- Email: [email protected]
J mouse
Physiology and Behavior, Vol. 14, pp. 825-832. Brain Research Publications Inc., 1975, Printed in the U.S.A.
Gonadectomy and the Development of Age-dependent Polydipsia and the Intake of NaC1 Solutions in the SWR/J Mouse NANCY L. SCHMALBACH AND CHARLES L. KUTSCHER
Department of Psychology, Syracuse University, Syracuse NY 13210 (Received 17 December 1974) SCHMALBACH, N. L. AND C. L. KUTSCHER. Gonadectomy and the development o f age-dependent polydipsia and the intake o f NaCl solutions in the SIVR/J mouse. PHYSIOL. BEHAV. 14(6) 825-832, 1975. - Gonadectomies were performed at 25 days of life in SWR/J mice, a strain known to develop age-dependent nephrogenic diabetes insipidus with females drinking far more than males. Effect of gonadectomy was different for each of the 3 nonreproductive behaviors studied. (1) Ovariectomy reduced the age-dependent polydipsia seen in females, but castration was without effect in males. (2) Castration of males resulted in a lower intake of an isotonic NaC1 solution, making the castrated males similar to the sham-operated and ovariectomized females, which did not differ. (3) For the short-term activity measurement sham-operated males did not differ from sham-operated females and castrated males did not differ from ovariectomized females. In each sex, gonadectomy reduced activity. Aging
Diabetes insipidus
NaC1
Polydipsia
Sex steroids
KUTSCHER and Miller [12] reported spontaneous polydipsia which increased in severity with age in the SWR/J mouse strain and was more severe in females than in males. Kidneys became refractory to exogenous antidiuretic hormone, resulting in low ceilings of renal concentrating ability. In some mice, large lesions were seen in the renal medulla [13]. In females, but not males, intake of hypertonic and even hypotonic NaC1 solutions, decreased with age as water intake increased [ 1 ]. It was suggested that the age-dependent renal deterioration with its behavioral, anatomical and physiological correlates might be a useful genetic model for the study of aging processes. Renal lesions have been observed with age in both germ-free and conventional mice of other strains [6]. One theory of aging suggests that age-dependent deterioration may be dependent not on spontaneous disruption of metabolic activities within the cells themselves, but upon some change in an epicellular factor which influences cell metabolism. For example, Furth [5] found that incidence of leukemia which usually develops in adult Ak mice was markedly reduced by thymectomy performed at 6 weeks of age.
study over the first 10 months of life was made on male and female mice gonadectomized at weaning. Tests with NaC1 solutions were also made in order to relate intake of hypotonic, isotonic and hypertonic solutions to changes in water intake and the removal of sex structures. METHOD Animals The 30 male and 30 female SWR/J mice used in this experiment were bred in the laboratory at Syracuse University from breeding stock obtained from Jackson Laboratory at Bar Harbor, Maine. Apparatus During the measurement of drinking, mice were housed in individual steel test cages 12.7 X 12.5 X 14.5 cm with tops and floors of 0.6 cm mesh hardware cloth. Fluid intakes were measured by means of 100 ml eudiometer tubes graduated in 0.2 ml units, attached vertically to the back of each cage with the spout protruding approximately 1 cm through an opening in the back of the cage. Purina Chow pellets were available in the cage at all times. Demineralized water was given in drinking tubes except when solution tests were being conducted. Between testing sessions mice were housed in the main animal colony in groups of 1 - 6 in standard 27 X 17 X 13 cm plastic mouse cages where they were given tap water to
EXPERIMENT 1 One possible explanation of the higher water intakes of female SWR/J mice [12] is that female sex steroids in circulation facilitate certain degenerative processes. To test this possibility that type of sex steroids may lead to sex differences in non-reproductive behaviors, a longitudinal 825
826
SCHMALBACH AND KUTSCHER
drink with Purina Chow pellets always available. In the test room and animal colony, lights were on for 12 hr and temperature was maintained at 21 +- I°C. Air was humidified during the winter months. Activity was measured by a Lehigh Valley Electronics activity drum utilizing the two banks of photocells, but not the tilt floor. The drum is a cylinder 44.4 cm in dia. and 41.9 cm high. Inside walls are painted black. Tests were run with lid in place thus producing a semidark test chamber. Procedure For each sex, 15 mice were gonadectomized and 15 were subjected to a sham operation at weaning, 2 5 - 3 0 days of life, under Nembutal anesthesia ( 6 5 - 7 0 mg/kg). Completeness of removal was verified by examination of the ovaries following surgery and by post-mortem examination when animals were sacrificed at 12 months. In the sham-operation condition, ovaries were exposed and visualized but not removed. In the males, testes were removed in the castrated group and merely exposed in the sham-operated group. In all animals incisions were closed with sutures of cotton thread. Mice were numbered by ear-punching at the time of the operation. At 3, 7 and 10 months mice were isolated in metal test cages in the test room. After 3 days of adaptation to the cages and drinking tubes, intake of demineralized water was measured for 7 days with body weight recorded at the beginning and end of the 7 day period. At 10 months food intake was also measured by placing new preweighed pellets in the cage at the beginning of each test day and weighing them at the end of the test day. Following the 7 day water intake measures, mice were given, one at a time in random order, the following NaC1 solutions: 0, 0.5, 1.0, and 2.0 percent. Solutions were presented for 23 hr, 1 hr being required to weigh animals and exchange solutions in tubes. Tests were made on alternate days with animals given water to drink on the intervening days. Body weight was measured at the beginning and end of each test day. Following the NaC1 test sequence, mice were tested in the same manner with 2.0 percent NaC1, 2.0 percent NaC1 + 5.0 percent glucose, and 5.0 percent glucose, given in random order on alternate days. Ten animals of each of the 4 groups were selected for activity measurement. Each mouse was placed in the apparatus and allowed 30 seconds to explore. Then activity (interruption of light beams) was measured for 10 min. Activity counts for both banks of photocells were summed to give the final activity measure. At approximately 12 months mice were sacrificed and both kidneys were removed and weighed. RESULTS Intakes of water, solutions and body weight were analyzed by a 3 × 4 split-plot factorial model of analysis of variance [29] in order to determine if significant differences were produced by the two main variables (1) age (3, 7 and 10 months) and (2) sex and operation, presumably relating to type and level of sex steroid produced and in circulation: sham-operated males, sham-operated females, castrated males and ovariectomized females. Comparisons between individual means were evaluated with Scheffe's test [291.
Water intakes Mean 7 day water intakes (ml) were influenced by age (p<0.01) and sex and operation (p<0.01). The interaction of these two variables was also significant (p<0.01). As may be seen in Fig. I, water intakes were not different for the 4 groups at 3 months. Thereafter, intakes increased with age, but at different rates. Ovariectomy greatly attenuated the development of polydipsia producing differences (p<0.01) in water intakes between the sham females and ovariectomized females at 7 months and at I0 months. Combining data over the 3 testing periods, intakes of the ovariectomized females were greater than intakes of the castrated males, but were not statistically distinguishable from intakes of the sham-operated males. Thus, the ovariectomy eradicated the sex difference in polydipsia development observed in this study and in the previous one [ 12]. The two male groups did not differ from each other. Body Weight This measure increased with age (p<0.01) and differed with sex and operation (p<0.01). The interaction was also significant (p<0.05) indicating that the rate of weight gain differed for the 4 groups (Fig. 1, center). Castration and ovariectomy had opposite effects on body weight. Ovariectomy at weaning produced a higher body weight level than sham-operated females at 3, 7 and 10 months. Castration of males at weaning resulted in body weights significantly lower than in sham-operated males at 7 and 10 months. Combining data over the 3 test periods, weights of castrated males were not different from those of ovariectomized females. Thus, gonadectomy eradicated the expected sex difference in body weight. Expressing water intakes as a proportion of body weight did not change any of the relationships mentioned above (Fig. 1, bottom). Mean daily water intake of the 10 month sham-operated females was considerably more than the body weight of the mice, indicating an extreme state of polydipsia. Food Intakes Food intakes were measured only at 10 months and were analyzed with a 1-way analysis of variance. Scheffe's test was employed to evaluate differences between individual means. Sham-operated males ate more (5.43 g) than castrated males (4.57 g, p<0.01), even though water intakes did not differ. Sham-operated females did not eat more (5.12 g) than ovariectomized females (4.78 g) even though water intakes were quite different at 10 months. Ovariectomized females ate less than sham-operated males (p<0.05), but sham-operated females did not. All other comparisons were not significant. NaCl Solution Intakes. Test solutions were hypotonic (0.0 and 0.5 percent), approximately isotonic (1.0 percent) and hypertonic (2.0 percent) to body fluids. Effects of age and sex and operation on NaC1 solution intake were quite different from water intake. For the 0.5 percent solution (intakes not shown), these variables were without significant effect even though water intakes were quite different. For the 1.0 percent solution, however, intakes differed as a function of age (p<0.01) and sex and operation (p<0.05); however, the interaction was also significant. It may be seen in Fig. 2
GONADECTOMY AND POLYDIPSIA
827
3S-
7
30 -
6
~ 25
S-F
~15
X-F
-
lo
~
Jl
../~__....It"- ~:.~ ~
I
I
I
7 AGE (MOS.)
::F
XF~I~",~
Z2
lO ~"
s....
