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Action of lead on early divisions of the mouse embryo
Toxicology, 6 (1976) 129--132 © Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
ACTION OF LEAD ON EARLY DIVISIONS OF THE MOUSE EMBRYO
P. JACQUET, A. LEONARD and G.B. GERBER Department of Radiobiology, C.E.N./S.C.K., B-2400 Mol (Belgium) (Received January 13th, 1976) (Accepted February 10th, 1976) SUMMARY The number of cells in mouse embryos 48 h after mating was studied in normal and lead-treated females (0.125, 0.25, 0.5, 1% of lead in the diet). The percentage of embryos in the 4-cell stage compared to those in the 8-cell stage increased after 0.125, 0.25 and 0.5% of dietary lead. After the highest dose an increase in nondivided eggs was also seen which occurred at the expense of 4-cell embryos. The different possibilities, delay of all divisions, delay of only the first division and action of lead on only a part of the embryonic population, are discussed.
INTRODUCTION Severe intoxication of w o m e n by lead delays intrauterine and postnatal growth of children and can cause abortion [1,2]. In rats, high doses of lead have a similar action whereas lower ones do n o t apparently affect development [3--6]. Recently we observed [7] that lead exposure of mice after successful mating (i.e. females displaying a vaginal plug) increases significantly the number of females having no implants. The embryos of such mothers, thus have failed to develop or were unable to become implanted, an effect which was t h o u g h t to be due to the action of lead on the mother rather than on the embryo, since in other females (with at least one implant), the preimplantation loss did n o t increase when compared to that of the controls. Moreover, on the 18th day of gestation, embryos of pregnant mothers exposed to high doses of lead had a lower weight than those from non-exposed ones, but this difference was much less noticeable at earlier times o f development (16 and 17th day). The present experiments were undertaken to study the action of lead on the very early development of the embryos. MATERIAL AND METHODS Mice of the C57B1 strain raised in our laboratory were utilized. Females were caged with males and examined daily for the presence of vaginal plugs.
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Females with a vaginal plug were r e m o v e d and given a diet c o n t a i n i n g 0, 0.125, 0.25, 0.5 or 1% o f lead. 48 h after o b s e r v a t i o n o f the vaginal plug, the mice were sacrificed, their oviducts placed in isotonic saline and carefully dissected u n d e r the microscope. The e m b r y o s o b t a i n e d were classified into f o u r categories a c c o r d i n g to their degree of d e v e l o p m e n t : n o n d i v i d e d e m b r y o s , 2-cell e m b r y o s , 4-cell e m b r y o s and 8-cell e m b r y o s . A t least 100 e m b r y o s were observed for each t r e a t m e n t . Statistical c o m p a r i s o n b e t w e e n c o n t r o l s and lead-treated e m b r y o s was based on the Chi square test. RESULTS AND DISCUSSION Table I shows data on the d e v e l o p m e n t o f e m b r y o s u n d e r the influence of d i f f e r e n t doses o f lead. In t h e c o n t r o l g r o u p a b o u t half the e m b r y o s had n o t divided at all, m o s t o f the rest had arrived at the 8-cell stage; only a few e m b r y o s had 2 or 4 cells. In the g r o u p s t r e a t e d with 0.125, 0.25 or 0.5% o f lead, an a p p r o x i m a t e l y equal n u m b e r o f e m b r y o s were seen in the 4- and 8-cell stage whereas the p r o p o r t i o n of e m b r y o s in the 1- and 2-cell stage was n o t altered. A f t e r t r e a t m e n t with 1% o f lead, the n u m b e r o f e m b r y o s u n d e r g o i n g no division at all had increased, and relatively m o r e e m b r y o s were seen at the 8-cell stage c o m p a r e d to the 4-cell stage t h a n in the case o f l o w e r doses o f lead. The early d e v e l o p m e n t o f the m o u s e e m b r y o can be s u m m a r i z e d as follows [8] : the first cleavage occurs 24 h after mating. S u b s e q u e n t divisions take place every 12 h, so t h a t after 48 h, the e m b r y o has arrived at the 8-cell
