CHRISTOPHER LAU,* GARY KUNEFELTER,* AND ANNIE M. CAMERON! * Reproductive Toxicology Division, National Health and Environmental Effects ...
FUNDAMENTAL AND APPLIED TOXICOLOGY 3 0 , 2 9 8 - 3 0 1 (1996) ARTICLE NO. 0 0 6 8
Reproductive Development and Functions in the Rat after Repeated Maternal Deprivation Stress1 CHRISTOPHER LAU,* GARY KUNEFELTER,* AND ANNIE M. CAMERON! * Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711; and tMantech Environmental Technology, Inc., Research Triangle Park, North Carolina 27709 Received October 6, 1995; accepted November 21, 1995
Reproductive Development and Functions in the Rat after Repeated Maternal Deprivation Stress. LAU, C, KUNEFELTER, G., AND CAMERON, A. M. (1996). Fundam. Appl. Toxicol. 30, 298-301.
Should Still be Considered.
© 1996 Society of Toxicology
Growth and development in mammals are intimately dependent upon support and stimuli from the nursing dam. When these maternal inputs are interrupted, biochemical and physiological stress responses are evoked acutely in the neonates, and altered behavioral development and maturation of organ function have been reported (Schanberg et al, 1984; Hofer, 1984; Domer et al, 1984; Laudenslager et al, 1985; Tonjes et al, 1986; Shimoda et al, 1986; Stanton et al, 1988; Vesell et al, 1989; Kuhn et al, 1990). These findings are of particular significance in applied research such as toxicology. For animal studies designed to assess the potential risks of chemicals to human health, dermal and inhalation exposure paradigms have been regarded as more realistic and appropriate than oral gavage. However, when the toxicity studies are extended to encompass postnatal development, these experimental procedures
METHODS Timed pregnant Sprague-Dawley rats from Charles River Laboratory (Raleigh, NC) were housed individually in breeding cages with food and water available ad libitum. All pups were randomized at birth and redistributed to the nursing dams with litter sizes kept at 12 pups. On postnatal Day 4, rats were divided into two groups. One group of neonates was removed from their nursing dams for 6 hr and placed in a "Servo-Care" isolation incubator (Ohio Medical Products, Madison. WI). This incubator is equipped with air circulation, a humidifier, handling ports, and a heating device with a thermostat so that temperature was maintained at 33°C. Temperature of the incubator was lowered to 29°C on Day 10 and to 27°C on Day 15 to avoid potential heat stress. Pups stayed with their own littermates in small plastic compartments and were segregated from those of other litters to minimize novel contact. At the end of the session, pups were returned to their respective dams who resumed the normal nursing and feeding behaviors within minutes. The other group of pups stayed with their dams without disturbance (except periodic handling during body weight measurements) throughout the entire study and served as control. The deprivation session was repeated daily at the same time of day until Day 21. Rats from both groups were weaned on Day 22, segregated by sex, and housed three to four per cage. Several male and female rats from every
' This paper has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the U.S. Environmental Protection Agency, and mention of trade names of commercial products does not constitute endorsement or recommendation for use. 0272-0590/96 $18.00 Copyright © 1996 by the Society of Toxicology. All nghts of reproduction in any form reserved.