~281-
1
,w" ~ ,," x-~.-~..i + ,," ,~-F
~ 271""
j
~26J--
2sl-
,_u v4
U0 3
I
../"
I 3
--
~.22t'-
~ 4
--
a_21 i-,.
~
_ . ~ -'~t_
t 7 Ao,: (Mos.)
I 10
t3
<3Z -
I
I I 7 (MOS.)
~
I
I I 10
I
....S-M "'".,ll
x-,
o
s-F
I
S-F
~ 2 -
.~1.0
I
AGE 5
I 3
I
S
_lj';~"+~
,
-M
Z 3
-
(.9 241 -
s-F
c'¢
I I I I I I I I I I I I o
3 AGE
~1~.0-5 ~¢~..~I~1~a__11 ~,~".~,,,.,~_~.~7 ~.-M-U] I',-
~0.1
I
3
I
7
AGE (MOS.)
I
!0
FIG. 1. Mean water intake (ml), body weight (g), and ratio of water intake to body weight of sham-operated female (S-F), ovarieetomized female (X-F), sham-operated male (S-M), and castrated male (X-M) animals plotted as a function of age (months).
7 (MOS.)
10
FIG. 2. Mean 23-hour 1.0 and 2.0 percent NaC1 intake (ml) for sham-operated female (S-F), ovariectomized female (X-F), sham-operated male (S-M), and castrated male (X-M) animals plotted as a function of age (months).
(top) that there are two distinct patterns of age-related change in 1.0 percent NaC1 intake. Sham-operated males show no change in intake with age, but the two female groups and the castrated male group decreased intake with age. Combining data over the 3 ages, intake of shamoperated males exceeded that of castrated males and ovariectomized females (p<0.01). For the 2.0 percent solution, there was a general decline in solution intake with age (p<0.01), but sex and operation condition had no significant effect.
828
SCHMALBACH AND KUTSCHER
25
7 -
0.5~0 NoCI
3 S-F
,-,6-
20
mm
Ol0
v
15 ~4-
10
~3-
ol0 o7
Z o
~2-
5-
mlO 07 r710
r13 03
1[11~17" 3
1-
3 I
I
I
I
I I 1 I I I I I I I 1 I 10 20 30 WATER INTAKE (ML)
I
0
1 36
20
40
2S
60
80
100
I.OTo NaCI
7el0
~20
n
~5
o10
~10
03 _
_
I01~°
03
X
UlO 03 7 I [] m3
_
3g u7
0
07 07
010 I10
~2
07 I
|
I
I
I I 10 WATER
010
07 0~o
i
i
I.
07
i
I 20 INTAKE
20
0 I
i
i
I
I 30
I
I
I 36
I
I
40
60
80
25
In Fig. 3, mean intakes of 1.0 and 2.0 percent NaC1 are shown plotted as a f u n c t i o n of mean 7 day water intake. It is obvious that in the two female groups and in the castrated males, there is an inverse relationship b e t w e e n water intake and 1.0 percent NaC1 intake, even t h o u g h the isotonic salt solution should produce no osmotic stress and d e h y d r a t i o n following ingestion. Apparently the presence o f male sex steroids prevents this inverse relationship since sham males show no decline in 1.0 percent NaC1 intake as water intake increased with age. (Lines shown in Fig. 3, top, were fitted visually). For the 2.0 percent NaC1 solution also, there is an obvious trend for NaC1 intake to decline as polydipsia develops with age. One possible, although unlikely, explanation for the inverse relationship b e t w e e n water and NaC1 intake is that with age the mice b e c o m e more able to extract free water
I 100
2.0% NoCI
(ML)
FIG. 3. Mean 23-hour 1.0 and 2.0 percent NaC1 intake (ml) for sham-operated female (e), ovariectomized female (o), sham-operated male (u), and castrated male (o) animals plotted as a function of mean water intake (ml) at the 3, 7, and 10 month (age) testing periods.
03
I
20 ol0 o!0
15 % f
10
0
-
mlO 07 m7 olo
t '20
I i I 40 60 80 SI/WI x I00
I 100
FIG. 4. Mean body weight loss (%) incurred following 23-hour presentation of 0.5, 1.0 and 2.0 percent NaCI of sham-operated female (e), ovarieetomized female (o), sham-operated male (e), and castrated male (o) animals, plotted as a function of percent ratio of salt intake to water intake at the 3, 7, and 10 month (age) testing periods.
GONADECTOMY AND POLYDIPSIA
829 TABLE 1
INTAKE OF GLUCOSE, NACL AND GLUCOSE-NACL SOLUTIONS Solution Intakes (Mean ± S.E.) 5% Glucose
2% NaCI
5% Glucose + 2% NaC1
Age
Treatment
3 Months
Sham-females Ovar-females Sham-males Cast-males
28.8 22.1 19.8 20.1
± 2.6 ± 1.4 ± 1.6 -+ 1.7
(3.56)* (3.11) (2.75) (3.24)
3.9 1.9 2.8 2.1
± 1.0 ± 0.3 ± 0.4 ± 0.3
3.5 1.9 2.7 2.2
-+ 0.8 -+ 0.3 ± 0.4 ± 0.3
7 Months
Sham-females Ovar-females Sham-males Cast-males
29.1 18.6 23.8 29.6
-+ 3.9 -+ 2.1 ± 2.3 ± 3.5
(1.41) (1.38) (2.45) (3.00)
1.4 0.7 2.3 0.8
+- 0.3 +- 0.2 -+ 0.4 ± 0.1
1.3 0.8 4.3 3.0
-+ 0.2 -+ 0.1 ± 1.0 ± 1.2
10 Months
Sham-females Ovar-females Sham-males Cast-males
55.0 26.9 31.9 35.3
± 5.8 ± 2.4 ± 3.0 ± 2.6
(1.72) (1.65) (2.31) (3.57)
3.0 1.1 4.2 2.4
± 0.6 ± 0.4 -+ 1.2 ± 0.7
3.1 1.6 2.5 2.4
± 0.6 ± 0.3 ± 0.5 -+ 0.5
*Number in parenthesis is ratio of mean glucose intake to mean water intake for each group. from the NaC1 solutions, thus needing less solution volume to secure their daily water requirements. In Fig. 4 are plotted mean body weight losses incurred while drinking the 3 NaC1 solutions, plotted as a function o f the ratio of the NaC1 intake to water intake. Ingestion of NaC1 solutions produced only mean weight losses, no weight gains and some of the losses are very substantial (> 20 percent). Two relationships emerge from these data. First, the higher the concentration of NaC1, the greater the weight losses. Secondly, the lower the intakes of each solution, the greater the weight loss, thus providing no support for the above hypothesis.
Glucose Intake These intakes were affected by age (p<0.01) and sex and operation (p<0.01). The interaction was also significant (p<0.01). As shown in Table 1, there was a general trend for glucose intakes unlike NaC1 intakes, to increase with age, as did water intakes. The only significant difference between individual means (Scheffe's test) were found to be between the female groups. At 10 months, and also combining the 3 test periods, sham-operated females drank more than did ovariectomized females. The ratios o f mean glucose intake to mean water intake shown in Table 1 indicate that offering the glucose solution instead of water increased fluid intake volume even in mice already very polydipsic. In spite of this demonstrated ability of glucose to produce palatability-induced polydipsia in this strain, mixing glucose with the 2.0 percent NaC1 solution did not relieve the strong aversion to the NaCI solution. In the hamster, an animal which has an aversion to a wide variety of NaC1 solutions, the addition o f sucrose or saccharin to 0.3 percent and 1.0 percent NaC1 solutions increased NaC1 intakes [23]. Failure to find such an effect in the present study may be due to the higher concentration of NaCI solution used (2.0 percent) or the possibility that the SWR/J mouse has a greater aversion to NaC1 than the hamster.
