m u t a g e n s , C a n c e r Res., 3 4 ( 1 9 7 4 ) 3 3 1 8 - - 3 3 2 5 . 15 R S h r b o r n , G., O. K u h n , I. H a n s m a n n a n d K. T h o n , 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 early e m b r y o g e n e s i s of m i c e , H u m a n g e n e t i k , 11 ( 1 9 7 1 ) 3 1 6 - - 3 2 2 . 16 Sega, G . A . , U n s c h e d u l e d D N A s y n t h e s i s in t h e g e r m cells of m a l e m i c e e x p o s e d in vivo to t h e c h e m ical m u t a g e n e t h y l m e t h a n e s u l f o n a t e , Proc. Natl. A c a d . Sci. U . S . A . , 71 ( 1 9 7 4 ) 4 9 5 5 - - 4 9 5 9 .
Mutation Research, 37 ( 1 9 7 6 ) 7 7 - - 8 2 © Elsevier S c i e n t i f i c 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
STUDIES ON CHEMICALLY INDUCED DOMINANT LETHALITY. II. CYTOGENETIC STUDIES OF MMS-INDUCED DOMINANT LETHALITY IN M A T U R I N G DICTYATE MOUSE OOCYTES *
J . G . B R E W E N a n d H.S. P A Y N E
Biology Division, Oak Ridge National Laboratory **, Oak Ridge, Tennessee 37830 (U.S.A.) (Received February 25th, 1976) (Accepted June 8th, 1976)
Summary Young adult female mice were injected intravenously with either 50- or 100mg/kg doses of methyl methanesulfonate. The females were superovulated and mated to untreated males at intervals ranging from 0.5 to 14.5 days after treatment. The fertilized ova were collected and cultured to the first cleavage mitosis, at which time the female chromosome complement was analyzed for structural chromosomal damage. Chromatid-type aberrations were observed, b u t at a much lower frequency than previously reported for treatment of post-meiotic male germ cells. The time after treatment at which chromosomal damage was observed and the frequency of affected cells agree, qualitatively, with existing dominant-lethal data derived from treatment of maturing oocytes. Parallel experiments in which metaphase I oocytes were analyzed indicate a lack of MMSinduced chromosomal damage in the meiotic stages. This observation is consistent with the suggestion that an intervening round of DNA synthesis is necessary for MMS-induced lesions to be translated into chromosomal damage. The low yield of chromosomal damage is consistent with the idea that maturing oocytes, unlike late spermatids and spermatozoa, are capable of performing macromolecular repair of premutational lesions.
* Research s u p p o r t e d b y the National Center for Toxicological Research under FDA Interagency Contract No. FDA 224-76-0020. ** Operated by Union Carbide Corporation for the U.S. Energy Research and Development Administrat.lon.
By acceptance of this article, the publisher or recipient a c k n o w l e d g e s the right of the U.S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article.
78
Introduction In a recent article Brewen et al. [1] presented data that correlated the frequency of chromosomal damage observed at the first zygotic cleavage division with the level of dominant lethality after treatment of post-meiotic male germ cells with MMS. The data indicated that the yields of both pre-and lJost-implanration death could be accounted for by simple structural damage to the male chromosome complement. Not only did the frequency of aberrant male chromosomes agree very well with the level of dominant lethality, but there was also an excellent correlation between the two events as a function of the time interval between treatment and mating. Similar results have been reported by Matter and Jaeger [5] using TEM as the mutagen. The induction of dominant lethality in male germ cells has been thoroughly studied for both ionizing radiations and a vast number of chemical agents. The data available on the dominant-lethal effects produced in females by chemicals, however, are much fewer in number. Generoso and co-workers [2--4] have analyzed dominant-lethal induction in maturing oocytes by eight chemicals, including MMS. They found a significant variation in the frequency of dominant lethality as a function of the strain of mice used as well as of the time interval between treatment and mating. The highest yields of dominant lethality were observed to occur during the 0.5- to 4.5-day interval after treatment for both MMS and EMS. There was also a very high level of pre-implantation death. In addition to the data on d o m i n a n t lethal induction in oocytes, there are also several reports on the cytogenetic analysis of metaphase-I and metaphaseII oocytes after treatment of the female with chemical mutagens [7--10], as well as analysis