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The present study examines whether interruptions of maternalneonatal behaviors may alter the maturation of reproductive organs and functions, thereby potentially confounding the interpretation of toxicity data with stress-induced responses. SpragueDawley rat neonates were removed from their dams for 6 hr daily beginning on Day 4 of postnatal age and continuing until Day 21. Vaginal opening and preputial separation were monitored as indices of puberty. Sperm production in the male rats, estrous cycles in the female rats, and reproductive tissues were weighed when the animals reached sexual maturity. In addition, rats were mated for the evaluation of pregnancy outcomes. Maternal separation stress in itself did not appear to alter these parameters, although potential interactions of stress responses with a toxicant
invariably require intermittent separation of the pups from their nursing dams. It is thus of considerable concern whether such maternal-neonatal disruptions might adversely affect growth and development of the animals, thereby introducing a confounding factor to the interpretation of toxicity data (Kimmel and Francis, 1990). Previous studies from our laboratories have shown that maternal separation during preweaning periods elicited stress responses, although brain growth and neuro-behavioral development were not affected (Lau et al, 1992; Stanton et al, 1992). The current study extends the investigation to the reproductive system. While prenatal stresses are known to cause reproductive dysfunction and aberrant sexual behaviors leading to reduced fertility (Herrenkohl, 1979; 1986; Pollard, 1984; Wiebold et al, 1986; Anderson et al, 1986; Kinsley and Bridges, 1988; Kinsley et al, 1989; Gutierrez et al, 1989), little information is available on the reproductive effects of neonatal stress. We therefore examined the effects of maternal deprivation stress on pubertal development of both male and female rats as well as their reproductive capability at the sexually mature stage.
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REPRODUCTIVE DEVELOPMENT AFTER MATERNAL DEPRIVATION litter within each treatment group were selected randomly and their body weights were recorded at intervals of several days. Female rats were monitored for vaginal opening from Day 24 to Day 40 and males rats for preputial separation from Day 35 to Day 48. In addition, estrous cycles of the female rats were monitored from Day 74 to Day 90 according to the method of Cooper el al. (1993). When the animals reached 90 days of age, some from each sex and treatment group were terminated, and their reproductive organs, liver, and kidney were weighed. Homogenization-resistant testicular spermatids and epididymal sperm counts were assessed in the male rats according to the method described by Robb et al. (1978). Cauda epididymal sperm counts were determined as a measure of sperm storage. In addition, the daily sperm production rate (number of sperm produced per gram of testis per day) was derived from the number of homogenization-resistant testicular spermatids per gram of testicular parenchyma, divided by a time factor representing the duration of spermatogenic cycle of these spermatids (6.1 days for the rat). An estimate of transit time through the cauda epididymis was obtained by dividing the number of sperm in the cauda by the daily sperm production rate. The remaining rats were mated within each treatment group for 10 days as a test for fertility. Pregnant rats were allowed to give birth. Litter size, number of implantations, and body weights of F2 pups at weaning were evaluated.
• Control 3 Deprived
.1
luii
29 30 31 32 33 34 35 36 37 38 39 AGE (days)
RESULTS AND DISCUSSION
TABLE 1 Effects of Maternal Deprivation on Body Weight Gains Male Age (days) 4 7 10 14 17 21 25 28 32 39 46 54 60 67 74 81 90
Female
Control (g)
Deprived (g)
Control (g)
Deprived (g)
9.6 16.4 19.5 27.8 34.3 46.9 68.5 89.8 123 189 258 324 378 421 450 478 493
9.7 ± 14.2 i 18.2 ; 25.8 j 33.8 j 46.1 i 67.0 i 87.8 j 122 j 184 j 254 H 327 H 374 ± 409 ± 455 ± 485 ± 497 ±
8.8 ± 15.6 ± 18.5 ± 27.1 ± 35.1 ± 47.1 ± 66.0 ± 84.2 ± 112 ± 162 ± 196 ± 223 ± 248 ± 264 ± 272 ± 288 ± 291 ±
8.9 ± 14.5 ± 17.3 : 26.0: 32.8 : 43.6 : 62.6 : 83.0: 109 : 157 : 191 : 223 : 245 ± 263 ± 274 ± 287 ± 292 ±
:t 0.3 :t 0.4 :t0.4 :t 0.7 :t 1.1 :t 1.4 :t 2.1 :t 2.9 :t 3 :t 3 :t 4 :t 5 ±4 ±6 ±6 ±7 ±7
0.3 0.4 0.5 0.8 1.3 1.2 2.2 2.5 3 4 5 5 5 7 6 7 8
0.3 0.5 0.4 0.5 0.9 1.3 1.7 1.9 2 2 3 4 3 4 4 5 5
0.2 0.3 0.5 0.8 1.6 1.4 2.8 2.9 2 3 3 3 5 5 5 5 6
Note. Values represent means and standard errors of 12 or more animals from 10 litters for each treatment group. Three-way ANOVA indicates age, sex, and treatment effects (p < 0.001) but no interaction.
