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Effects of adrenalectomy on maternal behavior in pregnancy-terminated rats
Physiology & Behavior, Vol. 21, pp. 831-833. Pergamon Press and Brain Research Publ., 1978. Printed in the U.S.A.
BRIEF COMMUNICATION Effects of Adrenalectomy on Maternal Behavior in Pregnancy-Terminated Rats I H A R O L D I. S I E G E L A N D J A Y S. R O S E N B L A T T
Institute of Animal Behavior, Rutgers-The State University, Newark, NJ 01702 (Received 12 June 1978) SIEGEL, H. I. AND J. S. ROSENBLATT. Effects of adrenalectomy on maternal behavior in pregnancy-terminated rats. PHYSIOL. BEHAV. 21(5) 831-833, 1978.--Latencies to the onset of maternal behavior were measured in 16 day pregnancy-terminated rats. Three conditions were tested and included hysterectomy-ovadectomy (HO) with testing beginning 24 hr after surgery and HO plus a concurrent subcutaneous injection of 5/~g/kg estradiol benzoate or hysterectomy only (I-I) with testing starting 48 hr post-operatively. Within each condition, one group was also adrenalectomized while another was sham-operated at the same time as HO or H. There were no differences in the distribution of latencies between the adrenalectomized and sham-operated controls in any of the conditions although a significantly greater percentage of the sham adrenalectomized H animals were maternal by Day 2 of testing. It was concluded that adrenal secretions are not essential for the onset of maternal behavior in pregnancy-terminated animals while they may add a facilitatory effect following H only.
Maternal behavior
Adrenalectomy
Femalerat
THE role of adrenal secretions in the onset and maintenance of maternal behavior in the rat has been studied in a variety of situations and the results have shown both facilitatory and inhibitory effects. Adrenalectomy prior to mating resulted in an increase in the amount of time the mothers spent in the nest and in the active suckling position during the first 16 days of lactation [11]. Animals adrenalectomized during the last trimester of gestation had longer latencies to retrieve pups on Days 1-4 postpartum and retrieved smaller percentages of their litters [2]. Intraperitoneal injections of saline around the time of caesarean section on Day 21 of gestation delayed the onset of maternal behavior towards foster pups while non-injected caesarean-sectioned rats were almost immediately maternal [10]. Adrenalectomy prior to mating or dexamethasone treatment on Day 21 protected caesarean sectioned animals from the inhibitory effects of the saline injections. Adrenal secretions may also act to lengthen the latencies to the onset of maternal care in nulliparous rats exposed to continuous pup stimulation. The normal 4-7 day latency found in intact and ovariectomized rats [4] was reduced to 40 hr in animals that had been both adrenalectomized and ovariectomized and tested four weeks post-operatively [3]. The present series of studies were aimed at elucidating the role of the adrenal glands in the onset of maternal behavior in 16 day pregnancy-terminated rats. Our previous re-
Pregnancytermination
Estradiolbenzoate
search [5,7] has shown that hysterectomy (H) on Day 16 results in shorter latencies than those found following combined hysterectomy-ovariectomy (HO). In addition, a single injection of estradiol benzoate (EB) at the time of HO restores short-latency maternal care equal to that found following H. These three experimental preparations, H, HO, and HO plus EB, were re-examined in adrenalectomized animals. MATERIALS AND METHOD
Animals Experimental animals consisted of Sprague-Dawley virgin rats obtained from Charles River Breeding Laboratory (Willmington, Mass.) at 60 days of age. The animals were mated 2-4 weeks after arrival; Day 1 of pregnancy refers to the day of mating. Immediately following surgery on Day 16 of gestation, the animals were placed into observation cages (46 × 51 ×42 cm) with Plexiglas walls and were provided with adequate supplies of paper towels for nesting material, food, and water. A 12 hr reversed light cycle was used with darkness beginning at 0800 hr.
Procedure Over a period of several weeks, three pairs of groups of
~This research was supported by USPHS Grant MH-08604 to J.S.R. and a Biomedical Research Support Grant. The authors wish to thank Ms. Marilyn Dotegowski for technical assistance and Ms. Cynthia Banas for technical assistance and preparation of the illustration. Publication No. 311 from the Institute of Animal Behavior, Rutgers---The State University.
