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Behavioral effects of ethynyl estrogens in the female rat
Physiology&Behavior,Vol. 25, pp. 409-415. PergamonPress
and Brain Research Publ., 1980. Printedin the U.S.A.
Behavioral Effects of Ethynyl Estrogens in the Female Rat I N E L L O J. S P I T E R I , 2 R O B E R T F. D R E W E T T A N D U N A P A D E L
Department o f Psychology, University of Durham, Science Laboratories, South Road Durham DHI 3 LE, England R e c e i v e d 22 F e b r u a r y 1980 SPITERI, N. J., R. F. DREWETT AND U. PADEL. Behavioral effects ofethynyl estrogens in thefemale rat. PHYSIOL. BEHAV. 25(3) 41)9-415, 1980.--The synthetic analogues of estradiol, ethynyl estradiol and mestranol, are used in oral contraceptives. Their effects on food intake and sexual behavior were evaluated in female rats, and compared with those of estradiol. It was found (Experiment 1) that both ethynyl estradiol and mestranol reduced food intake reliably, and more than estradiol. Water intake and body weight followed similar trends. Ethynyl estradiol, but not mestranol, plus progesterone stimulated proceptive and receptive behavior in ovariectomized female rats. Daily administration of ethynyl estrogens without progesterone showed similar trends (Experiment 3). It is hoped that these studies provide guidelines for further work on the effects of synthetic estrogens on sub-primate and primate behavior. Ethynyl estrogens Food and water intake Contraceptive steroids
Body weight
THE two synthetic estrogens, ethynyl estradioi (EE: 17ctethynyl- 1,3,5(10)-estratrien-3,17/3-diol) and Mestranol (MEE: 17a-ethynyl-3-methoxy-l,3,5(10)-estratrien-17/3-ol) have an important role in contraceptive steroids. One or the other, in combination with a progestogen, is an essential component in nearly all combination-type oral contraceptives currently in use. Knowledge of the pharmacological and physiological properties of ethynyl estrogens is accumulating gradually [16]; but studies of their behavioral effects are sadly lacking, which is surprising considering the wide use of ethynyl estrogens in oral contraceptives [5]. This lack of knowledge was the stimulus for the series of experiments described in this paper. EXPERIMENT 1 Estradiol has an anorexic effect [7, 8, 23, 25]. This experiment investigates the effects of ethynyl estrogens on food intake, water intake and body weight. Estradiol-17/3 (E) was used for comparison. METHOD
Experimental Animals and Housing Eighteen experimentally and sexually naive female Wistar rats, about 120 days of age, were used. They were kept under a 12 hr light-dark reversed cycle (lights out at 12.00 hr). Food (Rat and Mouse maintenance diet No. 1--BP
Sexual behavior
Behavioral effects
Nutrition, Witham) and water were available ad lib. The temperature was kept constant at 21°C. Ten days before ovariectomy, the rats were housed singly in plastic cages (42 x 30 x 17 cm). Coprophagia was prevented by a removable galvanized wire grid, 2 cm above the bottom of the cage. Dry, powdered food (Diet No. 1) was available in a metal food hopper which was attached to the top of the cage. This hopper was designed to keep spillage down to a minimum (about 0.2 g daily). This spillage was collected on paper towelling placed beneath the grid under the hopper. Body weight, food and water intake were measured to the nearest 1.0 g, 0.1 g and 0.1 ml, respectively, on four consecutive days prior to ovariectomy, irrespective of the stage of the estrous cycle. The rats were ovariectomized under ether anesthesia during the course of one day. They were then returned to their respective cages and body weight, food and water consumption were monitored postoperatively in order to determine when nutrient intake and body weight had returned to pre-operative levels. At this point, the experiment was started.
Hormone Administration Equimolar solutions of estradiol-17fl (5/~g/0.1 ml arachis oil), ethynyl estradiol (5.44/~g) and mestranol (5.69/zg) were prepared. The hormones were kept in blackened bottles and maintained at room temperature (21°C) to avoid decomposition. Fresh solutions of the hormones were prepared before each treatment period.