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40
41
42
43
44
45
46
AGE (days)
FIG. 1. Effects of maternal deprivation on reproductive pubertal indices: vaginal opening (top) and preputial separation (bottom). Bars represent the number of animals when these landmarks were observed. ANOVA does not indicate any treatment effect in either of these parameters.
during the preweaning period when the body weight gains revealed a subtle (ranging from 3 to 7%), but statistically significant lag (Table 1); however, even these small deficits were no longer apparent when the rats reached young adulthood. One of the most difficult tasks in risk assessment is to ascertain the "biological" significance of small deviations from a control population. For instance, does the small lag of growth evinced by deprivation stress reflect physiological deficiencies in the developing rat? More importantly, would stressful manipulations (with the associated elevations of circulating corticosteroid hormone) during preweaning periods influence maturation of the reproductive system? Data from the present study suggest that repeated maternal deprivation did not affect the pubertal indices for either female or male rats as the mean ages for vaginal opening and preputial separation were 34.2 and 41.6 days, respectively, for controls and 34.4 and 41.8 days for deprived rats (Fig. 1). Correspondingly, little difference was seen in the reproductive organ weights between control and stressed animals when they reached adulthood (Table 2). Liver and kidney weights were also similar between the two groups. The sole exception is testicular weight, which is about 8% lower in
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In a previous report, separation of preweaning rats from their nursing dams for 6 hr has been shown to produce an acute elevation of circulating corticosterone and a reduction of liver weight, though the latter deficit was quickly recovered upon refeeding (Lau et al., 1992). When the episode of maternal deprivation was repeated daily in the current study, a cumulative effect of stress on growth rate was noted
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TABLE 2 Organ Weights and Sperm Evaluation of Male Rats, and Organ Weights of Female Rats that Have Been Stressed Daily by Maternal Deprivation during the Preweaning Period Male Control Pituitary (mg) Adrenal glands (mg) Epididymis (g) Testis (g) Seminal vesicle (g) Epididymal sperm (million/cauda) Testicular sperm (million/testis) Daily testicular sperm production (million/testis) Sperm transit time through epididymis (day) Ovary (mg) Uterus (mg) Kidney (g) Liver (g)
13.0 ± 0.3 28.8 ± 1.0 0.587 ±0.015 1.81 ±0.03 1 46 ± 0.06 164 ± 19 201 ± 10 18.7 ± 0.9 8.02 ± 0.50 — — 2.13 ± 0.05 21.9 ± 0.7
Female Deprived 13.1 29.3 0.568 1.66 1.49 169 186 18.7 8.15
± 0.3 ± 0.9 ±0.010 ± 0.03* ± 0.05 ± 22 ± 9 ± 0.9 ± 0.39 — — 2.07 ± 0.04 21.3 ± 0.5
Control
Deprived
15.6 ± 0.5 45.4 ± 1.3 — — — — — — — 66.9 ± 2.0 504 ± 20 1.21 ±0.02 11.3 ± 0 . 2
14.8 ± 0 4 41.0 ± 2.1 — — — — — — — 66.0 ± 1.6 496 ± 26 1.19 ±0.02 11.2 ± 0.3
Note. Data represent means and standard errors of 68 or more rats obtained from 10 litters for each treatment group for organ weights and 12 rats from 6 litters for sperm evaluation. Astensk denotes significant differences between control and deprived rats (p < 0.001) determined by a two-tailed ( test.
