The Relation between Maternal Restraint and Food Deprivation, Plasma Co rticoste rone, and Induction of Cleft Palate in the Offspring of Mice SUSAN M. FRANK M. Depurtment London S E l
BARLOW, PATRICIA R. McELHATTON A N D SULLIVAN of Phurmacology, Guy's Hospitul Medicul School,
9RT
ABSTRACT The blood level of corticosterone was measured in mice following the injection on day 14 of pregnancy of a dose of corticosterone sufficient to cause a low frequency of cleft palate in the fetuses. This was compared with the blood levels present during maternal restraint and food deprivation that produced a similar frequency of cleft palate. The mean blood level over the 24 h following injection of corticosterone was 660 @lo0 ml, and during a similar period of restraint was 485 g g / l O O ml. Other mice were subjected either to restraint or food deprivation for 24 h beginning day 14 of pregnancy, the plasma corticosterone levels measured during that time, and the frequency of cleft palate in late fetuses compared with the individual plasma corticosterone levels during treatment. There was a significant (P < 0.025) correlation between high maternal corticosteroid levels and the frequency of cleft palate in the offspring of the restrained mice but not in the food-deprived animals. I t is suggested that in some stressed mice endogenous plasma corticosterone can reach levels sufficient to account for the development of cleft palate.
In some strains of mice injection of pregnant females with cortisone (Fraser and Fainstat, '51), cortisol (Kalter and Fraser, '52), or corticosterone (Blaustein et al., '71) during the sensitive period causes cleft palate in the offspring. Injection of adrenocorticotrophic hormone (ACTH) also caused cleft palate (Heiberg et al., '59; Kalter, '62; Blaustein et al., '71). It is unlikely that ACTH can cross the placenta (Allen et al., '73; Dokumov et al., '74; Eguchi et al., '73) and act directly on the fetus. Heiberg and coworkers ('59) suggested that pregnant mice may produce teratogenic amounts of corticosteroids in response to ACTH, and Velardo ('57) showed that the embryolethal effect of ACTH given in early pregnancy in rats is definitely mediated via the maternal adrenal glands rather than by a direct action on the embryo. "Stress" is difficult to define since it is essentially a subjective phenomenon. However it is conventionally considered to have occurred in animal experiments if there is a raised plasma corticosteroid level following the application of a "stressor." Many such stressors including restraint (Rosenzweig and Blaustein, '70), food or water TERATOLOGY, 12: 97-104.
deprivation (Kalter, '60; Peters and Strassburg, '69; Rosenzweig and Blaustein, '70; Miller, '73), avoidance conditioning (Rosenzweig, '66), and transportation (Brown et al., '72) induce cleft palate in mice. Restraint stress was also synergistic with cortisone in inducing cleft palate (Rosenzweig, '69). It is known that during stress in pregnant mice, plasma corticosterone reaches very high levels, around 400 times greater than in resting nonpregnant mice (Brain and Nowell, '70; Barlow et al., '74). It would be interesting to know whether the plasma corticosterone levels reached during stress in pregnant mice would be safficient by themselves to account for the development of cleft palate. We decided to test this by measuring the plasma levels of corticosterone reached following the injection of a dose of corticosterone known to produce cleft palate. Other mice were subjected to stress and the plasma corticosterone levels measured to see whether they were similar to those observed following corticosterone injection. Finally, both the corticosterone levels during stress and Received May 20, '75. Accepted June 20, '75
97
98
S. M. BARLOW, P. R. McELHATTON A N D F. M. SULLIVAN
the frequency of cleft palate were measured in the same mice to see if these were correlated. MATERIALS A N D METHODS
Animals Mice derived from the pathogen-free ICI randombred stock, bred under conventional conditions in the animal house at Guy's Hospital Medical School were used. Animal rooms were maintained at 21 rt_ 1 "C, humidity 5&60%, with 10 air changes per hour, under controlled lighting conditions with 14 h of light and 10 h of darkness. The diet used was FFG(M), Dixons. Males 12 weeks of age or over were placed overnight with virgin females 10 weeks of age, averaging 25 g body weight, and vaginal plugs looked for next morning. The day of finding the plug was designated day 1 of pregnancy, and pregnant females were removed from the mating cages and rehoused in groups of 4 or 5 until required for experiment. Injections To measure the frequency of cleft palate following corticosterone injection mice on day 14 of pregnancy were injected sc in the scruff of the neck with 1.25, 2.5, 5.0, or 10.0 mg (equivalent to 50, 100, 200, or 400 mg/kg) of corticosterone (Organon, England) suspended in arachis oil. The injection volume was standardized to 0.1 ml. Control mice were injected with arachis oil alone. In the study of the plasma levels of corticosterone following injection, mice on day 14 of pregnancy were injected with 2.5 mg (100 mg/kg) corticosterone and killed 0, 0.5, 1, 2, 5, 10, and 24 h later, 6 mice being lulled at 0 h and 3 mice at each of the other times. Stressor techniques Restraint. Mice were immobilized for 24 h from 10 AM on day 14 to 10 A M on day 15 of pregnancy using a method described by Renaud ('59). The mice were strapped down by their limbs in a prone position, onto a special board, using zinc oxide plaster, in such a manner that their paws were undamaged. Animals were observed to drink but do not normally eat while restrained in this way, so water but no food was provided during the period of
immobilization. The temperature of the room in which the animals were kept was 21 "C. To measure the frequency of cleft palate following restraint 16 mice were used. In the study of the plasma levels of corticosterone following restraint mice were killed 0, 0.3, 1, 2, 4, 8, 12, 17, and 24 h after the commencement of restraint, 8 mice being killed at each time. In the experiments relating the plasma corticosterone levels during stress with the subsequent presence of cleft palate, 20 mice were restrained and blood samples taken 7 and 24 h after commencement of restraint. After 24 h of restraint the mice were returned to the animal house until day 19 of pregnancy when they were killed and the offspring examined for palatal defects. Food deprivation. Mice were kept singly in cages at an ambient temperature of 21 "C. Water was provided and they were free to move about within their cages but, like the immobilized mice, they were without food for 24 h and unable to huddle for warmth. I t was known from previous experiments (Barlow, '72) that body temperature may drop by up to 2 "C under these conditions. Collection of blood In nonrecovery experiments mice were killed by cervical dislocation and 0.5 ml of blood taken by cardiac puncture using a heparinized syringe. In recovery experiments, where mice were allowed to continue until term so that the fetuses could be examined, 0.25 ml of blood was taken from the retroorbital sinus using a heparinized syringe and a plastic cannula during 2 min of ether anesthesia. The blood was immediately centrifuged and duplicate 100 or 50 pl aliquots of plasma taken for assay. Corticosterone assay Plasma corticosterone levels were measured using the microfluorimetric technique of Glick et al. ('64). The precision of the method was estimated as +-1ng/100 pI of plasma. All corticosterone values are expressed as pg +- SE/100 ml of plasma. Ter ato logy Mice were killed on day 19 of pregnancy and the contents of the uterus examined. The number of live and full-term dead fe-