Activity Measurement T h e s e measurements were made approximately 2 months subsequent to the 10 month water intake and solution intake tests. Data were analyzed by a 2 X 2 analysis of variance (sex × operation). Activity scores did not differ as a function of sex, but were reduced significantly by gonadectomy (p<0.05). Castrated males were less active (656 counts/10 min) than sham-operated males (794). Ovariectomized females were less active (685) than sham-operated females (796).
Kidney Weights These measurements were also submitted to a 2 × 2 analysis of variance. Significant differences were observed due to sex (p<0.05), operation (p<0.01) and interaction (/9<0.01). Mean kidney weight of castrated males (1.15 g/100 g body weight) was much less than kidney weight of sham-operated males (1.71, p<0.05). In the females, however, the kidney weight of ovariectomized females (1.45) was not different from that of sham-operated females ( 1 . 5 8 ) , a l t h o u g h kidneys of ovariectomized females weighed less than those of sham-operated males (p<0.05). EXPERIMENT 2 Clearly the development of polydipsia in adult SWR mice is greatly attenuated by ovariectomy performed at weaning. At least two possible modes of action can be suggested. (1) Female sex steroids may have a long-term, cumulative action by facilitating a degenerative process in the kidney over the lifespan of the animal, or by setting in motion some degenerative processes during a critical period of the lifespan. (2) Alternatively, female sex steroids may have a dipsogenic effect in the SWR/J mouse, facilitating drinking proportionately to momentary levels of steroids in circulation. If the second alternative is true, ovariectomy of adult, polydipsic mice should result in an immediate de-
830
SCHMALBACH AND KUTSCHER
crease in drinking, compared to sham-operated mice, since the source of sex steroids will be removed. METHOD
Animals Twenty naive, polydipsic, female mice, 14 months of age were used in this experiment.
Apparatus Cages were the same as those used in Experiment 1, except that they had solid metal floors covered with Sterolit bedding.
Procedure Ten mice were ovariectomized and 10 were given a sham operation. Prior to surgery mice were given 3 days to adapt to the cages followed by 7 days of water intake measurements. Mice were weighed on the first and last day of this 7 day period, Mice were ovariectomized under Nembutal anesthesia ( 6 5 - 7 0 mg/kg). After 1--2 days of postoperative rest, water intakes were measured for 7 consecutive days with body weights measured on the first and last days of this period. RESULTS Mean preoperative water intakes were 32.6 ml for the Sham-operated Group and 34.4 ml for the Ovariectomized Group. This difference was not significant. Following surgery, mean water intakes were 31.0 ml for the Shamoperated Group and 30.1 ml for mice in the Ovariectomized Group, a difference which was not significant. DISCUSSION In this experiment, three types of non-reproductive behaviors were altered by gonadectomy, but the resultant patterns of change produced were different in each case. For water intake, ovariectomy did not prevent the development of polydipsia with age, but attenuated it, such that the ovariectomized females did not differ in intake statistically from the sham-operated males, but were slightly, but significantly higher in intake than the castrated males. For intake of 1.0 percent NaC1, two basic intake patterns emerged. Sham-operated males did not change NaC1 intake as water intake increased with age. In the other 3 groups, NaC1 intake decreased as water intake increased. For general activity, gonadectomy decreased activity in both males and females. It is unlikely that one simple hypothesis can account for all these findings. It is well-known that castration of male rats results in a reduction in weight gain with age and ovariectomy produces an increase in weight gain [17,26] even when the operation is performed in rats as old as 100 days. A comprehensive literature review showed that castration also produced an increase in weight gain in baboons, cattle, guinea pigs and turkeys [7]. Rate of weight change is responsive to short-term changes in circulating levels of steroids in rats [26], e.g. the expected weight gain in adult, ovariectomized rats was prevented by injection of estrogen [16]. Similarly, the decrease in kidney size caused by castration in the SWR mouse with diabetes insipidus is similar to
changes in nondiabetic mouse strains. Castration of male mice resulted in lower kidney weight in DBA mice [ 8 ] and Holtzman-Swiss mice [9]. Testosterone has a well-known renotrophic influence [20] on kidney protein metabolism, DNA and RNA, by means of action in the ribosomal particulates [8,9]. Thus, the presence and development of nephrogenic diabetes insipidus did not alter the expected action of testosterone to facilitate and maintain growth of the kidney in the male. Female sex steroids do not appear to have this renotrophic effect since in the present study kidneys of ovariectomized females are not different from those of sham females. The interpretation of water intakes is not so easily guided by precedents in the literature. It is clear in this experiment that the large differences in water intakes between sham-operated females and ovariectomized females do not relate to kidney size since these two groups do not differ on this dimension. Furthermore, the kidneys of castrated males were 33 percent smaller than those of shamoperated males yet water intakes did not differ from this group. Significant correlations between food intake and water intake and between body weight and water intake have been found in 6 nonpolydipsic mouse strains [11]. However, it is clear in the present study that the large difference in water intakes between the two female groups was not due to a difference in food intake, since none was found, or to body weight differences, since sham-operated females weighed less, but drank more, than ovariectomized females. In studies of polydipsia it is important to determine to what extent the magnitude of water intake reflects the water needs of the animal, i.e. polyuria is primary and polydipsia is an obligatory adjustment required to maintain normal levels of body water. Rats selectively bred for high water intakes [22] could tolerate restriction of water intake to 50 percent of normal and suffer only a transitory effect on feeding and weight gain. In the SWR mouse strain, however, all indications are that the high water intakes indicate a high water need. Polydipsic female SWR/J mice have very limited renal concentrating ability and can produce urine only slightly hypertonic to plasma even when challenged with water deprivation because kidney tubules are refractory to antidiuretic hormone [13]. Although some mouse strains can survive without water for up to 10 days (Kutscher, unpublished study), polydipsic SWR mice are markedly weakened by only one day of deprivation, sometimes showing piloerection and cessation of temperature regulation. In the present experiment, the greater water intakes of the sham-operated females, compared to the ovariectomized females, probably indicate a greater water need. When given 1.0 percent NaC1, weight loss (an approximation of dehydration) of 10 month sham-operated females (19.9 percent) exceeded that of ovariectomized females (13.0%; p<0.05). When 2.0 percent NaC1 was offered, again, sham-operated females showed greater weight loss (22.7 percent) than ovariectomized females (16.6%; p<0.01) even though fluid intakes did not differ for the two groups under these conditions. Experiment 2 provided no indication of a short-term facilitatory effect of sex steroids on drinking and there is no clear precedent in the literature for such an effect. Ovariectomy produced no significant change in water intake in one study [15], but increased it in another [30]. Possibly the best indication of a mild dipsogenic effect of
GONADECTOMY AND POLYDIPSIA
831