1The authors wish to thank Professor F. V. Smith and Professor M. J. Morgan for laboratory facilities in the Department of Psychology, University of Durham; Dr. A. Still and Dr. D. Wastell for statistical advice, and Dr. K. Kendrick and D. Harper for technical assistance. 2Reprint requests should be sent to: Nello J. Spiteri, Department of Zoology, State University of Groningen, Kerklaan 30, 9751 NN HAREN, The Netherlands.
C o p y r i g h t © 1980 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/80/090409-07502.00/0
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FIG. 1. Food intake (mean _+ SEM) of ovariectomized rats before, during and after subcutaneous injections of either E (5/zg) or EE (5.44/zg) or MEE (5.69/zg). Injections were started on Day 1, after food intake measurements were made, and continued up to and including Day 4. Days 9 to 12 represent 'recovery' days.
Experimental Design All rats were treated with all 3 hormones. The treatment order for the 18 rats was counterbalanced to give three replications of the six possible orders. Three "treatment periods" were separated by ten "treatment-free" days, which allowed body weight, food and water intake to return to normal. Each treatment period consisted of four days during which the animals were given hormone injections. The animals were injected with one of the three hormones from Monday to Thursday of each treatment week. Food and water intake were measured from Monday to Friday of each week: the food hoppers and water bottles were filled and weighed daily. The animals were weighed daily and all measurements made between 09.00 hr and 10.00 hr, 2 hours before lights-out. On four days during the recovery period (from Tuesday to Friday) body weight, food and water intake were measured as for the treatment period. Thus records were kept for both treatment and recovery periods; the overall duration of the experiment was six weeks.
Statistical Analyses The results were analysed using analysis of variance (0between, two-within factors). The Least Significant Difference test [10] was used for post-hoc comparisons between means. RESULTS AND DISCUSSION
Food Intake Figure 1 shows the effect of estrogen treatment on daily food intake. As expected, estrogen treatment depressed food intake (when compared with pre-treatment levels). Although E caused a decrease in food intake, MEE caused a greater decrease and EE caused the greatest depression in food intake. The overall difference in food intake was significant, F(2,34)=54.24, p<0.001, and both individual comparisons were also significant, E vs MEE: F(1,34)=15.17, p<0.001; MEE vs EE: F(1,34)=41.30, p<0.001.
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Water Intake Figure 2 shows that the depression of water consumption by estrogen administration was similar to the depression in food intake, and the overall difference was significant, F(2,34)=26.36, p<0.01. Individual comparisons, however, showed that there was no difference in water consumption between E and MEE treated rats, F(1,34)=4.08, 0.06> p>0.05. However, comparisons between these two treatment groups (between means on the fourth day of treatment), using the appropriate error term, revealed that there was reliable difference in water intake, F(1,102)=7.63, p<0.01, on that day. Rats treated with EE drank significantly less than MEE treated rats, F(i,34)=25.26, p<0.001.
Body Weight The results were expressed as % change in body weight. Figure 3 shows that there was a significant change in weight with estrogen treatment, F(2,34)=20.23, p<0.001. Rats treated with MEE and E did not show any difference in percentage weight change, F(1,34)<1, p = n s , although by Day 5 a difference appeared, F(I,102)=5.23, p<0.05. Rats treated with EE significantly lost more weight than MEE treated rats, F(1,34)=25.26, p<0.001. These rats also showed a greater weight loss over days than either E or MEE treated rats, F(6,102)= 10.80, p<0.001. During the recovery period, the body weight of all animals approximated normal levels. The experiment reported here shows that the two synthetic estrogens used in oral contraceptive formulations, MEE and EE, depress food and water intake as well as body weight. Other estrogens are known to have similar effects |251. MEE and E had similar effects, but MEE treated rats showed a slower recovery to control levels of food intake, water intake and body weight. EE treatment produced the greatest depression of all three measures which were recorded during the experiment. These various effects may reflect differences in the pharmaco-kinetic properties of these synthetic estrogens.