99
STRESS, CORTICOSTERONE, AND CLEFT PALATE IN MICE TABLE 1
wfects on fetul development of corticosterone injection on day 14 of pregnuncy Corticosterone dose
No. of litters
Live fetuses
Resorbing and dead fetuses
Litters with cleft palate
Live fetuses with cleft palate (%)
mg
0 1.25 3 2.5 5.0 10.0
22 14 10 11
5
230 156 1
iig
2
132 53
27 21 15 4 8
0 1 3 10 4
0 (0.0) 1 10.6) 3 i2.5j 43 (32.3) 16 (30.2)
Included 1 fetus with exencephaly. Included 1 fetus with kyphosis and 1 fetus with fused ribs. 3 Equivalent to 50, 100, 200, and 400 mglkg. 1
2
tuses (no signs of maceration), late resorptions (maceration, death occurring at the fetal stage), and early resorptions (death occurring at the embryonic stage) were recorded. Fetuses were removed and examined for gross external morphological defects, dissected to check for internal defects, then stained with alizarin red S and inspected for skeletal defects. Particular attention was paid to the formation of the pal ate.
Statistical analysis Differences in malformation rates, as assessed on a per fetus basis, were analyzed by the Fisher two-tailed exact probability test unless otherwise stated. Differences assessed on a per litter basis were analyzed by analysis of variance of normal scores (Kendall and Stuart, '67), which implies no assumption as to the normality of the data. RESULTS
of data collected from untreated controls over a 3-year period, the total number of fetuses examined being 988. Other abnormalities observed were exencephaly (l), kyphosis (l), fused ribs (l), and poor ossification of the sternum (10-12% of all fetuses in treated groups, 2% in vehicle controls), but these abnormalities were not dose-related. Corticosterone injections caused no increase in the frequency of resorptions and had no effect on fetal body weight . Stressors. The frequency of cleft palate due to 24 h of maternal restraint or food deprivation beginning on day 14 was determined. Controls were left undisturbed in the animal house for the whole duration of pregnancy until day 19 when they were killed and the offspring examined for palatal defects. The results are shown in table 2 . There was a significant increase in the frequency of cleft palate in the offspring of both restrained ( P = 0.04) and food-deprived (P = 0.002) animals in comparison with untreated controls. The difference between restraint and food deprivation was not significant (P = 0.42).
Comparison of frequency of cleft palate following corticosterone injection and stressors Corticosterone injections. Preliminary Comparison of plasma corticosterone experiments established that day 14 of levels following corticosterone pregnancy was the most sensitive day for injections and restraint induction of cleft palate in the stock used. Mice were injected with various doses of Corticosterone injection. Maternal corticosterone on this day, and the results plasma corticosterone levels were measured are shown in table 1. over a period of 24 h following the injecThe frequency of the defect was dose- tion on day 14 of pregnancy of 2.5 mg related, reaching a plateau of around 30% (100 mg/kg) of corticosterone, a dose that at 5.0 mg (200 mg/kg). Although no cleft produced a small but significant ( P = palate was observed in the controls the 0.039, single-tailed) increase in the frespontaneous frequency of cleft palate in quency of cleft palate in the offspring apthis stock of mouse is 0.2%, a malforma- proximately equal to that caused by the tion rate calculated from the pooled results stressors, i.e., 2.5 and 5% respectively.
100
S. M. BARLOW, P. R. McELHATTON AND F. M. SULLIVAN TABLE 2 wfects on fetal development
of 24 h of maternal treatment beginning o n day 14 of pregnancy No. of
Maternal tcea tmen t
Resorbing and dead fetuses
Litters with cleft palate
Fetuses with cleft palate
148 305
39
107
21
3 9 0
7 (5) 22 (7) 0 (0)
Live fetuses
litters
(%I Restraint Food deprivation Untreated control
16 31 12
1000
-
500
-
100
-
50
8
1
1
I
L
0
4
8
1
12
16
20
24
Hours Fig. 1 Plasma corticosterone levels following subcutaneous injection of 2.5 mg (100 mg/kg) of corticosterone on day 14 of pregnancy (00 ) or during 24 h of restraint begun on - - -0). Vertical bars indicate SE. day 14 of pregnancy (0-
The results are shown in figure 1 . Mice killed at 0 h had a mean plasma corticosterone level of 542 k 42 pg/ml, reflecting the stress of being taken from the animal house to the laboratory half an hour before blood sampling (see Barlow et d.,'75). Following the injection of corticosterone, plasma levels rose to a peak of 1208 +- 57 pg/100 ml at 2 h, were maintained at around 800 p g / l O O ml up to at least 10 h after injection, but by 24 h had fallen to 95 k 5 pg/100 ml, which is a normal resting level for day 15 of pregnancy (Barlow et al., '74). Restraint. Maternal plasma corticosterone levels were measured at various intervals during 24 h of restraint beginning on day 14 of pregnancy. The results are shown in figure 1. Levels increased from a mean resting level of 79.9 6.3
*
pg/100 ml at 0 h, these mice being killed in the animal house, to reach a maximum of 733 t 56 pg/ml 1 h after the commencement of restraint. Thereafter levels were maintained at 500-600 p g / l O O ml plasma during the next 16 h of restraint. By 24 h levels had fallen to 268 ? 44 pg1100 ml but were still elevated in comparison with nonstressed pregnant controls on day 15 of pregnancy. It should be noted however that at any given time during the 24 h of restraint there was a wide variation between individuals. For example, at 17 h individual values ranged from 2 7 6 7 0 0 pg/lOO ml. The plasma corticosterone levels following restraint or corticosterone injection (fig. 1) were compared and gave a mean value of 485 p g / l O O ml of plasma during restraint and 660 pg/100 ml of plasma following corticosterone injection.