female sex steroids was shown by Bell and Zucker [2] who injected estradiol benzoate and progesterone into adult male rats which had been castrated at birth, and noted an increase in water intake at the same time as a decrease in food intake occurred, even though intakes of food and water are usually positively correlated in this species [ 10]. Estradiol was found to have an inhibitory effect on drinkhag in the rabbit polydipsic from food deprivation [19], but polydipsic drinking during food deprivation seems to be governed by different variables than drinking under ad lib feeding conditions [18]. It seems more likely that in the SWR/J mouse strain, female sex steroids facilitate a degenerative process in the kidneys which results in renal tubules becoming refractory to ADH thus producing primary polyuria and secondary polydipsia [ 13]. Kutcher and Miller [ 12] reported that intake of various NaC1 solutions decreased with age as water intake increased in females, but not in males. It was suggested that this sex difference might be accounted for by the higher water intakes of the females. The present study suggests, however, that for 1.0 percent NaC1, the presence or absence of male sex steroids may be the critical determinant of whether or not NaCI intake declines with age, irrespective of water intake. Here, the two male groups did not differ in water intake, but they did differ in 1.0 percent NaC1 intake. An important question still remains. Why do the ovariectomized and sham-operated females and the castrated males show aversion to relatively dilute NaC1 solutions at a time when polydipsia is well-developed, presumably indicating that the animal's water needs are high? Kutscher and Schmalbach [14] found that intraperitoneal injection of 2.0 percent NaCL into intact, polydipsic, female SWR mice did produce significantly more drinking than seen following a mock injection, but injection of 0.5 and 1.0 percent did not produce drinking, as expected. Rejection of isotonic and hypotonic NaC1 solutions is probably not based on feedback from dehydration consequent to ingestion and absorption of these solutions. It seems more likely that the aversion of SWR mice to these solutions (see salt/water intake ratios in Fig. 4), is the result of the development of a taste aversion. Kutscher and Schmalbach [14] found that polydipsic SWR females, compared to nonpolydipsic
C3H/He and C57BL/6 females, show strong rejection of 6 other relatively dilute solutions including quinine which is markedly hypotonic, even though large weight losses were incurred during the abstinence from drinking. Burke, Mook and Blass [3] found that water-deprived rats readily ingested water and dilute quinine solutions when offered, but when hypertonic NaC1 injections were given to waterdeprived rats, they became finicky. The injection increased water intakes, but decreased intake of quinine solutions, possibly in response to cellular dehydration produced by the NaC1 injection. Further experiments are needed to determine if the high rates of water exchange seen in the development of polydipsia produced finicky drinking in a manner similar to NaC1 injection and whether such a response pattern is a concomitant of any condition which serves to increase rate of water exchange; e.g. familial diabetes insipidus or injection of diurectics or dipsogens. If such an interpretation turns out to be tenable, it is clear that in the SWR mouse, at least, male sex steroids in some way attenuate the development of finicky drinking as water intake increases with age, or the steroid prevents the internal stimulus conditions which trigger such a response pattern. One major problem with the taste-aversion hypothesis as an explanation of the 1.0 percent NaC1 data is that the aversion seems to be prevented or attenuated by testosterone and appears in the absence of testosterone whether the female sex steroids are present or not. In rats, male sex steroids seem to have little influence on reactivity to stimuli, but female steroids make the female more reactive to saccharin [27], quinine [28], footshock [16], and glucose [ 25]. It is obvious that if the taste-aversion hypothesis is correct there may be a significant species difference in the effect of sex steroids on reactivity [31]. The activity measurements were made only on 12 month mice so no statement can be made regarding change over time, but it is clear that this activity measurement is influenced by gonadectomy in identical fashion in the two sexes. This study is consistent with experiments on a very different kind of activity measurement, wheel running, in which both male and female sex steroids potentiate activity [1, 4, 7, 24, 281.
REFERENCES 1. Asdell, S. A., H. Doornenbal and G. A. Speding. Sex steroid hormones and voluntary exercise in rats. J. Reprod. Fertil. 3: 26-32, 1962. 2. Bell, D. D. and I. Zucker. Sex differences in body weight and eating: organization and activation by gonadal hormones in the rat. Physiol. Behav. 7: 27-34, 1971. 3. Burke, G. H., D. G. Mook and E. M. Blass. Hyperreactivity to quinine associated with osmotic thirst in the rat. J. comp. physiol. Psychol. 78: 32-39, 1972. 4. Finger, F. W. Estrus and general activity in the rat. J. comp. physiol. Behav. 68: 461-466, 1969. 5. Furth, J. Leukemia by thymectomy in mice. J. Geront. 1: 46-54, 1946. 6. Gordon, H. A., E. Bruckner-Kardoss and B. S. Wostmann. Aging in germ-free mice: life tables and lesions observed at natural death. £ Geront. 21: 380-387, 1966. 7. Kakolewski, J. W., V. C. Cox and E. S. Valenstein. Sex differences in body-weight change following gonadectomy of rats. Psychol. Rep. 22: 547-554, 1968.
8. Kochakian, C. D. Histochemical study of 'alkaline' phosphatase of the kidney of the castrated mouse after stimulation with various androgens. Am. J. Physiol. 152: 257-262, 1948. 9. Kochakian, C. D. Intracellular regulation of nucleic acids of mouse kidney by androgens. Gen. comp. endocr. 13: 146150, 1969. 10. Kutscher, C. L. Species differences in the interaction of feeding and drinking. Ann. N. Y. Acad. Sci. 157: 539-552, 1969. 11. Kutscher, C. L. Strain differences in drinking in inbred mice during ad libitum feeding and food deprivation. Physiol. Behav. 13: 63-70, 1974. 12. Kutscher, C. L. and D. G. Miller. Age-dependent polydipsia in the SWR/J mouse. Physiol. Behav. 13: 71-79, 1974. 13. Kutscher, C. L, M. Miller, N. L. Schmalbach. Renal deficiency associated with diabetes insipidus in the SWR/J mouse. Physiol. Behav. 14: 815-818, 1975. 14. Kutscher, C. L. and Schmalbach, N. L. Effects of water deprivation, NaCI injection, and seven aversive taste stimulion drinking in two normal mouse strains and one with diabetes insipidus. Physiol. Behav. 15: 1975, in press.
832 15. Marks, H. E. Reactivity to different saccharin concentrations as a function of testing procedure and alterations in body weight of intact and oophorectomized female rats. Physiol. Behav. 12: 2 9 - 3 8 , 1974. 16. Marks, H. E., B. D. Fargason, and S. H. Hobbs. Reactivity to aversive stimuli as a function of alterations in body weight in normal and gonadectomized female rats. Physiol. Behav.9: 5 3 9 - 5 4 4 , 1972. 17. Marks, H. E. and S. H. Hobbs. Changes in stimulus reactivity following gonadectomy in male and female rats of different ages. Physiol. Behav. 8: 1113-1119, 1972. 18. Miller, D. G. and C. L. Kutscher. Effect of nutritive bulk on the suppression of food-deprivation polydipsia in the gerbil. Physiol. Behav. 14: 791-794, 1975. 19. Nocenti, M. R. and L. J. Cizek. Influence of estrogen on renal function and water intake in male rabbits rendered polyuricpolydipsic by food deprivation. Endocrinol. 93: 9 2 5 - 9 3 1 , 1973. 20. Pfeiffer, C. A., V. M. Emmel, and W. U. Gardner. Renal hypertrophy in mice receiving estrogens and androgens. Yale Z Biol. Med. 12: 4 9 3 - 5 0 1 , 1940. 21. Richter, C. P. The effect of early gonadectomy on the gross body activity of rats. Endocrinol. 17: 4 4 5 - 4 5 0 , 1933. 22. Roubicek, C. B., D. E. Ray and M. E. Douglas. Genetic regulation of water intake in the rat. Physiol. Behav. 8 : 1 1 0 7 - 1 1 1 2 , 1972.
SCHMALBACH AND KUTSCHER 23. Salber, P. and I. Zucker. Absence of salt appetite in adrenalectomized and DOCA-treated hamsters. Behav. Biol. 10: 295-311, 1974. 24. Stern, J. J. and M. Murphy. The effects of cyproterone acetate on the spontaneous activity and seminal vesicle weight of male rats. J. Endocr. 50: 4 4 1 - 4 4 3 , 1971. 25. Valenstein, E. S., J. W. Kakolewski and V, C. Cox. Sex differences in taste preference for glucose and saccharin solutions. Science 156: 9 4 2 - 9 4 3 , 1967. 26. Wade, G. N. Gonadal hormones and behavioral regulation of body weight. Physiol Behav. 8: 5 2 3 - 5 3 4 , 1972. 27. Wade, G. N. and I. Zucker. Hormonal and developmental influences on rat saccharin preferences. J. comp. physiol. Psychol. 69: 2 9 1 - 3 0 0 , 1969. 28. Wade, G. N. and I. Zucker. Hormonal modulation of responsiveness to an aversive taste stimulus in rats. Physiol. Behav. 5: 269-273, 1970. 29. Winer, B. J. Statistical principles in Experimental Design. New York: McGraw-Hill, 1971, p. 191f. 30. Zucker, I. Hormonal determinants of sex differences in saccharin preference, food intake, and body weight. Physiol. Behav. 4: 5 9 5 - 6 0 2 , 1969. 31. Zucker, I., G. N. Wade and R. Ziegler. Sexual and hormonal influences on eating, taste preferences, and body weight of hamsters. Physiol. Behav. 8: 101-111, 1972.