E T H Y N Y L E S T R O G E N S A N D F E M A L E RAT B E H A V I O R
411 either estradiol or ethynyl estradiol [2,3], could account for the slower recovery to normal levels of feeding, drinking and body weight in mestranol treated rats.
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Apart from the metabolism of estradiol to estrone leading to catecholestrogen formation, competitive hydroxylation also occurs at the C-16 position giving rise to estriol which is highly uterotrophic [12,19]. On the other hand, hydroxylation at the C-2 position, and therefore catecholestrogen formation, is the major metabolic process for ethynyl estradiol. In the rat, this process is in fact 51)% higher than for estradiol [4]. Recently it has been observed that catecholestrogens significantly reduced food intake in rats (13 cited in [11]). It is possible, therefore, that an increase in catecholestrogen formation may have caused partly, the greater decrease in food intake shown by rats treated with ethynyl estradiol. A second possibility is that ethynyl estradiol may have peripheral metabolic effects, for example by inducing changes in triglyceride clearance and fat metabolism, leading to reduced food intake. This has been recently suggested for the naturally occurring ovarian estrogens [27]. The observed depression in water intake by ethynyl estradiol treated rats may also be due to a reduction in /3-adrenergic responsiveness. It has been shown that chronic treatment with ethynyl estradiol plus norethynodrel (a progestogen with estrogenic properties) attenuates the responses to both hypovolemic and osmotic thirst mechanisms; and that these responses are mediated by EE alone [24]. The changes in food and water intake observed in rats treated with mestranol are probably due to the action of its major metabolite ethynyl estradioi, since mestranol itself is of very low "estrogenicity". Further, since mestranol is capable of binding to estrogen receptors in a form with low estrogenic activity [15], the number of receptors available for the uptake of its active metabolite (EE) would be reduced. The " a c t i v a t i o n " of such receptors would depend, to a large extent, on firstly the high lipophilicity of mestranol leading to its greater storage (and slower release rate), by adipose, adrenal and brain tissue [2,3], and secondly the metabolism Of mestranol to ethynyl estradiol. These properties, associated with the different pattern of excretion of mestranoi from
Treatment of ovariectomized rats with estradiol benzoate induces high levels of sexual behavior within five days of treatment [6]. Nearly all contraceptive steroids of the combination-type contain synthetic estrogens, either ethynyl estradiol or mestranol. We have therefore investigated the effects of ethynyl estrogens on sexual behavior of female rats. There is some evidence that ethynyl estradiol increases the receptivity of intact female rats [28]; but no detail of measures of sexual receptivity or proceptivity is reported. This experiment, therefore, investigates the effects of ethynyl estradioi and mestranol in combination with progesterone on the receptivity and proceptivity of ovariectomized female rats. Experiment 3 examines the effect of daily administration of ethynyl estrogens in the absence of exogenous progesterone. Estradiol-17/3 was used for comparison. METHOD
Experimental Animals and Housing Twenty-four female Wistar rats, 150 days of age, were housed in large group cages, in batches of 8 animals per cage, under a 12 hr light-dark reversed cycle (lights out at 12.00 hr). F o o d and water were available ad lib. The temperature was thermostatically controlled at 21°C. All the females were ovariectomized under ether anaesthesia 3 weeks before the start of any experimental procedures. Estrus was reinstated with injections of estradiol benzoate (5/xg in 0.1 ml arachis oil, 48 and 24 hr before the test) and progesterone (500/zg in 0.1 ml arachis oil, 6 hr before testing), and all females were given sexual experience by allowing them ten mounts with intromissions, by active males on three separate occasions in a testing arena. The females were randomly divided into three groups, consisting of eight rats per group. Twenty sexually experienced Wistar males (proven copulators) were used in the tests for sexual behavior. The testing arena was 90 cm in diameter and had a circular blackened wall 30 cm high. Experimental testing was carried out during the dark period of the animals' light-dark cycle in a room lit by a 60 W red light-bulb. Animals were habituated to the arena before the test. A female was placed with a sexually active male until the male performed 10 mounts with pelvic thrusting. Lordosis was recorded when a female showed pronounced concave arching of the back in response to mounting by a male. In addition to this measure, a general measure of proceptivity (or soliciting behavior, see [1]) was obtained by counting the number of inter-mount intervals during which soliciting behaviors such as darting and hopping, ear wiggling or nipping the male's face occurred [17].