101
STRESS, CORTICOSTERONE, AND CLEFT PALATE IN MICE
The ratio of the area under the restraint curve to the area under the injection curve was 0.75.
Correlation between plasma corticosterone leuels during application of stressors and cleft palate In the above-described experiments animals were killed to obtain the plasma for corticosterone assays, and therefore other animals were used to ascertain the fiequency of cleft palate following application of stressors or injection of corticosterone. It was decided to determine whether in individual mice there was any correlation between plasma corticosterone levels during stressing and subsequent development of cleft palate. Mice were subjected to 24 h of restraint or food deprivation on day 14 of pregnancy, blood samples were taken at 7 h and 24 h, and the offspring examined at term. The plasma corticosterone levels at 7 h and the presence or absence of cleft palate in the litters subsequently are shown in figure 2. There were 8 fetuses with cleft palate in 5 of the 20 litters in the restrained group and 29 fetuses with cleft palate in 12 of the 35 litters in the food-deprived group. In the restrained group there was a significant correlation between high maternal plasma corticosterone levels at 7 h and the presence of cleft palate in the offspring as assessed on a litter basis ( P < 0.025, analysis of varianceof normal scores, Kendall and Stuart, '67). Values at 24 h are not shown but there was also a significant correlation at that time (P < 0.025). In the food-deprived group, however, there was no significant correlation between maternal plasma corticosterone levels and palatal defects in the offspring at either 7 or 24 h (P < 0.35). There was no correlation between the number of affected fetuses per litter and maternal plasma corticosterone levels. DISCUSSION
Considering first maternal plasma corticosterone levels the present investigations showed that the levels found during restraint on day 14 of pregnancy were of a similar order of magnitude to those observed following injection of 2.5 mg of corticosterone, i.e., a mean of 485 p g / l O O ml and 660 pg/lOO ml, respectively. Although
a
8 0
8
0
a
b eo 0
0
0 0
9,
8J ?5
oa
%
0
8 8
Rest r a i n t
Food deprivation
Fig. 2 Relation between maternal plasma corticosterone levels at 7 h during 24 h of restraint or food deprivation begun on day 14 of pregnancy and the development of cleft palate in the offspring. Litters with cleft palate (a),normal litters (0).
the peak levels reached in the restrained group were lower than in the injected group the levels in the former remained elevated above control values for a longer period of time than in the latter. The frequency of cleft palate was similar in the 2 groups of mice. Thus it would appear that restraint may cause sufficient corticosterone to be released to cause a low frequency of cleft palate in these mice. To test this hypothesis more rigorously both maternal plasma corticosterone levels and the frequency of cleft palate in off-
102
S. M. BARLOW, P. R. McELHATTON AND F. M. SULLIVAN
spring were investigated in the same individuals subjected to stressors. In the restrained group there was a significant correlation between these 2 phenomena. Thus it seems that in some mice during restraint plasma corticosterone levels were high enough to account for the presence of cleft palate in the offspring. In the food-deprived group, however, some mice with relatively low plasma corticosterone levels during deprivation also had offspring with cleft palate. So in these instances some other mechanisms are presumably involved in the production of cleft palate. There remains some debate about the mode of action of corticosteroids as teratogens. They may have a direct action on the fetus (Larsson, '62; Dostal, '71) or may act indirectly, e.g., by reducing amnioticfluid volume (Harris, '64). If the action is a direct one, measurement of maternal plasma corticosterone levels may not accurately reflect the amount of corticosterone reaching the fetus, since placental transfer of the hormone is also influenced by factors other than concentration differences, e.g., by changes in placental blood flow. In some, but not all, mice during restraint pooling of blood in the placenta has been observed, accompanied by a reduction in placental transfer function (Barlow and Sullivan, '73). Presumably the placental transfer of corticosterone would also be reduced under such conditions. Thus, in some stressed mice with high maternal corticosterone levels fetal levels could be relatively low. In other mice, with lower maternal corticosterone levels but unimpaired placental transfer function, fetal levels might be relatively high, resulting in an unpredictable relation between maternal corticosterone levels and palatal development. Another factor that may interact with maternal corticosterone levels in determining whether palatal development is normal or abnormal is the genetic susceptibility of the fetus to cleft palate induction (Smithberg, '67). Brown and coworkers ('72) suggested that this interaction may explain the extreme sensitivity of a purebred strain such as A/J mice to stress-induced cleft palate since they have embryos that are very sensitive to cortisone-induced cleft palate and also have high corticosterone levels following stress. In the present