Copyright © 1978 Brain Research Publications Inc.--0031-9384/78/110831-03502.00/0
832
SIEGEL AND ROSENBLATT
rats underwent surgery on Day 16 of pregnancy. One pair was hysterectomized and tested 48 hr following surgery ( n = l l / g r o u p ) while another pair of groups were hysterectomized-ovariectomized and tested 24 hr after surgery (n=10/group). The final 2 groups were also hysterectomized-ovariectomized but were injected subcutaneously with 5 /zg/kg EB at surgery and tested 48 hr post-operatively (n= 11/group). One group of each pair was adrenalectomized (and provided with an ad lib supply of salt) while the other group of each pair was shamadrenaiectomized on Day 16. All surgery was performed under ether anesthesia using a ventral incision for the H and HO and bilateral dorsal incisions for the adrenalectomy and sham-surgery. Maternal Behavior Testing
Test pups consisted of three young between the ages of 3-8 days obtained from donor females bred in advance. The first day of testing (either 24 or 48 hr post-operatively) was counted as Day 0 in the presentation of results. Testing began between 1000 and 1100 hr when the test pups were placed in that corner of the cage that was diagonally opposite from the corner in which the female had established her sleeping nest. The animals were observed for 15 min and spot-checked during the next 4 hr. The pups were replaced by freshly nourished young every 24 hr until the females retrieved all pups to the nest and adopted a lactating position over them or until they failed to respond during five days of testing. The animals were required to behave in a similar manner on two consecutive days although the first day was counted as the latency to the onset of maternal behavior. For statistical analysis, latencies of 6 days were assigned to females that did not become maternal during the 5 days of testing. Animals that displayed maternal behavior on Day 5 were re-tested on Day 6 to satisfy the two day criterion.
All of our animals were tested at either 24 or 48 hr following the removal of both the adrenals and the fetuses, also allowing for the decline of maternal and fetal corticoids. Nevertheless, the animals in the present study appeared as maternally responsive as sham-operated controls. However, in the present study, the latency to retrieve within each test was not measured and only three pups were presented although all maternal animals were required to retrieve all three pups. Other differences between the earlier study [2] and the current one (e.g., the use of pups from adrenaiectomized mothers in the former study) preclude direct comparison of the results. The present study also answers related questions regarding the onset of maternal behavior in 16-day pregnancyterminated rats. HO animals are more maternally responsive to pups 24 hr post-operatively than animals that are initially presented with pups at 48 hr [ 1,9]. Because exogenous estrogen stimulates short-latency maternal behavior in HO animals tested 48 and 72 hr after surgery ([7,9]; Fig. 1), it was hypothesized that post-operative adrenal estrogen secretion [6] might be responsible for the increased maternal responsiveness in 16 day HO animals tested at 24 hr. The data in Fig. 1A do not support this possibility and also do not show any effect of adrenalectomy on EB-induced maternal care. A.
HO
C.I-I
B. H 0 + E B
I00 -
o
f /7
o
--o--o
80-
/
4O
20
RESULTS AND DISCUSSION The present study indicated that adrenal secretions do not play an essential role in the onset of maternal behavior in pregnancy-terminated rats (Fig. 1). The distribution of maternal latencies between adrenaiectomized and shamadrenaiectomized groups within each experimental condition (HO tested at 24 hr and H and HO plus EB tested at 48 hr) did not differ significantly (Mann Whitney U test, p > 0 . 1 , two-tailed). Although it is difficult to compare our results with those of previous investigations (see introduction) because of the variety of procedures and reproductive conditions of the animals, our data agree with those of Thoman and Levine [1 1]. The present results do not support the inhibitory effects of EB [3] in either pregnancy-terminated animals or in nuiliparous animals reported in an earlier study [8]. Hennessy et al. [2] showed that parturient rats that were adrenalectomized between Days 14-18 of pregnancy required longer latencies to retrieve and retrieved fewer pups on Days 1-4 post partum. These investigators suggested that the deficits in maternal behavior may have been due to the clearance of fetal corticoids occurring between parturition and the initial tests beginning 24 hr later.
124 hr)
0
I
2
3
5
(4(3 hr) l
,
r
i
O
I
2
3
H
,
(48hr) ,
,
,
v H
i
5
0
I
2
3
5
DAYS
FIG. 1. Cumulative percentage of 16 day pregnant adrenalectomized (ADX) and sham-adrenalectomized rats displaying maternal behavior. Groups were: (A) hysterectomized-ovariectomized (HO) and either tested 24 hr after surgery, or (B) injected subcutaneously with 5/zg/kg estradiol benzoate (EB) at surgery and tested at 48 hr. Two other groups were (C) hysterectomized only (H) and tested 48 hr post-operatively.
Similarly, we have suggested that endogenous ovarian estrogen secretion is responsible for the short-latency maternal behavior displayed by H animals [5,7]. In this case, adrenal secretions may contribute to the expression of maternal care because by Day 2 of testing, 91% of the sham-operated and only 46% of the adrenalectomized H females were maternal. However, the delay in the onset of maternal behavior was not especially dramatic and the Mann Whitney U test failed to reveal a statistical difference.