Experimental Design Each group of females received all three hormone treatments (3×3 latin square design). The three experimental periods were separated by ten days during which no treatment was given. This period minimized any possible interaction between estrogens. The rats were given four daily subcutaneous injections of either estradiol (5 p.g/0.1 ml arachis oil), or ethynyl estradiol (5.44/xg), or mestranol (5.69 ptg).
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Twenty-four hours after the 4th injection and 6 hours before testing began at 15.00 hr, all the rats were injected with 500 tzg of progesterone in 0.1 ml arachis oil per rat. All testing during the experiment was run "blind".
Statistical Analyses Data were analysed with a Friedman two-way analysis of variance, and the Wilcoxon matched-pairs signed-ranks test with a z-transformation was used for post-hoc comparisons between groups [9]. All tests were two-tailed.
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Number of inter-mount intervals with soliciting behavior FIG. 5. The number of inter-mount intervals during which ovariectomized rats, injected with equimolar doses of either E or EE or MEE (for 4 days before testing), and progesterone (6 hr before testing), showed soliciting behavior. also shown by sexually receptive females, but were remarkably passive during mounts with pelvic thrusting by the males. Some females sniffed or chewed the sawdust, and continued doing so during mounts. Others investigated the males' anogenital region quite thoroughly, but did not show any soliciting behavior. This passivity is uncharacteristic of diestrous females, which show active rejection of the male [12].
RESULTS
The overall difference in lordosis behavior was significant (~(2) =37,02, p <0.001). The difference in the number of lordosis to ten mounts by a male, between the E and EE treated females and the MEE treated females is evident from Fig. 4. Females treated with EE, however, showed a higher number of lordosis responses than females treated with E (z=2.551, p<0.01). Proceptivity was also different between the groups (X2(2)=36.02; p<0.001); and the rats treated with EE showed significantly more soliciting behavior than females treated with E (z=2.229, p<0.05). Rats treated with MEE showed no soliciting behavior (Fig. 5). However, these rats did not actively reject the males' attempts to mount. They all showed a noticeable stiffening of the tail, a characteristic
EXPERIMENT 3 METHOD
Experimental Animals Forty-two female Wistar rats, 150 to 200 days of age, were ovariectomized at least two weeks before the start of any experimental procedures.
Experimental Design Estrus was reinstated in all the rats and they were given sexual experience as in Experiment 2. Each rat was randomly allocated to one of three equal groups: one group received 5/.tg of E in 0.1 ml arachis oil per
E T H Y N Y L E S T R O G E N S A N D F E M A L E RAT B E H A V I O R
413 100
rat per day. A second group received 5.44/xg of EE in 0.1 ml oil; and a third group received 5.69/zg of M E E in 0.1 ml oil (n= 14 per group). All injections were administered subcutaneously, at 09.00 hr every morning. The room, testing arena and testing procedure were the same as those used in Experiment 2. In addition to measures of lordosis and proceptivity, a rejection measure was also obtained. This was done by counting the number of intermount intervals during which either kicking away the male, or fighting or " b o x i n g " [22] were observed to occur. These behaviors are also shown by diestrous females in response to mounts or attempted mounts by a male. The lordosis quotient was derived by applying the following formula:
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Statistical Analyses
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The lordosis, proceptivity and rejection quotients for each day were analysed using a Kruskal-Wallis one-way analysis of variance. Mann-Whitney U-tests, with a z-transformation were used in conducting post-hoc comparisons between groups [9]. All tests were two-tailed. RESULTS
The results are presented in Fig. 6. Lordosis quotients for Day 1 were the same for all three groups (0%), and were therefore excluded from statistical analysis. However, there was an overall significant difference in the performance of lordosis between groups from Day 3 to Day 9 inclusive (p<0.01). The difference in lordosis responses between EE and E treated females over M E E treated females was obvious and large (Fig. 6(A)). Females treated with E and EE did not show a difference in lordosis behavior up to and including Day 3; but EE treated rats showed significantly (p<0.05) more lordosis in response to mounts by a male thereafter, with the exception of Day 8 (z= 1.86; p~<0.06). These results followed a similar trend to the one observed in Experiment 2. There appeared a significant change in soliciting behavior (Fig. 6(B)) after Day 3 of testing (p<0.001). The M E E treated females showed significantly less soliciting behavior than the two other estrogen groups (p<0.01). A difference in proceptivity between E and EE females did not appear until the 5th day of testing and lasted until the 7th day (p<0.05). Females from both these groups showed a similar amount of soliciting behavior on Days 8 and 9. The rejection of a male by a female rat was frequent and similar for all three groups during the first three days of treatment (Fig. 6(C)). During the next 6 days, however, there
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FIG. 6. (A) Lordosis quotients, (B) proceptivity quotients and (C) rejection quotients of ovariectomized rats treated with equimolar doses of either estradiol (5 /zg standard), or ethynyl estradiol or mestranol daily, for 9 days; n= 14 per group.