experiments, randombred mice with variation in genotype were used, and the interaction of resistant and susceptible fetal genotypes with maternal corticosterone levels may explain the variability of the teratogenic effects of the stressors observed. Other factors that have been shown to affect frequency of cleft palate are maternal and fetal weight (Kalter, '56, '67), uterine crowding (Loevy, '63), and uterine position (Trader, '60), all of which might be expected to contribute to between- and within-litter variation in the response to stressors. The overall conclusion from the present experiments however is clear. In some mice during restraint plasma corticosterone can rise to levels similar to those seen after the injection of slightly teratogenic doses of corticosterone. It is thus reasonable to assume that in these animals the teratogenic action of restraint may be mediated via the release of endogenous corticosterone. But in mice subjected to food deprivation, other factors may play a major role. ACKNOWLEDGMENTS
We are grateful to K. Brame, J. Cannon, D. Carman, M. Lilley, and S. West who assisted with some of these experiments, and to the Foundation for Child Development (New York) for financial support. LITERATURE CITED Allen, J. P., D. M . Cook, J. W . Kendall and R. McGilvra 1973 Maternal-fetal ACTH relationship in man. J. Clin. Endocr., 37: 230-234. Barlow, S. M. 1972 Ph.D. Thesis, Univ. of London. Barlow, S . M., P. J. Morrison and F. M. Sullivan 1974 Plasma corticosterone levels during pregnancy in the mouse: the relative contributions of the adrenal glands and foeto-placental units. J. Endocr., 60: 4 7 3 4 8 3 . ___ 1975 Effects of acute and chronic stress on plasma corticosterone levels in the pregnant and non-pregnant mouse. J. Endocr., 66: 9399. Barlow, S . M . , and F. M . Sullivan 1973 Effects of maternal stress on prenatal and postnatal development. 4th International Congress on Birth Defects, Vienna. A. G. Motulsky and F. J. G . Ebling, eds. Excerpta Medica, Amsterdam, p. 86 (abstract). Blaustein, F. M., R. Feller and S. Rosenzweig 1971 Effect of ACTH and adrenal hormones on cleft palate frequency in CD-1 mice. J. Dent. Res., 50: 609-612. Brain, P. F., and N. W . Nowell 1970 Adrenal
STRESS, CORTICOSTERONE, AND CLEFT PALATE IN MICE function in pregnant and lactating mice. J . Endocr., 48: xvii-xviii (abstract). Brown, K. S., M. C . Johnston and J. D. Niswander 1972 Isolated cleft palate in mice after transportation during gestation. Teratology, 5 : 119124. Dokumov, S . I., S . C. Milanov and S. P. Trepetshov 1974 Adrenocorticotrophic hormones in plasma of mother and newborn. J. Obs. Gyn. Br. Comm., 81: 220-221. DostBl, M. 1971 Morphogenesis of cleft palate induced by exogenous factors. 111. Intraamniotic application of hydrocortisone in mice. Teratology, 4 : 63-68. Eguchi, Y., 0. Hirai, Y. Morikawa and Y. Hashimoto 1973 Critical time in the hypothalamic control of the pituitary-adrenal system in fetal rats: observations in fetuses subjected to hypervitaminosis A and hypothalamic destruction. Endocrinology, 93: 1-1 1 . Fraser, F. C., and T. D. Fainstat 1951 Production of congenital defects in the offspring of pregnant mice treated with cortisone. Pediatrics, 8: 527-533. Glick, D., D. von Redlich and S. Levine 1964 Fluorometric determination ofcorticosterone and cortisol in 0.02-0.05 milliliters of plasma or submilligram samples of adrenal tissue. Endocrinology, 7 4 : 65-55, Harris, J . W. S. 1964 Oligohydraminos and cortisone-induced cleft palate. Nature, 203: 533-534. Heiberg, K., H. Kalter and F. C. Fraser 1959 Production of cleft palate in the offspring of mice treated with ACTH during pregnancy. Biol. Neonat., 1 : 33-37. Kalter, H. 1956 Modification of teratogenic action of cortisone i n mice by maternal age, maternal weight and litter size. Am. J. Physiol., 185: 65-68. - 1957 Factors influencing the frequency of cortisone-induced cleft palate in mice. J. Exp. Zool., 134: 4 4 9 4 6 8 . __ 1960 Teratogenic action of a hypocaloric diet and small doses of cortisone. Proc. SOC.Exp. Biol. Med., 104: 518-520.
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1962 ACTH, congenital malformations and “stress.” Ann. Zool., 2 : 419 (abstract). Kalter, H., and F. C. Fraser 1952 Production or congenital defects in the offspring of pregnant mice treated with compound F. Nature, 169: 665. Kendall, M. G., and S. Stuart 1967 Analysis of variance of normal scores. Ch. 31. In: The Advanced Theory of Statistics, Vol. 2. Charles Griffin, London. Larsson, K. S. 1962 Studies on the closure of the secondary palate. IV. Autoradiographic and histochemical studies of mouse embryos from cortisone-treated mothers. Acta Morph. Neerl. Scand., 4: 3 6 9 3 7 5 . Loevy, H. 1963 Genetic influences o n induced cleft palate in different strains of mice. Anat. Rec., 145: 117-122, Miller, T. J . 1973 Cleft palate formation: the effects of fasting and iodoacetic acid in mice. Teratology, 7: 177-181. Peters, V. S . , and M. Strassburg 1969 Stress als teratogener Faktor. Arzneimittelforschung, 19: 1 1 0 6 1 111. Renaud, S. 1959 Improved restraint technique for producing stress and cardiac necrosis in rats. J. Appl. Physiol., 1 4 : 868-869. Rosenzweig, S. 1966 Psychological stress and cleft palate etiology. J. Dent. Res., 45: 1 5 8 5 1593. 1969 Cleft palate in A/Jax mice: synergistic effect of restraint fast and cortisone. Int. Ass. Dent. Res., General Meeting No. 151, p. 78 (abstract). Rosenzweig, S., and F. M. Blaustein 1970 Cleft palate in A/J mice resulting from restraint and deprivation of food and water. Teratology, 3: 4 7-52. Smithberg, M. 1967 Teratogenesis in inbred strains of mice. Adv. Terat., 2: 257-288. Trader, D. G. 1960 Influence of uterine site on occurrence of spontaneous cleft lip in mice. Science, 132: 4 2 0 4 2 1 . Velardo, J. T. 1957 Action of adrencorticotrophin on pregnancy and litter size in rats. Am. J. Physiol., 191 : 319-322.