Gonadectomy and the Development of Age-dependent Polydipsia and the Intake of NaC1 Solutions in the SWR/J Mouse NANCY L. SCHMALBACH AND CHARLES L. KUTSCHER
Department of Psychology, Syracuse University, Syracuse NY 13210 (Received 17 December 1974) SCHMALBACH, N. L. AND C. L. KUTSCHER. Gonadectomy and the development o f age-dependent polydipsia and the intake o f NaCl solutions in the SIVR/J mouse. PHYSIOL. BEHAV. 14(6) 825-832, 1975. - Gonadectomies were performed at 25 days of life in SWR/J mice, a strain known to develop age-dependent nephrogenic diabetes insipidus with females drinking far more than males. Effect of gonadectomy was different for each of the 3 nonreproductive behaviors studied. (1) Ovariectomy reduced the age-dependent polydipsia seen in females, but castration was without effect in males. (2) Castration of males resulted in a lower intake of an isotonic NaC1 solution, making the castrated males similar to the sham-operated and ovariectomized females, which did not differ. (3) For the short-term activity measurement sham-operated males did not differ from sham-operated females and castrated males did not differ from ovariectomized females. In each sex, gonadectomy reduced activity. Aging
Diabetes insipidus
NaC1
Polydipsia
Sex steroids
KUTSCHER and Miller [12] reported spontaneous polydipsia which increased in severity with age in the SWR/J mouse strain and was more severe in females than in males. Kidneys became refractory to exogenous antidiuretic hormone, resulting in low ceilings of renal concentrating ability. In some mice, large lesions were seen in the renal medulla [13]. In females, but not males, intake of hypertonic and even hypotonic NaC1 solutions, decreased with age as water intake increased [ 1 ]. It was suggested that the age-dependent renal deterioration with its behavioral, anatomical and physiological correlates might be a useful genetic model for the study of aging processes. Renal lesions have been observed with age in both germ-free and conventional mice of other strains [6]. One theory of aging suggests that age-dependent deterioration may be dependent not on spontaneous disruption of metabolic activities within the cells themselves, but upon some change in an epicellular factor which influences cell metabolism. For example, Furth [5] found that incidence of leukemia which usually develops in adult Ak mice was markedly reduced by thymectomy performed at 6 weeks of age.
study over the first 10 months of life was made on male and female mice gonadectomized at weaning. Tests with NaC1 solutions were also made in order to relate intake of hypotonic, isotonic and hypertonic solutions to changes in water intake and the removal of sex structures. METHOD Animals The 30 male and 30 female SWR/J mice used in this experiment were bred in the laboratory at Syracuse University from breeding stock obtained from Jackson Laboratory at Bar Harbor, Maine. Apparatus During the measurement of drinking, mice were housed in individual steel test cages 12.7 X 12.5 X 14.5 cm with tops and floors of 0.6 cm mesh hardware cloth. Fluid intakes were measured by means of 100 ml eudiometer tubes graduated in 0.2 ml units, attached vertically to the back of each cage with the spout protruding approximately 1 cm through an opening in the back of the cage. Purina Chow pellets were available in the cage at all times. Demineralized water was given in drinking tubes except when solution tests were being conducted. Between testing sessions mice were housed in the main animal colony in groups of 1 - 6 in standard 27 X 17 X 13 cm plastic mouse cages where they were given tap water to
EXPERIMENT 1 One possible explanation of the higher water intakes of female SWR/J mice [12] is that female sex steroids in circulation facilitate certain degenerative processes. To test this possibility that type of sex steroids may lead to sex differences in non-reproductive behaviors, a longitudinal 825
826
SCHMALBACH AND KUTSCHER
drink with Purina Chow pellets always available. In the test room and animal colony, lights were on for 12 hr and temperature was maintained at 21 +- I°C. Air was humidified during the winter months. Activity was measured by a Lehigh Valley Electronics activity drum utilizing the two banks of photocells, but not the tilt floor. The drum is a cylinder 44.4 cm in dia. and 41.9 cm high. Inside walls are painted black. Tests were run with lid in place thus producing a semidark test chamber. Procedure For each sex, 15 mice were gonadectomized and 15 were subjected to a sham operation at weaning, 2 5 - 3 0 days of life, under Nembutal anesthesia ( 6 5 - 7 0 mg/kg). Completeness of removal was verified by examination of the ovaries following surgery and by post-mortem examination when animals were sacrificed at 12 months. In the sham-operation condition, ovaries were exposed and visualized but not removed. In the males, testes were removed in the castrated group and merely exposed in the sham-operated group. In all animals incisions were closed with sutures of cotton thread. Mice were numbered by ear-punching at the time of the operation. At 3, 7 and 10 months mice were isolated in metal test cages in the test room. After 3 days of adaptation to the cages and drinking tubes, intake of demineralized water was measured for 7 days with body weight recorded at the beginning and end of the 7 day period. At 10 months food intake was also measured by placing new preweighed pellets in the cage at the beginning of each test day and weighing them at the end of the test day. Following the 7 day water intake measures, mice were given, one at a time in random order, the following NaC1 solutions: 0, 0.5, 1.0, and 2.0 percent. Solutions were presented for 23 hr, 1 hr being required to weigh animals and exchange solutions in tubes. Tests were made on alternate days with animals given water to drink on the intervening days. Body weight was measured at the beginning and end of each test day. Following the NaC1 test sequence, mice were tested in the same manner with 2.0 percent NaC1, 2.0 percent NaC1 + 5.0 percent glucose, and 5.0 percent glucose, given in random order on alternate days. Ten animals of each of the 4 groups were selected for activity measurement. Each mouse was placed in the apparatus and allowed 30 seconds to explore. Then activity (interruption of light beams) was measured for 10 min. Activity counts for both banks of photocells were summed to give the final activity measure. At approximately 12 months mice were sacrificed and both kidneys were removed and weighed. RESULTS Intakes of water, solutions and body weight were analyzed by a 3 × 4 split-plot factorial model of analysis of variance [29] in order to determine if significant differences were produced by the two main variables (1) age (3, 7 and 10 months) and (2) sex and operation, presumably relating to type and level of sex steroid produced and in circulation: sham-operated males, sham-operated females, castrated males and ovariectomized females. Comparisons between individual means were evaluated with Scheffe's test [291.
Water intakes Mean 7 day water intakes (ml) were influenced by age (p<0.01) and sex and operation (p<0.01). The interaction of these two variables was also significant (p<0.01). As may be seen in Fig. I, water intakes were not different for the 4 groups at 3 months. Thereafter, intakes increased with age, but at different rates. Ovariectomy greatly attenuated the development of polydipsia producing differences (p<0.01) in water intakes between the sham females and ovariectomized females at 7 months and at I0 months. Combining data over the 3 testing periods, intakes of the ovariectomized females were greater than intakes of the castrated males, but were not statistically distinguishable from intakes of the sham-operated males. Thus, the ovariectomy eradicated the sex difference in polydipsia development observed in this study and in the previous one [ 12]. The two male groups did not differ from each other. Body Weight This measure increased with age (p<0.01) and differed with sex and operation (p<0.01). The interaction was also significant (p<0.05) indicating that the rate of weight gain differed for the 4 groups (Fig. 1, center). Castration and ovariectomy had opposite effects on body weight. Ovariectomy at weaning produced a higher body weight level than sham-operated females at 3, 7 and 10 months. Castration of males at weaning resulted in body weights significantly lower than in sham-operated males at 7 and 10 months. Combining data over the 3 test periods, weights of castrated males were not different from those of ovariectomized females. Thus, gonadectomy eradicated the expected sex difference in body weight. Expressing water intakes as a proportion of body weight did not change any of the relationships mentioned above (Fig. 1, bottom). Mean daily water intake of the 10 month sham-operated females was considerably more than the body weight of the mice, indicating an extreme state of polydipsia. Food Intakes Food intakes were measured only at 10 months and were analyzed with a 1-way analysis of variance. Scheffe's test was employed to evaluate differences between individual means. Sham-operated males ate more (5.43 g) than castrated males (4.57 g, p<0.01), even though water intakes did not differ. Sham-operated females did not eat more (5.12 g) than ovariectomized females (4.78 g) even though water intakes were quite different at 10 months. Ovariectomized females ate less than sham-operated males (p<0.05), but sham-operated females did not. All other comparisons were not significant. NaCl Solution Intakes. Test solutions were hypotonic (0.0 and 0.5 percent), approximately isotonic (1.0 percent) and hypertonic (2.0 percent) to body fluids. Effects of age and sex and operation on NaC1 solution intake were quite different from water intake. For the 0.5 percent solution (intakes not shown), these variables were without significant effect even though water intakes were quite different. For the 1.0 percent solution, however, intakes differed as a function of age (p<0.01) and sex and operation (p<0.05); however, the interaction was also significant. It may be seen in Fig. 2
GONADECTOMY AND POLYDIPSIA
827
3S-
7
30 -
6
~ 25
S-F
~15
X-F
-
lo
~
Jl
../~__....It"- ~:.~ ~
I
I
I
7 AGE (MOS.)
::F
XF~I~",~
Z2
lO ~"
s....