TABLE I NUMBER OF CELLS IN NORMAL AND LEAD-TREATED EMBRYOS 48 H AFTER THE VAGINAL PLUG The X2' values for the comparison of divided embryos of controls with lead-treated embryos are 15.56, 14.8, 24.5, 1.98 for 0.125, 0.25, 0.5 and 1% of lead respectively. Comparison of divided with nondivided embryos yields for the 1% group X2 = 4.00. Treatment (%) 0 0.125 0.250 0.500 1
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Embryos examined 115 119 108 124 154
Stages reached One-cell
Two-cell
Four-cell
Eight-cell
60 60 50 64 99
2 (1.7%) 1 (0.8%) 2(1.85%) 1 (0.8%) 1(0.65%)
6 (5.3%) 26 (21.8%) 25 (23.15%) 34 (27.4%) 11(7.14%)
47 32 31 25 43
(52%) (50%) (46.3%) (51.6%) (64.29%)
(41%) (27.4%) (28.7%) (20.2%) (27.92%)
stage and is placed near the end of the oviduct ready to be discharged into the uterus. A certain percentage of eggs, however, do not divide at all either because they are not fertilized or because they are incapable of developing. The following possibilities should be considered to explain the delay in divisions caused by lead. Lead could cause: (a) a delay in all cell divisions following conception; (b) a delay in the first cell division only and eventually a block; (c) a delay of division in part of the population which would then be eliminated before implantation. Although the data presented do not allow an unequivocal decision between these possibilities, it appears worthwhile to speculate on the different implications of the alternate hypothesis. A delay in all cell divisions caused by lead appears unlikely since it would mean an even greater delay in develo p m e n t from 5 h after 2 days to about 2 days after 18 days. The weight difference f o u n d in embryos on day 18 is not sufficiently great to account for such an effect and, in addition, seems to develop only during the final part of pregnancy [7]. With respect to possibility (c) that only part of the embryonic population is affected, arguments in favour and against can be presented. Normally early cell divisions are synchronized so that most cells will be in the same stage as is shown by the (truncated) distribution in control embryos whose variance is much smaller than that of a Poisson distribution (standard variation found = 0.47 - - t h e o r e t i c a l Poisson variation = 1.7). In embryos from mothers treated with 0.125% to 0.5% of lead, this synchronisation seems to be lost since about half the developing embryos are in the 4-ceU, the other half in the 8-cell stage, suggesting the presence of two populations with different characteristics of division. On the other hand, one may argue that in this case an excess of nearly 50% of the developing embryos should be lost before implantation, a postulate which is disproved by our earlier data [7] where no significant preimplantation loss has been found in pregnant females. It must be pointed out, however, that information on developing embryos comes from all females with vaginal plugs and n o t only from the pregnant ones. The percentage of non-pregnant females which have displayed a vaginal plug augments indeed from 50 to over 80 after high doses of lead but is still normal after 0.125% of lead [7] when the delay in early development is already fully expressed. Thus, by exclusion possibility (b) seems most likely. A variable delay in the first cell division only could also result in a distribution function of the form seen for lead-treated embryos. The fact that the percentage of eggs not undergoing any division at all increases slightly at the highest dose of lead suggests a particular action of lead on this division. ACKNOWLEDGEMENTS This work was supported by the Research Contract EURATOM-CEN 038-74-7 ENV.B.
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REFERENCES 1 2 3 4 5
C.R. Angle and M.S. McIntire, Amer. J. Dis. Child., 108 (1964) 436. P.A. Palmisano, R.C. Sneed and G. Cassady, J. Pediat., 75 (1969) 869. R.M. McClain and B.A. Becker, Toxicol. Appl. Pharmacol., 21 (1972) 265. A. Pentschew and F. Garro, Acta Neuropathol., 6 (1966) 266. A. Azar, H.J. Trochimowicz and M.E. Maxfield, Proc. Intern. Symp. Environ. Health Aspects of Lead, Amsterdam, EUR 5004 d-e-f- (1973) 199. 6 P.E. Fournier and E. Rosenberg, Proc. Intern. Syrup. Environ. Health Aspects of Lead, Amsterdam, EUR 5004 d-e-f (1973) 287. 7 P. Jacquet, A. Leonard and G.B. Gerber, Experientia, 31 (1975) 1312. 8 G.D. Snell and L.C. Stevens, Early embryology, in E.L. Green (Ed.), Biology of the Laboratory Mouse, McGraw-Hill, New York, 1966, p. 205.