BARLOW, PATRICIA R. McELHATTON A N D SULLIVAN of Phurmacology, Guy's Hospitul Medicul School,
9RT
ABSTRACT The blood level of corticosterone was measured in mice following the injection on day 14 of pregnancy of a dose of corticosterone sufficient to cause a low frequency of cleft palate in the fetuses. This was compared with the blood levels present during maternal restraint and food deprivation that produced a similar frequency of cleft palate. The mean blood level over the 24 h following injection of corticosterone was 660 @lo0 ml, and during a similar period of restraint was 485 g g / l O O ml. Other mice were subjected either to restraint or food deprivation for 24 h beginning day 14 of pregnancy, the plasma corticosterone levels measured during that time, and the frequency of cleft palate in late fetuses compared with the individual plasma corticosterone levels during treatment. There was a significant (P < 0.025) correlation between high maternal corticosteroid levels and the frequency of cleft palate in the offspring of the restrained mice but not in the food-deprived animals. I t is suggested that in some stressed mice endogenous plasma corticosterone can reach levels sufficient to account for the development of cleft palate.
In some strains of mice injection of pregnant females with cortisone (Fraser and Fainstat, '51), cortisol (Kalter and Fraser, '52), or corticosterone (Blaustein et al., '71) during the sensitive period causes cleft palate in the offspring. Injection of adrenocorticotrophic hormone (ACTH) also caused cleft palate (Heiberg et al., '59; Kalter, '62; Blaustein et al., '71). It is unlikely that ACTH can cross the placenta (Allen et al., '73; Dokumov et al., '74; Eguchi et al., '73) and act directly on the fetus. Heiberg and coworkers ('59) suggested that pregnant mice may produce teratogenic amounts of corticosteroids in response to ACTH, and Velardo ('57) showed that the embryolethal effect of ACTH given in early pregnancy in rats is definitely mediated via the maternal adrenal glands rather than by a direct action on the embryo. "Stress" is difficult to define since it is essentially a subjective phenomenon. However it is conventionally considered to have occurred in animal experiments if there is a raised plasma corticosteroid level following the application of a "stressor." Many such stressors including restraint (Rosenzweig and Blaustein, '70), food or water TERATOLOGY, 12: 97-104.
deprivation (Kalter, '60; Peters and Strassburg, '69; Rosenzweig and Blaustein, '70; Miller, '73), avoidance conditioning (Rosenzweig, '66), and transportation (Brown et al., '72) induce cleft palate in mice. Restraint stress was also synergistic with cortisone in inducing cleft palate (Rosenzweig, '69). It is known that during stress in pregnant mice, plasma corticosterone reaches very high levels, around 400 times greater than in resting nonpregnant mice (Brain and Nowell, '70; Barlow et al., '74). It would be interesting to know whether the plasma corticosterone levels reached during stress in pregnant mice would be safficient by themselves to account for the development of cleft palate. We decided to test this by measuring the plasma levels of corticosterone reached following the injection of a dose of corticosterone known to produce cleft palate. Other mice were subjected to stress and the plasma corticosterone levels measured to see whether they were similar to those observed following corticosterone injection. Finally, both the corticosterone levels during stress and Received May 20, '75. Accepted June 20, '75
97
98
S. M. BARLOW, P. R. McELHATTON A N D F. M. SULLIVAN
the frequency of cleft palate were measured in the same mice to see if these were correlated. MATERIALS A N D METHODS
Animals Mice derived from the pathogen-free ICI randombred stock, bred under conventional conditions in the animal house at Guy's Hospital Medical School were used. Animal rooms were maintained at 21 rt_ 1 "C, humidity 5&60%, with 10 air changes per hour, under controlled lighting conditions with 14 h of light and 10 h of darkness. The diet used was FFG(M), Dixons. Males 12 weeks of age or over were placed overnight with virgin females 10 weeks of age, averaging 25 g body weight, and vaginal plugs looked for next morning. The day of finding the plug was designated day 1 of pregnancy, and pregnant females were removed from the mating cages and rehoused in groups of 4 or 5 until required for experiment. Injections To measure the frequency of cleft palate following corticosterone injection mice on day 14 of pregnancy were injected sc in the scruff of the neck with 1.25, 2.5, 5.0, or 10.0 mg (equivalent to 50, 100, 200, or 400 mg/kg) of corticosterone (Organon, England) suspended in arachis oil. The injection volume was standardized to 0.1 ml. Control mice were injected with arachis oil alone. In the study of the plasma levels of corticosterone following injection, mice on day 14 of pregnancy were injected with 2.5 mg (100 mg/kg) corticosterone and killed 0, 0.5, 1, 2, 5, 10, and 24 h later, 6 mice being lulled at 0 h and 3 mice at each of the other times. Stressor techniques Restraint. Mice were immobilized for 24 h from 10 AM on day 14 to 10 A M on day 15 of pregnancy using a method described by Renaud ('59). The mice were strapped down by their limbs in a prone position, onto a special board, using zinc oxide plaster, in such a manner that their paws were undamaged. Animals were observed to drink but do not normally eat while restrained in this way, so water but no food was provided during the period of
immobilization. The temperature of the room in which the animals were kept was 21 "C. To measure the frequency of cleft palate following restraint 16 mice were used. In the study of the plasma levels of corticosterone following restraint mice were killed 0, 0.3, 1, 2, 4, 8, 12, 17, and 24 h after the commencement of restraint, 8 mice being killed at each time. In the experiments relating the plasma corticosterone levels during stress with the subsequent presence of cleft palate, 20 mice were restrained and blood samples taken 7 and 24 h after commencement of restraint. After 24 h of restraint the mice were returned to the animal house until day 19 of pregnancy when they were killed and the offspring examined for palatal defects. Food deprivation. Mice were kept singly in cages at an ambient temperature of 21 "C. Water was provided and they were free to move about within their cages but, like the immobilized mice, they were without food for 24 h and unable to huddle for warmth. I t was known from previous experiments (Barlow, '72) that body temperature may drop by up to 2 "C under these conditions. Collection of blood In nonrecovery experiments mice were killed by cervical dislocation and 0.5 ml of blood taken by cardiac puncture using a heparinized syringe. In recovery experiments, where mice were allowed to continue until term so that the fetuses could be examined, 0.25 ml of blood was taken from the retroorbital sinus using a heparinized syringe and a plastic cannula during 2 min of ether anesthesia. The blood was immediately centrifuged and duplicate 100 or 50 pl aliquots of plasma taken for assay. Corticosterone assay Plasma corticosterone levels were measured using the microfluorimetric technique of Glick et al. ('64). The precision of the method was estimated as +-1ng/100 pI of plasma. All corticosterone values are expressed as pg +- SE/100 ml of plasma. Ter ato logy Mice were killed on day 19 of pregnancy and the contents of the uterus examined. The number of live and full-term dead fe-