~281-
1
,w" ~ ,," x-~.-~..i + ,," ,~-F
~ 271""
j
~26J--
2sl-
,_u v4
U0 3
I
../"
I 3
--
~.22t'-
~ 4
--
a_21 i-,.
~
_ . ~ -'~t_
t 7 Ao,: (Mos.)
I 10
t3
<3Z -
I
I I 7 (MOS.)
~
I
I I 10
I
....S-M "'".,ll
x-,
o
s-F
I
S-F
~ 2 -
.~1.0
I
AGE 5
I 3
I
S
_lj';~"+~
,
-M
Z 3
-
(.9 241 -
s-F
c'¢
I I I I I I I I I I I I o
3 AGE
~1~.0-5 ~¢~..~I~1~a__11 ~,~".~,,,.,~_~.~7 ~.-M-U] I',-
~0.1
I
3
I
7
AGE (MOS.)
I
!0
FIG. 1. Mean water intake (ml), body weight (g), and ratio of water intake to body weight of sham-operated female (S-F), ovarieetomized female (X-F), sham-operated male (S-M), and castrated male (X-M) animals plotted as a function of age (months).
7 (MOS.)
10
FIG. 2. Mean 23-hour 1.0 and 2.0 percent NaC1 intake (ml) for sham-operated female (S-F), ovariectomized female (X-F), sham-operated male (S-M), and castrated male (X-M) animals plotted as a function of age (months).
(top) that there are two distinct patterns of age-related change in 1.0 percent NaC1 intake. Sham-operated males show no change in intake with age, but the two female groups and the castrated male group decreased intake with age. Combining data over the 3 ages, intake of shamoperated males exceeded that of castrated males and ovariectomized females (p<0.01). For the 2.0 percent solution, there was a general decline in solution intake with age (p<0.01), but sex and operation condition had no significant effect.
828
SCHMALBACH AND KUTSCHER
25
7 -
0.5~0 NoCI
3 S-F
,-,6-
20
mm
Ol0
v
15 ~4-
10
~3-
ol0 o7
Z o
~2-
5-
mlO 07 r710
r13 03
1[11~17" 3
1-
3 I
I
I
I
I I 1 I I I I I I I 1 I 10 20 30 WATER INTAKE (ML)
I
0
1 36
20
40
2S
60
80
100
I.OTo NaCI
7el0
~20
n
~5
o10
~10
03 _
_
I01~°
03
X
UlO 03 7 I [] m3
_
3g u7
0
07 07
010 I10
~2
07 I
|
I
I
I I 10 WATER
010
07 0~o
i
i
I.
07
i
I 20 INTAKE
20
0 I
i
i
I
I 30
I
I
I 36
I
I
40
60
80
25
In Fig. 3, mean intakes of 1.0 and 2.0 percent NaC1 are shown plotted as a f u n c t i o n of mean 7 day water intake. It is obvious that in the two female groups and in the castrated males, there is an inverse relationship b e t w e e n water intake and 1.0 percent NaC1 intake, even t h o u g h the isotonic salt solution should produce no osmotic stress and d e h y d r a t i o n following ingestion. Apparently the presence o f male sex steroids prevents this inverse relationship since sham males show no decline in 1.0 percent NaC1 intake as water intake increased with age. (Lines shown in Fig. 3, top, were fitted visually). For the 2.0 percent NaC1 solution also, there is an obvious trend for NaC1 intake to decline as polydipsia develops with age. One possible, although unlikely, explanation for the inverse relationship b e t w e e n water and NaC1 intake is that with age the mice b e c o m e more able to extract free water
I 100
2.0% NoCI
(ML)
FIG. 3. Mean 23-hour 1.0 and 2.0 percent NaC1 intake (ml) for sham-operated female (e), ovariectomized female (o), sham-operated male (u), and castrated male (o) animals plotted as a function of mean water intake (ml) at the 3, 7, and 10 month (age) testing periods.
03
I
20 ol0 o!0
15 % f
10
0
-
mlO 07 m7 olo
t '20
I i I 40 60 80 SI/WI x I00
I 100
FIG. 4. Mean body weight loss (%) incurred following 23-hour presentation of 0.5, 1.0 and 2.0 percent NaCI of sham-operated female (e), ovarieetomized female (o), sham-operated male (e), and castrated male (o) animals, plotted as a function of percent ratio of salt intake to water intake at the 3, 7, and 10 month (age) testing periods.
GONADECTOMY AND POLYDIPSIA
829 TABLE 1
INTAKE OF GLUCOSE, NACL AND GLUCOSE-NACL SOLUTIONS Solution Intakes (Mean ± S.E.) 5% Glucose
2% NaCI
5% Glucose + 2% NaC1
Age
Treatment
3 Months
Sham-females Ovar-females Sham-males Cast-males
28.8 22.1 19.8 20.1
± 2.6 ± 1.4 ± 1.6 -+ 1.7
(3.56)* (3.11) (2.75) (3.24)
3.9 1.9 2.8 2.1
± 1.0 ± 0.3 ± 0.4 ± 0.3
3.5 1.9 2.7 2.2
-+ 0.8 -+ 0.3 ± 0.4 ± 0.3
7 Months
Sham-females Ovar-females Sham-males Cast-males
29.1 18.6 23.8 29.6
-+ 3.9 -+ 2.1 ± 2.3 ± 3.5
(1.41) (1.38) (2.45) (3.00)
1.4 0.7 2.3 0.8
+- 0.3 +- 0.2 -+ 0.4 ± 0.1
1.3 0.8 4.3 3.0
-+ 0.2 -+ 0.1 ± 1.0 ± 1.2
10 Months
Sham-females Ovar-females Sham-males Cast-males
55.0 26.9 31.9 35.3
± 5.8 ± 2.4 ± 3.0 ± 2.6
(1.72) (1.65) (2.31) (3.57)
3.0 1.1 4.2 2.4
± 0.6 ± 0.4 -+ 1.2 ± 0.7
3.1 1.6 2.5 2.4
± 0.6 ± 0.3 ± 0.5 -+ 0.5
*Number in parenthesis is ratio of mean glucose intake to mean water intake for each group. from the NaC1 solutions, thus needing less solution volume to secure their daily water requirements. In Fig. 4 are plotted mean body weight losses incurred while drinking the 3 NaC1 solutions, plotted as a function o f the ratio of the NaC1 intake to water intake. Ingestion of NaC1 solutions produced only mean weight losses, no weight gains and some of the losses are very substantial (> 20 percent). Two relationships emerge from these data. First, the higher the concentration of NaC1, the greater the weight losses. Secondly, the lower the intakes of each solution, the greater the weight loss, thus providing no support for the above hypothesis.
Glucose Intake These intakes were affected by age (p<0.01) and sex and operation (p<0.01). The interaction was also significant (p<0.01). As shown in Table 1, there was a general trend for glucose intakes unlike NaC1 intakes, to increase with age, as did water intakes. The only significant difference between individual means (Scheffe's test) were found to be between the female groups. At 10 months, and also combining the 3 test periods, sham-operated females drank more than did ovariectomized females. The ratios o f mean glucose intake to mean water intake shown in Table 1 indicate that offering the glucose solution instead of water increased fluid intake volume even in mice already very polydipsic. In spite of this demonstrated ability of glucose to produce palatability-induced polydipsia in this strain, mixing glucose with the 2.0 percent NaC1 solution did not relieve the strong aversion to the NaCI solution. In the hamster, an animal which has an aversion to a wide variety of NaC1 solutions, the addition o f sucrose or saccharin to 0.3 percent and 1.0 percent NaC1 solutions increased NaC1 intakes [23]. Failure to find such an effect in the present study may be due to the higher concentration of NaCI solution used (2.0 percent) or the possibility that the SWR/J mouse has a greater aversion to NaC1 than the hamster.