was an overall significant change in rejection behavior (p<0.001). Females treated with MEE rejected the males' attempts to mount more often than E treated females (range of z=2.41 to 4.61, p=0.05 to 0.001); but female rats treated with EE rejected males on a fewer number of occasions than E treated females. This difference proved significant between Days 4 and 7 of hormone treatment (p<0.01). DISCUSSION Two main points have emerged from the present experiments. First, the quality of female sexual behavior is differentially affected by the estrogens. Second, daily treatment with these estrogens leads to the differential appearance of pre-copulatory and copulatory components of sexual behavior. The most prominent female sexual behaviors seen in response to daily administration of estradiol or ethynyl estradiol were (1) a decrease in male rejection, (2) soliciting behavior and (3) lordosis behavior.
414
SPITERI E T A L .
While female aggression towards a male was obvious during the initial days of hormone treatment, females gradually showed soliciting behavior and then lordosis behavior in response to a male's mount (with the exception of mestranol treated rats). This type of lordosis behavior seemed similar to " f o r c e d " lordosis, the female sometimes moving away as the male dismounted [30]. With continued hormone treatment, the elicited lordoses were more intense, with deeper arching of the back, and the posture was kept after the male dismounted. Females which demonstrated intense lordosis showed this behavior when a male bumped into the back of a female prior to mounting. The degree of concavity of the back during lordosis is more pronounced as the estrous period progresses [21], and there is a similar effect with increasing, exogenously administered estrogen doses [30]. In this study, high proceptivity quotients were associated with high lordosis levels. The spontaneous occurrence of dart-hop movements, ear wiggling and lordosis is indicative of the very highest levels of receptivity at estrus [17]. Spontaneous dart-hop movements and ear wiggling were seen most frequently in females treated with ethynyl estradiol. Ethynyl estradiol enhanced lordosis behavior and reduced rejection behavior in females to a greater extent than estradiol. The transient difference in soliciting behavior between the two female groups appeared more gradually and, by the final day, all the females treated with estradiol or ethynyl estradiol showed high and similar levels of soliciting behavior. One point that is of interest is that during the development of sexual behavior in females which were treated with either estradiol or ethynyl estradiol, females showed lordosis and soliciting behavior as well as rejecting the males. This suggests that although a female rat showed lor-
dosis to mounts by a male, she did not readily accept all his mounting attempts. Lordosis and soliciting behavior are consistent with enhanced receptivity while rejection behavior suggests the opposite. Mestranol treated rats, however, did not exhibit any signs of receptivity. They did show some passivity to attempted mounts by males, particularly during the last two days of treatment; but generally they were not as passive as the mestranol + progesterone treated females in Experiment 2, frequently rejecting and fighting with the males. Although this comparison is somewhat subjective, it supports findings which suggest that progesterone has sedative or depressant effects on central nervous system activity [14]. Differences in the uptake and metabolism of estradiol17/3, ethynyl estradiol and mestranol may have also led to the differential appearance of appetitive and consummatory components of sexual behavior. The finding that mestranol suppressed food intake reliably more than estradiol (Experiment 1), but failed to stimulate sexual behavior (Experiment 2 and 3) may be attributable, in part, to the different "response thresholds" of these two behavioral systems to the different estrogens. For example, it has been demonstrated that doses of estradiol which reliably depressed food intake [8] were considerably lower than those which stimulated either running activity [10,29] or sexual behavior [30] in the female rat. Clearly, animal studies, particularly those involving subprimate species are of limited relevance to any studies of the effects of synthetic estrogens on human behavior. They could, however, provide useful guidelines for further work in this field.