of early cleavage divisions [5,6,15]. The studies on cleavage stages, however, include later stages than the first division and thus make quantitative comparisons to dominant lethal effects difficult due to the loss of "unstable" chromosome aberrations during the intervening mitoses. To better understand the mechanism of dominant lethality in females we undertook the following experiments. Materials and methods Adult female CD1/CR mice, aged 8--12 weeks, were intravenously injected with MMS at doses of 50 or 100 mg/kg body weight. Superovulation was induced by injections of gonadotropin (5 i.u.) from pregnant mare's serum, followed in 48 h by injections of chorionic gonadotropin from human pregnancy urine (5 i.u.). The superovulated females were mated, in single-pair matings, to young male mice at various intervals after treatment with MMS. The mating intervals were so designed that in two instances the MMS was administered between the two hormone treatments used to induce superovulation. 15 to 18 h after the females were caged with the males, they were checked for vaginal plugs: those with plugs were killed and the ova were removed; those without plugs were discarded. The isolated ova were cultured in the presence of 1 × 10 .7 M colchicine for 24 h, at which time slides were made of the first cleavage mitosis. The details
79 of the culture procedure and slide preparation have been published previously [6]. In addition to the study on the first cleavage zygotes, metaphase I oocytes from superovulated females treated with 100 mg/kg b o d y weight of MMS were also analyzed. The intervals between treatment of the females and ovulation were 0.5, 2.5 and 14.5 days. The slide preparation of the metaphase I oocytes is described in the same reference that details the first cleavage mitosis technique [6]. All of the slides were stained with aceto-orcein, and suitable cells were analyzed for chromosomal aberrations at 1360 × magnification using bright-field optics. Female pronuclei were adjudged as such on the basis of the differential contraction of the chromosome complements. In those rare instances in which an aberration occurred in a zygote undergoing karyogamy and the male and female chromosomes were n o t distinguishable, the aberration was assumed to be female in origin. Results
The results of the cytological analyses of the first cleavage divisions and of the metaphase I oocytes are presented in Tables I and II, respectively. The first cleavage division data are presented as the number of female pronuclei with the different numbers of chromatid aberrations. A phenomenon observed in these experiments that was n o t observed in the studies on post-meiotic male germ cells was a shattering effect (Fig. 1B), or almost total fragmentation of the chromosomes. As was the case when post-meiotic male cells were treated, chromatid-type aberrations were observed which were predominantly isochromatid deletions and chromatid deletions (Fig. 1C). The experiments in which metaphase I oocytes were analyzed yielded negative results in that only 1 isochromatid deletion was seen in 300 treated cells (Table II). TABLE I CHROMOSOME ABERRATION YIELDS AT THE FIRST CLEAVAGE MITOSIS AFTER TREATMENT O F D I C T Y A T E O O C Y T E S W I T H MMS Dose (mg/kg)
Control
Interval (days)
--
N o . o f cells scored
100
N o . o f cells w i t h a b e r r a t i o n s
Expected l e t h a l i t y (%)
0
1
2
3
)3
Shattered
100
0
0
0
0
0
0
50 50 50 50
2.5 6.5 10.5 14.5
75 50 50 50
70 a 2 47 3 49 1 50 0
2 0 0 0
0 0 0 0
0 0 0 0
1 0 0 0
8.0 a 6.0 2.0 0
100 100 100 100 100 100
0.5 1.5 2.5 6.5 10.5 14.5
50 50 50 50 50 50
40 4 43 3 40 5 43 a 5 47 3 50 0
0 1 1 0 0 0
0 0 1 0 0 0
0 1 0 1 0 0
6 2 3 1 0 0
20.0 14.0 20.0 16.0 a 6.0 0
a I n c l u d e s a t r i P l o i d z y g o t e ( S e e Fig. 1).
T A B L E II C H R O M O S O M E A B E R R A T I O N Y I E L D S IN M E T A P H A S E I O O C Y T E S F O L L O W I N G A 1 0 0 m g / k g D O S E O F MMS Interval (days)
No. o f cells s c o r e d
No. o f d e l e t i o n s
No. of e x c h a n g e s
Control 0.5 2.5 14.5
200 100 100 100
0 1 0 0
0 0 0 0
l~ - , ~ t ~ t$
,,
B
I ~-d -.-
o~~ "cry"
2',
Fig. 1. E x a m p l e s of first cleavage m i t o s e s in c u l t u r e d m o u s e ova. (A) N o r m a l f e m a l e and m a l e c h r o m o somes~ (B) s h a t t e r e d f e m a l e c h r o m o s o m e c o m p l e m e n t (arrow)~ (C) f e m a l e c h r o m o s o m e s w i t h a c h o m a t i d d e l e t i o n ( a r r o w ) (D) triploid z y g o t e .