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ACTION OF LEAD ON EARLY DIVISIONS OF THE MOUSE EMBRYO
P. JACQUET, A. LEONARD and G.B. GERBER Department of Radiobiology, C.E.N./S.C.K., B-2400 Mol (Belgium) (Received January 13th, 1976) (Accepted February 10th, 1976) SUMMARY The number of cells in mouse embryos 48 h after mating was studied in normal and lead-treated females (0.125, 0.25, 0.5, 1% of lead in the diet). The percentage of embryos in the 4-cell stage compared to those in the 8-cell stage increased after 0.125, 0.25 and 0.5% of dietary lead. After the highest dose an increase in nondivided eggs was also seen which occurred at the expense of 4-cell embryos. The different possibilities, delay of all divisions, delay of only the first division and action of lead on only a part of the embryonic population, are discussed.
INTRODUCTION Severe intoxication of w o m e n by lead delays intrauterine and postnatal growth of children and can cause abortion [1,2]. In rats, high doses of lead have a similar action whereas lower ones do n o t apparently affect development [3--6]. Recently we observed [7] that lead exposure of mice after successful mating (i.e. females displaying a vaginal plug) increases significantly the number of females having no implants. The embryos of such mothers, thus have failed to develop or were unable to become implanted, an effect which was t h o u g h t to be due to the action of lead on the mother rather than on the embryo, since in other females (with at least one implant), the preimplantation loss did n o t increase when compared to that of the controls. Moreover, on the 18th day of gestation, embryos of pregnant mothers exposed to high doses of lead had a lower weight than those from non-exposed ones, but this difference was much less noticeable at earlier times o f development (16 and 17th day). The present experiments were undertaken to study the action of lead on the very early development of the embryos. MATERIAL AND METHODS Mice of the C57B1 strain raised in our laboratory were utilized. Females were caged with males and examined daily for the presence of vaginal plugs.
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Females with a vaginal plug were r e m o v e d and given a diet c o n t a i n i n g 0, 0.125, 0.25, 0.5 or 1% o f lead. 48 h after o b s e r v a t i o n o f the vaginal plug, the mice were sacrificed, their oviducts placed in isotonic saline and carefully dissected u n d e r the microscope. The e m b r y o s o b t a i n e d were classified into f o u r categories a c c o r d i n g to their degree of d e v e l o p m e n t : n o n d i v i d e d e m b r y o s , 2-cell e m b r y o s , 4-cell e m b r y o s and 8-cell e m b r y o s . A t least 100 e m b r y o s were observed for each t r e a t m e n t . Statistical c o m p a r i s o n b e t w e e n c o n t r o l s and lead-treated e m b r y o s was based on the Chi square test. RESULTS AND DISCUSSION Table I shows data on the d e v e l o p m e n t o f e m b r y o s u n d e r the influence of d i f f e r e n t doses o f lead. In t h e c o n t r o l g r o u p a b o u t half the e m b r y o s had n o t divided at all, m o s t o f the rest had arrived at the 8-cell stage; only a few e m b r y o s had 2 or 4 cells. In the g r o u p s t r e a t e d with 0.125, 0.25 or 0.5% o f lead, an a p p r o x i m a t e l y equal n u m b e r o f e m b r y o s were seen in the 4- and 8-cell stage whereas the p r o p o r t i o n of e m b r y o s in the 1- and 2-cell stage was n o t altered. A f t e r t r e a t m e n t with 1% o f lead, the n u m b e r o f e m b r y o s u n d e r g o i n g no division at all had increased, and relatively m o r e e m b r y o s were seen at the 8-cell stage c o m p a r e d to the 4-cell stage t h a n in the case o f l o w e r doses o f lead. The early d e v e l o p m e n t o f the m o u s e e m b r y o can be s u m m a r i z e d as follows [8] : the first cleavage occurs 24 h after mating. S u b s e q u e n t divisions take place every 12 h, so t h a t after 48 h, the e m b r y o has arrived at the 8-cell
TABLE I NUMBER OF CELLS IN NORMAL AND LEAD-TREATED EMBRYOS 48 H AFTER THE VAGINAL PLUG The X2' values for the comparison of divided embryos of controls with lead-treated embryos are 15.56, 14.8, 24.5, 1.98 for 0.125, 0.25, 0.5 and 1% of lead respectively. Comparison of divided with nondivided embryos yields for the 1% group X2 = 4.00. Treatment (%) 0 0.125 0.250 0.500 1