99
STRESS, CORTICOSTERONE, AND CLEFT PALATE IN MICE TABLE 1
wfects on fetul development of corticosterone injection on day 14 of pregnuncy Corticosterone dose
No. of litters
Live fetuses
Resorbing and dead fetuses
Litters with cleft palate
Live fetuses with cleft palate (%)
mg
0 1.25 3 2.5 5.0 10.0
22 14 10 11
5
230 156 1
iig
2
132 53
27 21 15 4 8
0 1 3 10 4
0 (0.0) 1 10.6) 3 i2.5j 43 (32.3) 16 (30.2)
Included 1 fetus with exencephaly. Included 1 fetus with kyphosis and 1 fetus with fused ribs. 3 Equivalent to 50, 100, 200, and 400 mglkg. 1
2
tuses (no signs of maceration), late resorptions (maceration, death occurring at the fetal stage), and early resorptions (death occurring at the embryonic stage) were recorded. Fetuses were removed and examined for gross external morphological defects, dissected to check for internal defects, then stained with alizarin red S and inspected for skeletal defects. Particular attention was paid to the formation of the pal ate.
Statistical analysis Differences in malformation rates, as assessed on a per fetus basis, were analyzed by the Fisher two-tailed exact probability test unless otherwise stated. Differences assessed on a per litter basis were analyzed by analysis of variance of normal scores (Kendall and Stuart, '67), which implies no assumption as to the normality of the data. RESULTS
of data collected from untreated controls over a 3-year period, the total number of fetuses examined being 988. Other abnormalities observed were exencephaly (l), kyphosis (l), fused ribs (l), and poor ossification of the sternum (10-12% of all fetuses in treated groups, 2% in vehicle controls), but these abnormalities were not dose-related. Corticosterone injections caused no increase in the frequency of resorptions and had no effect on fetal body weight . Stressors. The frequency of cleft palate due to 24 h of maternal restraint or food deprivation beginning on day 14 was determined. Controls were left undisturbed in the animal house for the whole duration of pregnancy until day 19 when they were killed and the offspring examined for palatal defects. The results are shown in table 2 . There was a significant increase in the frequency of cleft palate in the offspring of both restrained ( P = 0.04) and food-deprived (P = 0.002) animals in comparison with untreated controls. The difference between restraint and food deprivation was not significant (P = 0.42).
Comparison of frequency of cleft palate following corticosterone injection and stressors Corticosterone injections. Preliminary Comparison of plasma corticosterone experiments established that day 14 of levels following corticosterone pregnancy was the most sensitive day for injections and restraint induction of cleft palate in the stock used. Mice were injected with various doses of Corticosterone injection. Maternal corticosterone on this day, and the results plasma corticosterone levels were measured are shown in table 1. over a period of 24 h following the injecThe frequency of the defect was dose- tion on day 14 of pregnancy of 2.5 mg related, reaching a plateau of around 30% (100 mg/kg) of corticosterone, a dose that at 5.0 mg (200 mg/kg). Although no cleft produced a small but significant ( P = palate was observed in the controls the 0.039, single-tailed) increase in the frespontaneous frequency of cleft palate in quency of cleft palate in the offspring apthis stock of mouse is 0.2%, a malforma- proximately equal to that caused by the tion rate calculated from the pooled results stressors, i.e., 2.5 and 5% respectively.
100
S. M. BARLOW, P. R. McELHATTON AND F. M. SULLIVAN TABLE 2 wfects on fetal development
of 24 h of maternal treatment beginning o n day 14 of pregnancy No. of
Maternal tcea tmen t
Resorbing and dead fetuses
Litters with cleft palate
Fetuses with cleft palate
148 305
39
107
21
3 9 0
7 (5) 22 (7) 0 (0)
Live fetuses
litters
(%I Restraint Food deprivation Untreated control
16 31 12
1000
-
500
-
100
-
50
8
1
1
I
L
0
4
8
1
12
16
20
24
Hours Fig. 1 Plasma corticosterone levels following subcutaneous injection of 2.5 mg (100 mg/kg) of corticosterone on day 14 of pregnancy (00 ) or during 24 h of restraint begun on - - -0). Vertical bars indicate SE. day 14 of pregnancy (0-
The results are shown in figure 1 . Mice killed at 0 h had a mean plasma corticosterone level of 542 k 42 pg/ml, reflecting the stress of being taken from the animal house to the laboratory half an hour before blood sampling (see Barlow et d.,'75). Following the injection of corticosterone, plasma levels rose to a peak of 1208 +- 57 pg/100 ml at 2 h, were maintained at around 800 p g / l O O ml up to at least 10 h after injection, but by 24 h had fallen to 95 k 5 pg/100 ml, which is a normal resting level for day 15 of pregnancy (Barlow et al., '74). Restraint. Maternal plasma corticosterone levels were measured at various intervals during 24 h of restraint beginning on day 14 of pregnancy. The results are shown in figure 1. Levels increased from a mean resting level of 79.9 6.3
*
pg/100 ml at 0 h, these mice being killed in the animal house, to reach a maximum of 733 t 56 pg/ml 1 h after the commencement of restraint. Thereafter levels were maintained at 500-600 p g / l O O ml plasma during the next 16 h of restraint. By 24 h levels had fallen to 268 ? 44 pg1100 ml but were still elevated in comparison with nonstressed pregnant controls on day 15 of pregnancy. It should be noted however that at any given time during the 24 h of restraint there was a wide variation between individuals. For example, at 17 h individual values ranged from 2 7 6 7 0 0 pg/lOO ml. The plasma corticosterone levels following restraint or corticosterone injection (fig. 1) were compared and gave a mean value of 485 p g / l O O ml of plasma during restraint and 660 pg/100 ml of plasma following corticosterone injection.