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9. Ferguson, G. A. Statistical Analysis in Psychology and Education. Int. student edition. Tokyo: McGraw Hill Kogakusha, 1976. 10. Finger, F. W. Estrus and general activity in the rat. J. comp. physiol. Psychol. 68: 461-466, 1969. 11. Fishman, J. Appetite and sex hormones. In: Appetite and Food Intake, edited by T. Silverstone. Berlin: Dahlem Konferenzen. Life Sciences Research Report, No. 2,207-218, 1976. 12. Fishman, J., H. Bradlow and T. F. Gallagher. Oxidative metabolism of estradiol. J. biol. Chem. 235: 3104-3107, 1%0.
13. Garattini, S. and J. Fishman. 2-Hydroxyestrone-induced reduction of food intake by rats. Unpublished observation, 1975, cited in [11]. 14. Gyermek, G., G. Genther and N. Fleming. Some effects of progesterone and related steroids on the central nervous system. Int. J. Neuropharmae. 6: 191-198, 1967. 15. Hahn, D. W., J. L. McGuire, F. C. Greenslade and G. D. Turner. Molecular parameters involved in the estrogenicity of mestranol and ethynylestradiol. Proe. Soc. exp. Biol. Med. 137: 1180-1185, 1971. 16. Helton, E. D. and J. W. Goldzieher. The pharmacokinetics of ethynyl estrogens: A review. Contraception 15: 255-284, 1977. 17. Hemmingsen, A. M. Studies on the oestrus-producing hormone (Oestrin). Skand. Arch Physiol. 65: 97-250, 1933. 18. Kappus, H., H. M. Bolt and H. Remmer. Demethylation of mestranol to ethynylestradiol in vitro and in vivo. Acta Endocr. 71: 374-384, 1972. 19. Kappus, H., H. M. Bolt and H. Remmer. Affinity of ethynylestradiol and mestranol for the uterine estrogen receptor and the microsomal mixed function oxidase of the liver. J. Steroid Biochem. 4: 121-128, 1973. 20. Keppel, G. Design and Analysis: A Researcher's Handbook. Englewood Cliffs, New Jersey: Prentice-Hall Inc., 1973. 21. Komisaruk, B. R. and C. Diakow. Lordosis reflex intensity in rats in relation to the estrous cycle, ovariectomy, estrogen administration and mating behavior. Endocrinology 93: 548-557, 1973. 22. Pfaff, D. W. and C. Lewis. Film analysis of lordosis in female rats. Hormones Behav. 5: 317-335, 1974. 23. Tarttelin, M. F. and R. A. Gorski. Varitions in food and water intake in the normal and acyclic female rat. Physiol. Behav. 7: 847-852, 1971.
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24. Thrasher, T. N. and M. J. Fregly. Effect of chronic treatment with an estrogen-progestogen combination on beta-adrenergic induced thirst. Pharmac. Biochem. Behav. 8: 177-183, 1978. 25. Wade, G. N. Some effects of ovarian hormones on food intake and body weight in female rats. J. comp. physiol. Psychol. 88: 183-193, 1975. 26. Wade, G. N. Sex hormones, regulatory behaviors, and body weight. In: Advances in the Study o f Behavior, Vol. 6, edited by J. S. Rosenblatt, R. A. Hinde, E. Shaw and C. G. Beer. New York: Academic Press, 1976, pp. 201-279.