ADRENALECTOMY
AND MATERNAL BEHAVIOR
833
REFERENCES 1. Bridges, R. S., H. H. Feder and J. S. Rosenblatt. Induction of maternal behaviors in primigravid rats by ovariectomy, hysterectomy, or ovariectomy plus hysterectomy: Effect of length of gestation. Hormones Behav. 9: 156--169, 1977. 2. Hennessy, M. B., K. S. I-Iarney, W. P. Smotherman, S. Coyle and S. Levine. Adrenalectomy-induced deficits in maternal retrieval in the rat. Hormones Behav. 9: 222-227, 1977. 3. Leon, M., M. Numan and A. Chan. Adrenal inhibition of maternal behavior in virgin female rats. Hormones Behav. 6: 165171, 1975. 4. Rosenblatt, J. S. Nonhormonal basis of maternal behavior in the rat. Science 156: 1512-1514, 1967. 5. Rosenblatt, J. S. and H. I. Siegel. Hysterectomy-induced maternal behavior during pregnancy in the rat. J. comp. physiol. Psychol. 89: 685-700, 1975.
6. Shaikh, A. A. and S. A. Shaikh. Adrenal and ovarian steroid secretion in the rat estrous cycle temporally related to gonadotropins and steroid levels found in peripheral plasma. Endocrinology 96: 37-44, 1975. 7. Siegel, H. I. and J. S. Rosenblatt. Hormonal basis of hysterectomy-induced maternal behavior during pregnancy in the rat. Hormones Behav. 6: 211-222, 1975. 8. Siegel, H. I. and J. S. Rosenblatt. Estrogen-induced maternal behavior in hysterectomized-ovariectomized virgin rats. Physiol. Behav. 14: 465--471, 1975. 9. Siegel, H. I. and J. S. Rosenblatt. Duration of estrogen stimulation and progesterone inhibition of maternal behavior in pregnancy-terminated rats. Hormones Behav., in press. 10. Steele, M., H. Moltz and D. Rowland. Adrenal disruption of maternal behavior in the Caesarean-sectioned rat. Hormones Behav. 7: 473--479, 1976. 11. Thoman, E. B. and S. Levine. Effects of adrenalectomy on maternal behavior in rats. Devl. Psychobiol. 3: 237-244, 1970.
BRIEF COMMUNICATION Effects of Adrenalectomy on Maternal Behavior in Pregnancy-Terminated Rats I H A R O L D I. S I E G E L A N D J A Y S. R O S E N B L A T T
Institute of Animal Behavior, Rutgers-The State University, Newark, NJ 01702 (Received 12 June 1978) SIEGEL, H. I. AND J. S. ROSENBLATT. Effects of adrenalectomy on maternal behavior in pregnancy-terminated rats. PHYSIOL. BEHAV. 21(5) 831-833, 1978.--Latencies to the onset of maternal behavior were measured in 16 day pregnancy-terminated rats. Three conditions were tested and included hysterectomy-ovadectomy (HO) with testing beginning 24 hr after surgery and HO plus a concurrent subcutaneous injection of 5/~g/kg estradiol benzoate or hysterectomy only (I-I) with testing starting 48 hr post-operatively. Within each condition, one group was also adrenalectomized while another was sham-operated at the same time as HO or H. There were no differences in the distribution of latencies between the adrenalectomized and sham-operated controls in any of the conditions although a significantly greater percentage of the sham adrenalectomized H animals were maternal by Day 2 of testing. It was concluded that adrenal secretions are not essential for the onset of maternal behavior in pregnancy-terminated animals while they may add a facilitatory effect following H only.