101
STRESS, CORTICOSTERONE, AND CLEFT PALATE IN MICE
The ratio of the area under the restraint curve to the area under the injection curve was 0.75.
Correlation between plasma corticosterone leuels during application of stressors and cleft palate In the above-described experiments animals were killed to obtain the plasma for corticosterone assays, and therefore other animals were used to ascertain the fiequency of cleft palate following application of stressors or injection of corticosterone. It was decided to determine whether in individual mice there was any correlation between plasma corticosterone levels during stressing and subsequent development of cleft palate. Mice were subjected to 24 h of restraint or food deprivation on day 14 of pregnancy, blood samples were taken at 7 h and 24 h, and the offspring examined at term. The plasma corticosterone levels at 7 h and the presence or absence of cleft palate in the litters subsequently are shown in figure 2. There were 8 fetuses with cleft palate in 5 of the 20 litters in the restrained group and 29 fetuses with cleft palate in 12 of the 35 litters in the food-deprived group. In the restrained group there was a significant correlation between high maternal plasma corticosterone levels at 7 h and the presence of cleft palate in the offspring as assessed on a litter basis ( P < 0.025, analysis of varianceof normal scores, Kendall and Stuart, '67). Values at 24 h are not shown but there was also a significant correlation at that time (P < 0.025). In the food-deprived group, however, there was no significant correlation between maternal plasma corticosterone levels and palatal defects in the offspring at either 7 or 24 h (P < 0.35). There was no correlation between the number of affected fetuses per litter and maternal plasma corticosterone levels. DISCUSSION
Considering first maternal plasma corticosterone levels the present investigations showed that the levels found during restraint on day 14 of pregnancy were of a similar order of magnitude to those observed following injection of 2.5 mg of corticosterone, i.e., a mean of 485 p g / l O O ml and 660 pg/lOO ml, respectively. Although
a
8 0
8
0
a
b eo 0
0
0 0
9,
8J ?5
oa
%
0
8 8
Rest r a i n t
Food deprivation
Fig. 2 Relation between maternal plasma corticosterone levels at 7 h during 24 h of restraint or food deprivation begun on day 14 of pregnancy and the development of cleft palate in the offspring. Litters with cleft palate (a),normal litters (0).
the peak levels reached in the restrained group were lower than in the injected group the levels in the former remained elevated above control values for a longer period of time than in the latter. The frequency of cleft palate was similar in the 2 groups of mice. Thus it would appear that restraint may cause sufficient corticosterone to be released to cause a low frequency of cleft palate in these mice. To test this hypothesis more rigorously both maternal plasma corticosterone levels and the frequency of cleft palate in off-
102
S. M. BARLOW, P. R. McELHATTON AND F. M. SULLIVAN
spring were investigated in the same individuals subjected to stressors. In the restrained group there was a significant correlation between these 2 phenomena. Thus it seems that in some mice during restraint plasma corticosterone levels were high enough to account for the presence of cleft palate in the offspring. In the food-deprived group, however, some mice with relatively low plasma corticosterone levels during deprivation also had offspring with cleft palate. So in these instances some other mechanisms are presumably involved in the production of cleft palate. There remains some debate about the mode of action of corticosteroids as teratogens. They may have a direct action on the fetus (Larsson, '62; Dostal, '71) or may act indirectly, e.g., by reducing amnioticfluid volume (Harris, '64). If the action is a direct one, measurement of maternal plasma corticosterone levels may not accurately reflect the amount of corticosterone reaching the fetus, since placental transfer of the hormone is also influenced by factors other than concentration differences, e.g., by changes in placental blood flow. In some, but not all, mice during restraint pooling of blood in the placenta has been observed, accompanied by a reduction in placental transfer function (Barlow and Sullivan, '73). Presumably the placental transfer of corticosterone would also be reduced under such conditions. Thus, in some stressed mice with high maternal corticosterone levels fetal levels could be relatively low. In other mice, with lower maternal corticosterone levels but unimpaired placental transfer function, fetal levels might be relatively high, resulting in an unpredictable relation between maternal corticosterone levels and palatal development. Another factor that may interact with maternal corticosterone levels in determining whether palatal development is normal or abnormal is the genetic susceptibility of the fetus to cleft palate induction (Smithberg, '67). Brown and coworkers ('72) suggested that this interaction may explain the extreme sensitivity of a purebred strain such as A/J mice to stress-induced cleft palate since they have embryos that are very sensitive to cortisone-induced cleft palate and also have high corticosterone levels following stress. In the present