Activity Measurement T h e s e measurements were made approximately 2 months subsequent to the 10 month water intake and solution intake tests. Data were analyzed by a 2 X 2 analysis of variance (sex × operation). Activity scores did not differ as a function of sex, but were reduced significantly by gonadectomy (p<0.05). Castrated males were less active (656 counts/10 min) than sham-operated males (794). Ovariectomized females were less active (685) than sham-operated females (796).
Kidney Weights These measurements were also submitted to a 2 × 2 analysis of variance. Significant differences were observed due to sex (p<0.05), operation (p<0.01) and interaction (/9<0.01). Mean kidney weight of castrated males (1.15 g/100 g body weight) was much less than kidney weight of sham-operated males (1.71, p<0.05). In the females, however, the kidney weight of ovariectomized females (1.45) was not different from that of sham-operated females ( 1 . 5 8 ) , a l t h o u g h kidneys of ovariectomized females weighed less than those of sham-operated males (p<0.05). EXPERIMENT 2 Clearly the development of polydipsia in adult SWR mice is greatly attenuated by ovariectomy performed at weaning. At least two possible modes of action can be suggested. (1) Female sex steroids may have a long-term, cumulative action by facilitating a degenerative process in the kidney over the lifespan of the animal, or by setting in motion some degenerative processes during a critical period of the lifespan. (2) Alternatively, female sex steroids may have a dipsogenic effect in the SWR/J mouse, facilitating drinking proportionately to momentary levels of steroids in circulation. If the second alternative is true, ovariectomy of adult, polydipsic mice should result in an immediate de-
830
SCHMALBACH AND KUTSCHER
crease in drinking, compared to sham-operated mice, since the source of sex steroids will be removed. METHOD
Animals Twenty naive, polydipsic, female mice, 14 months of age were used in this experiment.
Apparatus Cages were the same as those used in Experiment 1, except that they had solid metal floors covered with Sterolit bedding.
Procedure Ten mice were ovariectomized and 10 were given a sham operation. Prior to surgery mice were given 3 days to adapt to the cages followed by 7 days of water intake measurements. Mice were weighed on the first and last day of this 7 day period, Mice were ovariectomized under Nembutal anesthesia ( 6 5 - 7 0 mg/kg). After 1--2 days of postoperative rest, water intakes were measured for 7 consecutive days with body weights measured on the first and last days of this period. RESULTS Mean preoperative water intakes were 32.6 ml for the Sham-operated Group and 34.4 ml for the Ovariectomized Group. This difference was not significant. Following surgery, mean water intakes were 31.0 ml for the Shamoperated Group and 30.1 ml for mice in the Ovariectomized Group, a difference which was not significant. DISCUSSION In this experiment, three types of non-reproductive behaviors were altered by gonadectomy, but the resultant patterns of change produced were different in each case. For water intake, ovariectomy did not prevent the development of polydipsia with age, but attenuated it, such that the ovariectomized females did not differ in intake statistically from the sham-operated males, but were slightly, but significantly higher in intake than the castrated males. For intake of 1.0 percent NaC1, two basic intake patterns emerged. Sham-operated males did not change NaC1 intake as water intake increased with age. In the other 3 groups, NaC1 intake decreased as water intake increased. For general activity, gonadectomy decreased activity in both males and females. It is unlikely that one simple hypothesis can account for all these findings. It is well-known that castration of male rats results in a reduction in weight gain with age and ovariectomy produces an increase in weight gain [17,26] even when the operation is performed in rats as old as 100 days. A comprehensive literature review showed that castration also produced an increase in weight gain in baboons, cattle, guinea pigs and turkeys [7]. Rate of weight change is responsive to short-term changes in circulating levels of steroids in rats [26], e.g. the expected weight gain in adult, ovariectomized rats was prevented by injection of estrogen [16]. Similarly, the decrease in kidney size caused by castration in the SWR mouse with diabetes insipidus is similar to
changes in nondiabetic mouse strains. Castration of male mice resulted in lower kidney weight in DBA mice [ 8 ] and Holtzman-Swiss mice [9]. Testosterone has a well-known renotrophic influence [20] on kidney protein metabolism, DNA and RNA, by means of action in the ribosomal particulates [8,9]. Thus, the presence and development of nephrogenic diabetes insipidus did not alter the expected action of testosterone to facilitate and maintain growth of the kidney in the male. Female sex steroids do not appear to have this renotrophic effect since in the present study kidneys of ovariectomized females are not different from those of sham females. The interpretation of water intakes is not so easily guided by precedents in the literature. It is clear in this experiment that the large differences in water intakes between sham-operated females and ovariectomized females do not relate to kidney size since these two groups do not differ on this dimension. Furthermore, the kidneys of castrated males were 33 percent smaller than those of shamoperated males yet water intakes did not differ from this group. Significant correlations between food intake and water intake and between body weight and water intake have been found in 6 nonpolydipsic mouse strains [11]. However, it is clear in the present study that the large difference in water intakes between the two female groups was not due to a difference in food intake, since none was found, or to body weight differences, since sham-operated females weighed less, but drank more, than ovariectomized females. In studies of polydipsia it is important to determine to what extent the magnitude of water intake reflects the water needs of the animal, i.e. polyuria is primary and polydipsia is an obligatory adjustment required to maintain normal levels of body water. Rats selectively bred for high water intakes [22] could tolerate restriction of water intake to 50 percent of normal and suffer only a transitory effect on feeding and weight gain. In the SWR mouse strain, however, all indications are that the high water intakes indicate a high water need. Polydipsic female SWR/J mice have very limited renal concentrating ability and can produce urine only slightly hypertonic to plasma even when challenged with water deprivation because kidney tubules are refractory to antidiuretic hormone [13]. Although some mouse strains can survive without water for up to 10 days (Kutscher, unpublished study), polydipsic SWR mice are markedly weakened by only one day of deprivation, sometimes showing piloerection and cessation of temperature regulation. In the present experiment, the greater water intakes of the sham-operated females, compared to the ovariectomized females, probably indicate a greater water need. When given 1.0 percent NaC1, weight loss (an approximation of dehydration) of 10 month sham-operated females (19.9 percent) exceeded that of ovariectomized females (13.0%; p<0.05). When 2.0 percent NaC1 was offered, again, sham-operated females showed greater weight loss (22.7 percent) than ovariectomized females (16.6%; p<0.01) even though fluid intakes did not differ for the two groups under these conditions. Experiment 2 provided no indication of a short-term facilitatory effect of sex steroids on drinking and there is no clear precedent in the literature for such an effect. Ovariectomy produced no significant change in water intake in one study [15], but increased it in another [30]. Possibly the best indication of a mild dipsogenic effect of
GONADECTOMY AND POLYDIPSIA
831