While prenatal stress has been shown to alter reproductive functions and disrupt sexual behaviors in the offspring (Herrenkohl, 1979; 1986; Pollard. 1984; Wiebold et al., 1986; Anderson et al., 1986; Kinsley and Bridges, 1988; Kinsley et al., 1989; Gutierrez et al., 1989), stress exerted during postnatal ages did not appear to elicit similar changes. Results from the current study are consistent with those reported by McGrady and Chakraborty (1983) and Murthy et al. (1988), in which no deleterious effect was observed in adult male rats and mice after chronic immobilization stress. On the other hand, Pellerin-Massicotte and co-workers (1987) reported that daily swimming exercise (up to 6 hr) from postnatal Day 11 to Day 50 delayed vaginal opening and first estrus in the rat, an effect accompanied by changes
in serum and pituitary gonadotropins. However, in conjunction with these alterations, marked deficits of body weight gain (as much as 23% of sedentary controls) were observed, suggesting that the rigor of exercise might have impeded the overall growth of the animals and delays in the onset of reproductive functions might be secondary to these interferences. Indeed, the maternal separation stress paradigm employed in the present study produced a much smaller growth deficit and hence a corresponding lack of change in the onset of puberty. More importantly, this milder form of stress did not appear to alter the reproductive capacity (sperm evaluations, estrous cyclicity, and fertility) nor the pregnancy outcomes of the animals. Thus, daily episodes of maternal deprivation stress in itself are not sufficient to affect the development of reproductive functions in the rat. However, caution should still be taken in the interpretation of toxicity data which involve stresses in the experimental design. The possibility of interactions between stress responses and reproductive toxicants cannot be ruled out. Additional studies to address this very issue are warranted to alleviate such concern. ACKNOWLEDGMENTS The authors thank Mr. David Ellis, Juan Suarez, Joseph Ostby, Ms. Deborah Hunter, Jennifer Merriman, Shannon Sawin, and Tammy Stoker for their technical assistance, and Ms. Judith Schmid for her help in the statistical analysis. REFERENCES Anderson, R. H., Fleming, D. E., Rhees, R. W., and Kinghom, E. (1986). Relationships between sexual activity, plasma testosterone, and the vol-
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the deprived animals than in the controls. Again, does this small weight deficit reflect functional deficiency of the male reproductive organ? Results from sperm evaluation suggest that this is not the case, as the rate of daily sperm production in the testis was unaffected. In addition, cauda epididymal sperm number, which denotes sperm storage capacity, as well as cauda epididymal transit time revealed no appreciable differences between control and deprived groups. Consistent with the organ weight data, the patterns of estrous cycle in female rats also did not reveal any significant differences between the two treatment groups (data not shown). Indeed, similar fertility rates and pregnancy outcomes were noted in control and deprived rats (data not shown), further suggesting a lack of significant adverse sequelae in the reproductive system after repeated stress episodes during neonatal development.
REPRODUCTIVE DEVELOPMENT AFTER MATERNAL DEPRIVATION
McGrady, A. V., and Chakraborty, J. (1983). Effects of stress on the reproductive system of male rats and mice. Arch. Androl. 10, 95-101. Murthy, N. V., Wray, S. R., Melville, G. N., Wynter, H. H., Ram, N. V., and Haran. N. V. (1988). Testicular function in rats following immobilization stress. Int. J. Gynecol. Obslet. 26, 297-299. Pellerin-Massicotte, J., Brisson, G. R., St. Pierre, C , Rioux, P., and Rajotte, D. (1987). Effect of exercise on the onset of puberty, gonadotropins, and ovarian inhibin. J. Appl. Physiol. 63, 1165-1173. Pollard, I. (1984). Effects of stress administered during pregnancy on reproductive capacity and subsequent development of the offspring of rats: Prolonged effects on the litters of a second pregnancy. J. Endocrinol. 