Maternal behavior
Adrenalectomy
Femalerat
THE role of adrenal secretions in the onset and maintenance of maternal behavior in the rat has been studied in a variety of situations and the results have shown both facilitatory and inhibitory effects. Adrenalectomy prior to mating resulted in an increase in the amount of time the mothers spent in the nest and in the active suckling position during the first 16 days of lactation [11]. Animals adrenalectomized during the last trimester of gestation had longer latencies to retrieve pups on Days 1-4 postpartum and retrieved smaller percentages of their litters [2]. Intraperitoneal injections of saline around the time of caesarean section on Day 21 of gestation delayed the onset of maternal behavior towards foster pups while non-injected caesarean-sectioned rats were almost immediately maternal [10]. Adrenalectomy prior to mating or dexamethasone treatment on Day 21 protected caesarean sectioned animals from the inhibitory effects of the saline injections. Adrenal secretions may also act to lengthen the latencies to the onset of maternal care in nulliparous rats exposed to continuous pup stimulation. The normal 4-7 day latency found in intact and ovariectomized rats [4] was reduced to 40 hr in animals that had been both adrenalectomized and ovariectomized and tested four weeks post-operatively [3]. The present series of studies were aimed at elucidating the role of the adrenal glands in the onset of maternal behavior in 16 day pregnancy-terminated rats. Our previous re-
Pregnancytermination
Estradiolbenzoate
search [5,7] has shown that hysterectomy (H) on Day 16 results in shorter latencies than those found following combined hysterectomy-ovariectomy (HO). In addition, a single injection of estradiol benzoate (EB) at the time of HO restores short-latency maternal care equal to that found following H. These three experimental preparations, H, HO, and HO plus EB, were re-examined in adrenalectomized animals. MATERIALS AND METHOD
Animals Experimental animals consisted of Sprague-Dawley virgin rats obtained from Charles River Breeding Laboratory (Willmington, Mass.) at 60 days of age. The animals were mated 2-4 weeks after arrival; Day 1 of pregnancy refers to the day of mating. Immediately following surgery on Day 16 of gestation, the animals were placed into observation cages (46 × 51 ×42 cm) with Plexiglas walls and were provided with adequate supplies of paper towels for nesting material, food, and water. A 12 hr reversed light cycle was used with darkness beginning at 0800 hr.
Procedure Over a period of several weeks, three pairs of groups of
~This research was supported by USPHS Grant MH-08604 to J.S.R. and a Biomedical Research Support Grant. The authors wish to thank Ms. Marilyn Dotegowski for technical assistance and Ms. Cynthia Banas for technical assistance and preparation of the illustration. Publication No. 311 from the Institute of Animal Behavior, Rutgers---The State University.
Copyright © 1978 Brain Research Publications Inc.--0031-9384/78/110831-03502.00/0
832
SIEGEL AND ROSENBLATT
rats underwent surgery on Day 16 of pregnancy. One pair was hysterectomized and tested 48 hr following surgery ( n = l l / g r o u p ) while another pair of groups were hysterectomized-ovariectomized and tested 24 hr after surgery (n=10/group). The final 2 groups were also hysterectomized-ovariectomized but were injected subcutaneously with 5 /zg/kg EB at surgery and tested 48 hr post-operatively (n= 11/group). One group of each pair was adrenalectomized (and provided with an ad lib supply of salt) while the other group of each pair was shamadrenaiectomized on Day 16. All surgery was performed under ether anesthesia using a ventral incision for the H and HO and bilateral dorsal incisions for the adrenalectomy and sham-surgery. Maternal Behavior Testing
Test pups consisted of three young between the ages of 3-8 days obtained from donor females bred in advance. The first day of testing (either 24 or 48 hr post-operatively) was counted as Day 0 in the presentation of results. Testing began between 1000 and 1100 hr when the test pups were placed in that corner of the cage that was diagonally opposite from the corner in which the female had established her sleeping nest. The animals were observed for 15 min and spot-checked during the next 4 hr. The pups were replaced by freshly nourished young every 24 hr until the females retrieved all pups to the nest and adopted a lactating position over them or until they failed to respond during five days of testing. The animals were required to behave in a similar manner on two consecutive days although the first day was counted as the latency to the onset of maternal behavior. For statistical analysis, latencies of 6 days were assigned to females that did not become maternal during the 5 days of testing. Animals that displayed maternal behavior on Day 5 were re-tested on Day 6 to satisfy the two day criterion.
All of our animals were tested at either 24 or 48 hr following the removal of both the adrenals and the fetuses, also allowing for the decline of maternal and fetal corticoids. Nevertheless, the animals in the present study appeared as maternally responsive as sham-operated controls. However, in the present study, the latency to retrieve within each test was not measured and only three pups were presented although all maternal animals were required to retrieve all three pups. Other differences between the earlier study [2] and the current one (e.g., the use of pups from adrenaiectomized mothers in the former study) preclude direct comparison of the results. The present study also answers related questions regarding the onset of maternal behavior in 16-day pregnancyterminated rats. HO animals are more maternally responsive to pups 24 hr post-operatively than animals that are initially presented with pups at 48 hr [ 1,9]. Because exogenous estrogen stimulates short-latency maternal behavior in HO animals tested 48 and 72 hr after surgery ([7,9]; Fig. 1), it was hypothesized that post-operative adrenal estrogen secretion [6] might be responsible for the increased maternal responsiveness in 16 day HO animals tested at 24 hr. The data in Fig. 1A do not support this possibility and also do not show any effect of adrenalectomy on EB-induced maternal care. A.