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Embryos examined 115 119 108 124 154
Stages reached One-cell
Two-cell
Four-cell
Eight-cell
60 60 50 64 99
2 (1.7%) 1 (0.8%) 2(1.85%) 1 (0.8%) 1(0.65%)
6 (5.3%) 26 (21.8%) 25 (23.15%) 34 (27.4%) 11(7.14%)
47 32 31 25 43
(52%) (50%) (46.3%) (51.6%) (64.29%)
(41%) (27.4%) (28.7%) (20.2%) (27.92%)
stage and is placed near the end of the oviduct ready to be discharged into the uterus. A certain percentage of eggs, however, do not divide at all either because they are not fertilized or because they are incapable of developing. The following possibilities should be considered to explain the delay in divisions caused by lead. Lead could cause: (a) a delay in all cell divisions following conception; (b) a delay in the first cell division only and eventually a block; (c) a delay of division in part of the population which would then be eliminated before implantation. Although the data presented do not allow an unequivocal decision between these possibilities, it appears worthwhile to speculate on the different implications of the alternate hypothesis. A delay in all cell divisions caused by lead appears unlikely since it would mean an even greater delay in develo p m e n t from 5 h after 2 days to about 2 days after 18 days. The weight difference f o u n d in embryos on day 18 is not sufficiently great to account for such an effect and, in addition, seems to develop only during the final part of pregnancy [7]. With respect to possibility (c) that only part of the embryonic population is affected, arguments in favour and against can be presented. Normally early cell divisions are synchronized so that most cells will be in the same stage as is shown by the (truncated) distribution in control embryos whose variance is much smaller than that of a Poisson distribution (standard variation found = 0.47 - - t h e o r e t i c a l Poisson variation = 1.7). In embryos from mothers treated with 0.125% to 0.5% of lead, this synchronisation seems to be lost since about half the developing embryos are in the 4-ceU, the other half in the 8-cell stage, suggesting the presence of two populations with different characteristics of division. On the other hand, one may argue that in this case an excess of nearly 50% of the developing embryos should be lost before implantation, a postulate which is disproved by our earlier data [7] where no significant preimplantation loss has been found in pregnant females. It must be pointed out, however, that information on developing embryos comes from all females with vaginal plugs and n o t only from the pregnant ones. The percentage of non-pregnant females which have displayed a vaginal plug augments indeed from 50 to over 80 after high doses of lead but is still normal after 0.125% of lead [7] when the delay in early development is already fully expressed. Thus, by exclusion possibility (b) seems most likely. A variable delay in the first cell division only could also result in a distribution function of the form seen for lead-treated embryos. The fact that the percentage of eggs not undergoing any division at all increases slightly at the highest dose of lead suggests a particular action of lead on this division. ACKNOWLEDGEMENTS This work was supported by the Research Contract EURATOM-CEN 038-74-7 ENV.B.
131
REFERENCES 1 2 3 4 5
C.R. Angle and M.S. McIntire, Amer. J. Dis. Child., 108 (1964) 436. P.A. Palmisano, R.C. Sneed and G. Cassady, J. Pediat., 75 (1969) 869. R.M. McClain and B.A. Becker, Toxicol. Appl. Pharmacol., 21 (1972) 265. A. Pentschew and F. Garro, Acta Neuropathol., 6 (1966) 266. A. Azar, H.J. Trochimowicz and M.E. Maxfield, Proc. Intern. Symp. Environ. Health Aspects of Lead, Amsterdam, EUR 5004 d-e-f- (1973) 199. 6 P.E. Fournier and E. Rosenberg, Proc. Intern. Syrup. Environ. Health Aspects of Lead, Amsterdam, EUR 5004 d-e-f (1973) 287. 7 P. Jacquet, A. Leonard and G.B. Gerber, Experientia, 31 (1975) 1312. 8 G.D. Snell and L.C. Stevens, Early embryology, in E.L. Green (Ed.), Biology of the Laboratory Mouse, McGraw-Hill, New York, 1966, p. 205.
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