female sex steroids was shown by Bell and Zucker [2] who injected estradiol benzoate and progesterone into adult male rats which had been castrated at birth, and noted an increase in water intake at the same time as a decrease in food intake occurred, even though intakes of food and water are usually positively correlated in this species [ 10]. Estradiol was found to have an inhibitory effect on drinkhag in the rabbit polydipsic from food deprivation [19], but polydipsic drinking during food deprivation seems to be governed by different variables than drinking under ad lib feeding conditions [18]. It seems more likely that in the SWR/J mouse strain, female sex steroids facilitate a degenerative process in the kidneys which results in renal tubules becoming refractory to ADH thus producing primary polyuria and secondary polydipsia [ 13]. Kutcher and Miller [ 12] reported that intake of various NaC1 solutions decreased with age as water intake increased in females, but not in males. It was suggested that this sex difference might be accounted for by the higher water intakes of the females. The present study suggests, however, that for 1.0 percent NaC1, the presence or absence of male sex steroids may be the critical determinant of whether or not NaCI intake declines with age, irrespective of water intake. Here, the two male groups did not differ in water intake, but they did differ in 1.0 percent NaC1 intake. An important question still remains. Why do the ovariectomized and sham-operated females and the castrated males show aversion to relatively dilute NaC1 solutions at a time when polydipsia is well-developed, presumably indicating that the animal's water needs are high? Kutscher and Schmalbach [14] found that intraperitoneal injection of 2.0 percent NaCL into intact, polydipsic, female SWR mice did produce significantly more drinking than seen following a mock injection, but injection of 0.5 and 1.0 percent did not produce drinking, as expected. Rejection of isotonic and hypotonic NaC1 solutions is probably not based on feedback from dehydration consequent to ingestion and absorption of these solutions. It seems more likely that the aversion of SWR mice to these solutions (see salt/water intake ratios in Fig. 4), is the result of the development of a taste aversion. Kutscher and Schmalbach [14] found that polydipsic SWR females, compared to nonpolydipsic
C3H/He and C57BL/6 females, show strong rejection of 6 other relatively dilute solutions including quinine which is markedly hypotonic, even though large weight losses were incurred during the abstinence from drinking. Burke, Mook and Blass [3] found that water-deprived rats readily ingested water and dilute quinine solutions when offered, but when hypertonic NaC1 injections were given to waterdeprived rats, they became finicky. The injection increased water intakes, but decreased intake of quinine solutions, possibly in response to cellular dehydration produced by the NaC1 injection. Further experiments are needed to determine if the high rates of water exchange seen in the development of polydipsia produced finicky drinking in a manner similar to NaC1 injection and whether such a response pattern is a concomitant of any condition which serves to increase rate of water exchange; e.g. familial diabetes insipidus or injection of diurectics or dipsogens. If such an interpretation turns out to be tenable, it is clear that in the SWR mouse, at least, male sex steroids in some way attenuate the development of finicky drinking as water intake increases with age, or the steroid prevents the internal stimulus conditions which trigger such a response pattern. One major problem with the taste-aversion hypothesis as an explanation of the 1.0 percent NaC1 data is that the aversion seems to be prevented or attenuated by testosterone and appears in the absence of testosterone whether the female sex steroids are present or not. In rats, male sex steroids seem to have little influence on reactivity to stimuli, but female steroids make the female more reactive to saccharin [27], quinine [28], footshock [16], and glucose [ 25]. It is obvious that if the taste-aversion hypothesis is correct there may be a significant species difference in the effect of sex steroids on reactivity [31]. The activity measurements were made only on 12 month mice so no statement can be made regarding change over time, but it is clear that this activity measurement is influenced by gonadectomy in identical fashion in the two sexes. This study is consistent with experiments on a very different kind of activity measurement, wheel running, in which both male and female sex steroids potentiate activity [1, 4, 7, 24, 281.
REFERENCES 1. Asdell, S. A., H. Doornenbal and G. A. Speding. Sex steroid hormones and voluntary exercise in rats. J. Reprod. Fertil. 3: 26-32, 1962. 2. Bell, D. D. and I. Zucker. Sex differences in body weight and eating: organization and activation by gonadal hormones in the rat. Physiol. Behav. 7: 27-34, 1971. 3. Burke, G. H., D. G. Mook and E. M. Blass. Hyperreactivity to quinine associated with osmotic thirst in the rat. J. comp. physiol. Psychol. 78: 32-39, 1972. 4. Finger, F. W. Estrus and general activity in the rat. J. comp. physiol. Behav. 68: 461-466, 1969. 5. Furth, J. Leukemia by thymectomy in mice. J. Geront. 1: 46-54, 1946. 6. Gordon, H. A., E. Bruckner-Kardoss and B. S. Wostmann. Aging in germ-free mice: life tables and lesions observed at natural death. £ Geront. 21: 380-387, 1966. 7. Kakolewski, J. W., V. C. Cox and E. S. Valenstein. Sex differences in body-weight change following gonadectomy of rats. Psychol. Rep. 22: 547-554, 1968.
8. Kochakian, C. D. Histochemical study of 'alkaline' phosphatase of the kidney of the castrated mouse after stimulation with various androgens. Am. J. Physiol. 152: 257-262, 1948. 9. Kochakian, C. D. Intracellular regulation of nucleic acids of mouse kidney by androgens. Gen. comp. endocr. 13: 146150, 1969. 10. Kutscher, C. L. Species differences in the interaction of feeding and drinking. Ann. N. Y. Acad. Sci. 157: 539-552, 1969. 11. Kutscher, C. L. Strain differences in drinking in inbred mice during ad libitum feeding and food deprivation. Physiol. Behav. 13: 63-70, 1974. 12. Kutscher, C. L. and D. G. Miller. Age-dependent polydipsia in the SWR/J mouse. Physiol. Behav. 13: 71-79, 1974. 13. Kutscher, C. L, M. Miller, N. L. Schmalbach. Renal deficiency associated with diabetes insipidus in the SWR/J mouse. Physiol. Behav. 14: 815-818, 1975. 14. Kutscher, C. L. and Schmalbach, N. L. Effects of water deprivation, NaCI injection, and seven aversive taste stimulion drinking in two normal mouse strains and one with diabetes insipidus. Physiol. Behav. 15: 1975, in press.
832 15. Marks, H. E. Reactivity to different saccharin concentrations as a function of testing procedure and alterations in body weight of intact and oophorectomized female rats. Physiol. Behav. 12: 2 9 - 3 8 , 1974. 16. Marks, H. E., B. D. Fargason, and S. H. Hobbs. Reactivity to aversive stimuli as a function of alterations in body weight in normal and gonadectomized female rats. Physiol. Behav.9: 5 3 9 - 5 4 4 , 1972. 17. Marks, H. E. and S. H. Hobbs. Changes in stimulus reactivity following gonadectomy in male and female rats of different ages. Physiol. Behav. 8: 1113-1119, 1972. 18. Miller, D. G. and C. L. Kutscher. Effect of nutritive bulk on the suppression of food-deprivation polydipsia in the gerbil. Physiol. Behav. 14: 791-794, 1975. 19. Nocenti, M. R. and L. J. Cizek. Influence of estrogen on renal function and water intake in male rabbits rendered polyuricpolydipsic by food deprivation. Endocrinol. 93: 9 2 5 - 9 3 1 , 1973. 20. Pfeiffer, C. A., V. M. Emmel, and W. U. Gardner. Renal hypertrophy in mice receiving estrogens and androgens. Yale Z Biol. Med. 12: 4 9 3 - 5 0 1 , 1940. 21. Richter, C. P. The effect of early gonadectomy on the gross body activity of rats. Endocrinol. 17: 4 4 5 - 4 5 0 , 1933. 22. Roubicek, C. B., D. E. Ray and M. E. Douglas. Genetic regulation of water intake in the rat. Physiol. Behav. 8 : 1 1 0 7 - 1 1 1 2 , 1972.
SCHMALBACH AND KUTSCHER 23. Salber, P. and I. Zucker. Absence of salt appetite in adrenalectomized and DOCA-treated hamsters. Behav. Biol. 10: 295-311, 1974. 24. Stern, J. J. and M. Murphy. The effects of cyproterone acetate on the spontaneous activity and seminal vesicle weight of male rats. J. Endocr. 50: 4 4 1 - 4 4 3 , 1971. 25. Valenstein, E. S., J. W. Kakolewski and V, C. Cox. Sex differences in taste preference for glucose and saccharin solutions. Science 156: 9 4 2 - 9 4 3 , 1967. 26. Wade, G. N. Gonadal hormones and behavioral regulation of body weight. Physiol Behav. 8: 5 2 3 - 5 3 4 , 1972. 27. Wade, G. N. and I. Zucker. Hormonal and developmental influences on rat saccharin preferences. J. comp. physiol. Psychol. 69: 2 9 1 - 3 0 0 , 1969. 28. Wade, G. N. and I. Zucker. Hormonal modulation of responsiveness to an aversive taste stimulus in rats. Physiol. Behav. 5: 269-273, 1970. 29. Winer, B. J. Statistical principles in Experimental Design. New York: McGraw-Hill, 1971, p. 191f. 30. Zucker, I. Hormonal determinants of sex differences in saccharin preference, food intake, and body weight. Physiol. Behav. 4: 5 9 5 - 6 0 2 , 1969. 31. Zucker, I., G. N. Wade and R. Ziegler. Sexual and hormonal influences on eating, taste preferences, and body weight of hamsters. Physiol. Behav. 8: 101-111, 1972.