81 Discussion In their study on the induction of dominant lethals in dictyate oocytes by MMS and EMS, Generoso and co-workers [2--4] reported that there is a restricted time period during which these c o m p o u n d s are effective. The highest frequency of dominant lethality was observed when matings occurred during the interval of 0.5--4.5 days after treatment. The interval of 10.5--14.5 days after treatment resulted in 21.6 and 10% dominant lethality after EMS and MMS, respectively, and the 10% frequency was not significant. The present data agree very well with Generoso's data in that no known cell-lethal chromosomal aberrations were observed after the 10.5-day interval after treatment. Generoso also observed a high frequency of sterile matings and pre-implantation loss in his studies, with a corresponding high level of zygotic arrest at the two-blastomere stage. He postulated that the failure of the zygote to develop b e y o n d the two-blastomere stage was the cause of pre-implantation loss. In fact, the level of pre-implantation loss at the most sensitive time interval was approx. 50%. In his studies, Gene~oso showed that for EMS the reduction in fertile matings could be attributed to death after fertilization rather than to a reduction in fertilization. Presumably this is also the case for MMS, and consequently a toxiCity effect can be ruled out. This interpretation is substantiated by the equal frequency of fertilized ova in the control and treated groups. The present data show that at the time interval corresponding to the peak yield of dominant lethality, the highest frequency of chromosome aberrations was also observed. Furthermore, at the highest dose, i.e. 100 mg/kg, approximately one third of the cells with aberrations had shattered female chromosomes. This shattering phenomenon most probably results in very early embryonic death, and conceivably can account for Generoso's observation of arrest at the two-blastomere stage. The discrepancy in absolute frequency in Generoso's and the present study can most likely be accounted for by the difference in dose (i.e. 150 vs 100 mg/kg, respectively) or by possible strain differences in sensitivity. That Generoso found equal numbers of corpora lutea in his MMS-treated and control females argues against the possibility that the damaged metaphase I oocytes were not ovulated. The lack of a significant level of structural aberrations in the metaphase I oocytes, and a corresponding significant level in the female complement at first cleavage after treatment at the stages of maturation, affirms the premise that an intervening round of DNA synthesis is required for an MMS-induced lesion to be translated into a structural aberration. That the aberrations were at the level of the chromatid confirms the observation made on post-meiotic male germ cells and lends further support to the argument that DNA synthesis is necessary for aberrations to be formed. The data of Jagiello [8,9] are exemplary of the opposite phenomenon, i.e. the non-requirement of an intervening round of DNA synthesis. In this work it is shown that streptonigrin induces classical chromatid aberrations at metaphase I when oocytes are treated. Streptonigrin does n o t require DNA synthesis to produce its clastogenic effect. The time-specificity for the eventual production of chromosome aberrations by MMS, and presumably EMS, in maturing oocytes carries certain implications
82
regarding repair of the pre-mutational lesion. Pederson and Masui [7] have reported that the enzymes necessary for unscheduled D N A synthesis are present in mouse o o c y t e s . In fact, they observed a significant increase in tritiated thymidine incorporation when metaphase I oocytes were irradiated. It has been shown by Regan and Setlow [8] that repair-competent cells do perform unscheduled D N A synthesis after MMS treatment, and Sega [9] has extended these observations to include spermatozoa and spermatid stages in the mouse. It seems reasonable to assume that the efficiency of repair is higher in early oocytes than in more mature oocytes (less than 6.5 days from ovulation), hence the higher aberration frequency could be due to more residual unrepaired lesions. The reason for this may be the more highly condensed nature of the chromosomes as the oocyte approaches the ovulation stage and the consequent inaccessibility of the lesion to attack by the repair enzymes. No attempt was made to analyze the first cleavage