100,301-306. Robb, G. W., Amann, R. P., and Killian, G. J. (1978). Daily sperm production and epididymal sperm reserves of pubertal and adult rats. J. Reprod. Fert. 54, 103-107. Schanberg, S. M., Evoniuk, G., and Kuhn, C. M. (1984). Tactile and nutritional aspects of maternal care: Specific regulators of neuroendocrine function and cellular development. Proc. Soc. Exp. Biol. Med. 175, 135— 146. Shimoda, K., Hanada, K., Yamada, N., Takahashi, K., and Takahashi, S. (1986). Periodic exposure to mother is potent zeitgeber of rat pups' rhythm. Physiol. Behav. 36, 723-730. Stanton, M. E., Gutierrez, Y. R., and Levine, S. (1988). Maternal deprivation potentiates pituitary—adrenal stress responses in infant rats Behav. Neurosci. 102, 692-700. Stanton, M. E., Crofton, M. E., and Lau, C. (1992). Behavioral development following daily episodes of mother—infant separation in the rat. Fundam. Appl. Toxicol. 19, 474-477. Tonjes, R., Hecht, K., Brautzsch, M., Lucius, R., and Dorner, G. (1986). Behavioral changes in adult rats produced by early postnatal maternal deprivation and treatment with choline chloride. Exp. Clin. Endocrinol. 88, 151-157. Vesell, E. S., Heubel, F., and Netter, K. J. (1989). Brief maternal deprivation of rats reduces hepatic mixed function oxidase activities. Life Sci. 45, 1755-1761. Wiebold, J. L., Stanfield, P. H., Becker, W. C , and Hillers, J. K. (1986). The effect of restraint stress in early pregnancy in mice. J. Reprod. Fert. 78, 185-192.
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ume of the sexually dimorphic nucleus of the preoptic area in prenatally stressed and non-stressed rats. Brain Res. 370, 1-10. Cooper, R. L., Goldman, J. M., and Vandenberg, J. G. (1993). Monitoring of the estrous cycle in the laboratory rodent by vaginal lavage. In Methods in Toxicology, Vol. 3, Part B: Female Reproductive Toxicology (J. J. Heindel and R. E. Chapin, Eds.), pp. 45-56. Academic Press, San Diego. Dorner, G., Plaschke, M., Tonjes, R., and Wenzel, J. (1984). Teratomorphogenic effects on the brain produced by neonatal maternal deprivation can be partly prevented by pyridostigmine administration. Exp. Clin. Endocrinol. 84, 352-355. Gutierrez, J., Alvarez-Ordas, I., Rojo, M., Marin, B., and Medendez-Patterson, A. (1989). Reproductive function and sexual behavior in female rats exposed to immobilization stress or ACTH injections during gestation. Physiol. Bohemoslov. 38, 13-20. Herrenkohl, L. R. (1979). Prenatal stress reduces fertility and fecundity in female offspring. Science 206, 1097-1099. Herrenkohl, L. R. (1986). Prenatal stress disrupts reproductive behavior and physiology in offspring. Ann. NY Acad. Sci. 474, 120-128. Hofer, M. A. (1984). Early stages in the organization of cardiovascular control. Proc. Soc. Exp. Biol. Med. 175, 147-157. Kimmel, C. A., and Francis, E. Z. (1990). Proceedings of the workshop on the acceptability and interpretation of dermal developmental toxicity studies. Fundam. Appl. Toxicol. 14, 386-398. Kinsley, C. H., and Bridges, R. S. (1988). Prenatal stress and maternal behavior in intact virgin rats: Response latencies are decreased in males and increased in females. Harm. Behav. 22, 76-89. Kinsley, C. H.. Mann, P E., and Bridges, R. S. (1989). Alterations in stressinduced prolactin release in adult female and male rats exposed to stress, in utero. Physiol. Behav. 45, 1073-1076. Kuhn, C. M., Pauk, J., and Schanberg, S. M. (1990). Endocrine responses to mother-infant separation in developing rats. Dev. Psychobiol. 23, 395-410. Lau, C , Cameron, A. M., Antolick, L. L., and Stanton, M. E. (1992). Repeated maternal separation in the neonatal rat: Cellular mechanisms contributing to brain growth sparing. J. Dev. Physiol. 17, 265-276. Laudenslager, M., Capitanio, J. P., and Reite, M. (1985). Possible effects of early separation experiences on subsequent immune function in adult macaque monkeys. Am. J. Psychiatry 142, 862-864.
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