experiments, randombred mice with variation in genotype were used, and the interaction of resistant and susceptible fetal genotypes with maternal corticosterone levels may explain the variability of the teratogenic effects of the stressors observed. Other factors that have been shown to affect frequency of cleft palate are maternal and fetal weight (Kalter, '56, '67), uterine crowding (Loevy, '63), and uterine position (Trader, '60), all of which might be expected to contribute to between- and within-litter variation in the response to stressors. The overall conclusion from the present experiments however is clear. In some mice during restraint plasma corticosterone can rise to levels similar to those seen after the injection of slightly teratogenic doses of corticosterone. It is thus reasonable to assume that in these animals the teratogenic action of restraint may be mediated via the release of endogenous corticosterone. But in mice subjected to food deprivation, other factors may play a major role. ACKNOWLEDGMENTS
We are grateful to K. Brame, J. Cannon, D. Carman, M. Lilley, and S. West who assisted with some of these experiments, and to the Foundation for Child Development (New York) for financial support. LITERATURE CITED Allen, J. P., D. M . Cook, J. W . Kendall and R. McGilvra 1973 Maternal-fetal ACTH relationship in man. J. Clin. Endocr., 37: 230-234. Barlow, S. M. 1972 Ph.D. Thesis, Univ. of London. Barlow, S . M., P. J. Morrison and F. M. Sullivan 1974 Plasma corticosterone levels during pregnancy in the mouse: the relative contributions of the adrenal glands and foeto-placental units. J. Endocr., 60: 4 7 3 4 8 3 . ___ 1975 Effects of acute and chronic stress on plasma corticosterone levels in the pregnant and non-pregnant mouse. J. Endocr., 66: 9399. Barlow, S . M . , and F. M . Sullivan 1973 Effects of maternal stress on prenatal and postnatal development. 4th International Congress on Birth Defects, Vienna. A. G. Motulsky and F. J. G . Ebling, eds. Excerpta Medica, Amsterdam, p. 86 (abstract). Blaustein, F. M., R. Feller and S. Rosenzweig 1971 Effect of ACTH and adrenal hormones on cleft palate frequency in CD-1 mice. J. Dent. Res., 50: 609-612. Brain, P. F., and N. W . Nowell 1970 Adrenal
STRESS, CORTICOSTERONE, AND CLEFT PALATE IN MICE function in pregnant and lactating mice. J . Endocr., 48: xvii-xviii (abstract). Brown, K. S., M. C . Johnston and J. D. Niswander 1972 Isolated cleft palate in mice after transportation during gestation. Teratology, 5 : 119124. Dokumov, S . I., S . C. Milanov and S. P. Trepetshov 1974 Adrenocorticotrophic hormones in plasma of mother and newborn. J. Obs. Gyn. Br. Comm., 81: 220-221. DostBl, M. 1971 Morphogenesis of cleft palate induced by exogenous factors. 111. Intraamniotic application of hydrocortisone in mice. Teratology, 4 : 63-68. Eguchi, Y., 0. Hirai, Y. Morikawa and Y. Hashimoto 1973 Critical time in the hypothalamic control of the pituitary-adrenal system in fetal rats: observations in fetuses subjected to hypervitaminosis A and hypothalamic destruction. Endocrinology, 93: 1-1 1 . Fraser, F. C., and T. D. Fainstat 1951 Production of congenital defects in the offspring of pregnant mice treated with cortisone. Pediatrics, 8: 527-533. Glick, D., D. von Redlich and S. Levine 1964 Fluorometric determination ofcorticosterone and cortisol in 0.02-0.05 milliliters of plasma or submilligram samples of adrenal tissue. Endocrinology, 7 4 : 65-55, Harris, J . W. S. 1964 Oligohydraminos and cortisone-induced cleft palate. Nature, 203: 533-534. Heiberg, K., H. Kalter and F. C. Fraser 1959 Production of cleft palate in the offspring of mice treated with ACTH during pregnancy. Biol. Neonat., 1 : 33-37. Kalter, H. 1956 Modification of teratogenic action of cortisone i n mice by maternal age, maternal weight and litter size. Am. J. Physiol., 185: 65-68. - 1957 Factors influencing the frequency of cortisone-induced cleft palate in mice. J. Exp. Zool., 134: 4 4 9 4 6 8 . __ 1960 Teratogenic action of a hypocaloric diet and small doses of cortisone. Proc. SOC.Exp. Biol. Med., 104: 518-520.
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1962 ACTH, congenital malformations and “stress.” Ann. Zool., 2 : 419 (abstract). Kalter, H., and F. C. Fraser 1952 Production or congenital defects in the offspring of pregnant mice treated with compound F. Nature, 169: 665. Kendall, M. G., and S. Stuart 1967 Analysis of variance of normal scores. Ch. 31. In: The Advanced Theory of Statistics, Vol. 2. Charles Griffin, London. Larsson, K. S. 1962 Studies on the closure of the secondary palate. IV. Autoradiographic and histochemical studies of mouse embryos from cortisone-treated mothers. Acta Morph. Neerl. Scand., 4: 3 6 9 3 7 5 . Loevy, H. 1963 Genetic influences o n induced cleft palate in different strains of mice. Anat. Rec., 145: 117-122, Miller, T. J . 1973 Cleft palate formation: the effects of fasting and iodoacetic acid in mice. Teratology, 7: 177-181. Peters, V. S . , and M. Strassburg 1969 Stress als teratogener Faktor. Arzneimittelforschung, 19: 1 1 0 6 1 111. Renaud, S. 1959 Improved restraint technique for producing stress and cardiac necrosis in rats. J. Appl. Physiol., 1 4 : 868-869. Rosenzweig, S. 1966 Psychological stress and cleft palate etiology. J. Dent. Res., 45: 1 5 8 5 1593. 1969 Cleft palate in A/Jax mice: synergistic effect of restraint fast and cortisone. Int. Ass. Dent. Res., General Meeting No. 151, p. 78 (abstract). Rosenzweig, S., and F. M. Blaustein 1970 Cleft palate in A/J mice resulting from restraint and deprivation of food and water. Teratology, 3: 4 7-52. Smithberg, M. 1967 Teratogenesis in inbred strains of mice. Adv. Terat., 2: 257-288. Trader, D. G. 1960 Influence of uterine site on occurrence of spontaneous cleft lip in mice. Science, 132: 4 2 0 4 2 1 . Velardo, J. T. 1957 Action of adrencorticotrophin on pregnancy and litter size in rats. Am. J. Physiol., 191 : 319-322.