HO
C.I-I
B. H 0 + E B
I00 -
o
f /7
o
--o--o
80-
/
4O
20
RESULTS AND DISCUSSION The present study indicated that adrenal secretions do not play an essential role in the onset of maternal behavior in pregnancy-terminated rats (Fig. 1). The distribution of maternal latencies between adrenaiectomized and shamadrenaiectomized groups within each experimental condition (HO tested at 24 hr and H and HO plus EB tested at 48 hr) did not differ significantly (Mann Whitney U test, p > 0 . 1 , two-tailed). Although it is difficult to compare our results with those of previous investigations (see introduction) because of the variety of procedures and reproductive conditions of the animals, our data agree with those of Thoman and Levine [1 1]. The present results do not support the inhibitory effects of EB [3] in either pregnancy-terminated animals or in nuiliparous animals reported in an earlier study [8]. Hennessy et al. [2] showed that parturient rats that were adrenalectomized between Days 14-18 of pregnancy required longer latencies to retrieve and retrieved fewer pups on Days 1-4 post partum. These investigators suggested that the deficits in maternal behavior may have been due to the clearance of fetal corticoids occurring between parturition and the initial tests beginning 24 hr later.
124 hr)
0
I
2
3
5
(4(3 hr) l
,
r
i
O
I
2
3
H
,
(48hr) ,
,
,
v H
i
5
0
I
2
3
5
DAYS
FIG. 1. Cumulative percentage of 16 day pregnant adrenalectomized (ADX) and sham-adrenalectomized rats displaying maternal behavior. Groups were: (A) hysterectomized-ovariectomized (HO) and either tested 24 hr after surgery, or (B) injected subcutaneously with 5/zg/kg estradiol benzoate (EB) at surgery and tested at 48 hr. Two other groups were (C) hysterectomized only (H) and tested 48 hr post-operatively.
Similarly, we have suggested that endogenous ovarian estrogen secretion is responsible for the short-latency maternal behavior displayed by H animals [5,7]. In this case, adrenal secretions may contribute to the expression of maternal care because by Day 2 of testing, 91% of the sham-operated and only 46% of the adrenalectomized H females were maternal. However, the delay in the onset of maternal behavior was not especially dramatic and the Mann Whitney U test failed to reveal a statistical difference.
ADRENALECTOMY
AND MATERNAL BEHAVIOR
833
REFERENCES 1. Bridges, R. S., H. H. Feder and J. S. Rosenblatt. Induction of maternal behaviors in primigravid rats by ovariectomy, hysterectomy, or ovariectomy plus hysterectomy: Effect of length of gestation. Hormones Behav. 9: 156--169, 1977. 2. Hennessy, M. B., K. S. I-Iarney, W. P. Smotherman, S. Coyle and S. Levine. Adrenalectomy-induced deficits in maternal retrieval in the rat. Hormones Behav. 9: 222-227, 1977. 3. Leon, M., M. Numan and A. Chan. Adrenal inhibition of maternal behavior in virgin female rats. Hormones Behav. 6: 165171, 1975. 4. Rosenblatt, J. S. Nonhormonal basis of maternal behavior in the rat. Science 156: 1512-1514, 1967. 5. Rosenblatt, J. S. and H. I. Siegel. Hysterectomy-induced maternal behavior during pregnancy in the rat. J. comp. physiol. Psychol. 89: 685-700, 1975.
6. Shaikh, A. A. and S. A. Shaikh. Adrenal and ovarian steroid secretion in the rat estrous cycle temporally related to gonadotropins and steroid levels found in peripheral plasma. Endocrinology 96: 37-44, 1975. 7. Siegel, H. I. and J. S. Rosenblatt. Hormonal basis of hysterectomy-induced maternal behavior during pregnancy in the rat. Hormones Behav. 6: 211-222, 1975. 8. Siegel, H. I. and J. S. Rosenblatt. Estrogen-induced maternal behavior in hysterectomized-ovariectomized virgin rats. Physiol. Behav. 14: 465--471, 1975. 9. Siegel, H. I. and J. S. Rosenblatt. Duration of estrogen stimulation and progesterone inhibition of maternal behavior in pregnancy-terminated rats. Hormones Behav., in press. 10. Steele, M., H. Moltz and D. Rowland. Adrenal disruption of maternal behavior in the Caesarean-sectioned rat. Hormones Behav. 7: 473--479, 1976. 11. Thoman, E. B. and S. Levine. Effects of adrenalectomy on maternal behavior in rats. Devl. Psychobiol. 3: 237-244, 1970.