divisions for aneuploidy. The reason for this is that the preparation techniques employed lead to a significant level of chromosome loss through cell rupture, which it is felt would mask any increase in non-disjunction. Two triploid zygotes were seen, both of which {Fig. 1D) appeared to contain a diploid female complement as adjudged by the differential degree of chromosome condensation. References 1 B r e w e n , J . G . , H.S. P a y n e , K.P. J o n e s a n d R.J. P r e s t o n , S t u d i e s on c h e m i c a l l y - i n d u c e d d o m i n a n t let h a l i t y . I. T h e c y t o g e n e t i c basis o f M M S - i n d u c e d d o m i n a n t l e t h a l i t y in p o s t - m e i o t i c m a l e g e r m cells, M u t a t i o n Res., 33 ( 1 9 7 5 ) 2 3 9 - - 2 5 0 . 2 G e n e r o s o , W.M.° C h e m i c a l i n d u c t i o n of d o m i n a n t lethals in f e m a l e m i c e , G e n e t i c s , 61 ( 1 9 6 9 ) 4 6 1 - 470. 3 G e n e r o s o , W.M. a n d W.L. Russell, Strain a n d sex v a r i a t i o n s in t h e sensitivity of m i c e to d o m i n a n t lethal i n d u c t i o n w i t h e t h y l m e t h a n e s u l f o n a t e , M u t a t i o n Res., 8 ( 1 9 6 9 ) 5 8 9 - - 5 9 8 . 4 G e n e r o s o , W.M., S.W. H u f f a n d S.K. S t o u t , C h e m i c a l l y i n d u c e d d o m i n a n t l e t h a l m u t a t i o n s and cell killing in m o u s e o o c y t e s in a d v a n c e d stages o f follicular d e v e l o p m e n t , M u t a t i o n Res., 11 ( 1 9 7 1 ) 4 1 1 - 420. 5 H a n s m a n n , I. a n d G. R S h r b o r n , C h r o m o s o m e a b e r r a t i o n s in p r c i m p l a n t a t i o n stages of m i c e a f t e r t r e a t m e n t w i t h t r i a z o q u i n o n e , H u m a n g e n e t i k , 18 ( 1 9 7 3 ) 1 0 1 - - 1 0 9 . 6 H a n s m a n n , I., I n d u c e d c h r o m o s o m a l a b e r r a t i o n s in p r o n u c l e i c , 2-cell stages, and m o r u l a e of m i c e , M u t a t i o n Res., 20 ( 1 9 7 3 ) 3 5 3 - - 3 6 7 . 7 H a n s m a n n , I., C h r o m o s o m e a b e r r a t i o n s in m e t a p h a s e I I - o o c y t e s stage s e n s t i t i v i t y in the m o u s e oogenesis to a m e t h o p t c r i n , M u t a t i o n Res., 22 ( 1 9 7 4 ) 1 7 5 - - 1 9 1 . 8 Jagiello, G., S t r e p t o n i g r i n : e f f e c t on t h e first m e i o t i c m e t a p h a s e of t h e m o u s e egg, Science 152 (1967) 453--454. 9 Jagiello, G., A c t i o n o f p h l e o m y c i n on t h e m e i o s i s of the m o u s e o v u m , M u t a t i o n Res., 6 ( 1 9 6 8 ) 2 8 9 - 295. 10 Jagiello, G. a n d P.E. Polani, M o u s e g e r m cells a n d L S D - 2 5 , C y t o g e n e t i c s ° 8 ( 1 9 6 9 ) 1 3 6 - - 1 4 7 . 11 M a t t e r , B.E. a n d I. J a e g e r , P r e m a t u r e c h r o m o s o m e c o n d e n s a t i o n , s t r u c t u r a l c h r o m o s o m e a b e r r a t i o n s a n d m i c r o n u c l e i in early m o u s e e m b r y o s o f f e r t r e a t m e n t of p a t e r n a l g e r m cells w i t h t r i e t h y l e n e m e l a m i n e : a possible m e c h a n i s m f o r c h e m i c a l l y - i n d u c e d d o m i n a n t l e t h a l m u t a t i o n s , M u t a t i o n Res., 33 (1975) 251--260. 12 P a y n e , H.S. a n d K.P. J o n e s , T e c h n i q u e for m a s s isolation a n d c u l t u r e of m o u s e ova f o r c y t o g e n e t i c analysis of the first cleavage m i t o s i s , M u t a t i o n Res., 33 ( 1 9 7 5 ) 2 3 9 - - 2 5 0 . 13 P e d e r s e n , R . A . a n d Y. Masui, U n s c h e d u l e d D N A s y n t h e s i s a f t e r U V i r r a d i a t i o n of m o u s e o o c y t e s , A b s t r a c t s of P a p e r s for the 2 3 r d Mtg. of the R a d i a t i o n R e s e a r c h S o c i e t y , May 1 1 - - 1 5 , 1 9 7 5 , A b s t r . Be-2. 14 R e g a n , J . D . a n d R.B. S e t l o w , T w o f o r m s of r e p a i r in the D N A o f h u m a n cells d a m a g e d b y c h e m i c a l caxcinogens a n d m u t a g e n s , C a n c e r Res., 3 4 ( 1 9 7 4 ) 3 3 1 8 - - 3 3 2 5 . 15 R S h r b o r n , G., O. K u h n , I. H a n s m a n n a n d K. T h o n , 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 early e m b r y o g e n e s i s of m i c e , H u m a n g e n e t i k , 11 ( 1 9 7 1 ) 3 1 6 - - 3 2 2 . 16 Sega, G . A . , U n s c h e d u l e d D N A s y n t h e s i s in t h e g e r m cells of m a l e m i c e e x p o s e d in vivo to t h e c h e m ical m u t a g e n e t h y l m e t h a n e s u l f o n a t e , Proc. Natl. A c a d . Sci. U . S . A . , 71 ( 1 9 7 4 ) 4 9 5 5 - - 4 9 5 9 .