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27. Wade, G. N. and J. M. Gray. Gonadal effects of food intake and adiposity: A metabolic hypothesis. Physiol. Behav. 22: 583-593, 1979. 28. Watnick, A. S., J. Gibson, M. Vinegra and S. Talksdorf. Ethinyl estradiol: A potent orally active contraceptive in rats. Proc. Soc. exp. Biol. Med. 116: 343-347, 1964. 29. Young, W. and W. R. Fish: The ovarian hormones and spontaneous running activity in the female rat. Endocrinology 36: 181-189, 1945. 30. Zemlan, F. P. and N. T. Adler. Hormonal control of female sexual behavior in the rat. Hormones Behav. 9: 345-357, 1977.
and Brain Research Publ., 1980. Printedin the U.S.A.
Behavioral Effects of Ethynyl Estrogens in the Female Rat I N E L L O J. S P I T E R I , 2 R O B E R T F. D R E W E T T A N D U N A P A D E L
Department o f Psychology, University of Durham, Science Laboratories, South Road Durham DHI 3 LE, England R e c e i v e d 22 F e b r u a r y 1980 SPITERI, N. J., R. F. DREWETT AND U. PADEL. Behavioral effects ofethynyl estrogens in thefemale rat. PHYSIOL. BEHAV. 25(3) 41)9-415, 1980.--The synthetic analogues of estradiol, ethynyl estradiol and mestranol, are used in oral contraceptives. Their effects on food intake and sexual behavior were evaluated in female rats, and compared with those of estradiol. It was found (Experiment 1) that both ethynyl estradiol and mestranol reduced food intake reliably, and more than estradiol. Water intake and body weight followed similar trends. Ethynyl estradiol, but not mestranol, plus progesterone stimulated proceptive and receptive behavior in ovariectomized female rats. Daily administration of ethynyl estrogens without progesterone showed similar trends (Experiment 3). It is hoped that these studies provide guidelines for further work on the effects of synthetic estrogens on sub-primate and primate behavior. Ethynyl estrogens Food and water intake Contraceptive steroids
Body weight
THE two synthetic estrogens, ethynyl estradioi (EE: 17ctethynyl- 1,3,5(10)-estratrien-3,17/3-diol) and Mestranol (MEE: 17a-ethynyl-3-methoxy-l,3,5(10)-estratrien-17/3-ol) have an important role in contraceptive steroids. One or the other, in combination with a progestogen, is an essential component in nearly all combination-type oral contraceptives currently in use. Knowledge of the pharmacological and physiological properties of ethynyl estrogens is accumulating gradually [16]; but studies of their behavioral effects are sadly lacking, which is surprising considering the wide use of ethynyl estrogens in oral contraceptives [5]. This lack of knowledge was the stimulus for the series of experiments described in this paper. EXPERIMENT 1 Estradiol has an anorexic effect [7, 8, 23, 25]. This experiment investigates the effects of ethynyl estrogens on food intake, water intake and body weight. Estradiol-17/3 (E) was used for comparison. METHOD
Experimental Animals and Housing Eighteen experimentally and sexually naive female Wistar rats, about 120 days of age, were used. They were kept under a 12 hr light-dark reversed cycle (lights out at 12.00 hr). Food (Rat and Mouse maintenance diet No. 1--BP
Sexual behavior
Behavioral effects
Nutrition, Witham) and water were available ad lib. The temperature was kept constant at 21°C. Ten days before ovariectomy, the rats were housed singly in plastic cages (42 x 30 x 17 cm). Coprophagia was prevented by a removable galvanized wire grid, 2 cm above the bottom of the cage. Dry, powdered food (Diet No. 1) was available in a metal food hopper which was attached to the top of the cage. This hopper was designed to keep spillage down to a minimum (about 0.2 g daily). This spillage was collected on paper towelling placed beneath the grid under the hopper. Body weight, food and water intake were measured to the nearest 1.0 g, 0.1 g and 0.1 ml, respectively, on four consecutive days prior to ovariectomy, irrespective of the stage of the estrous cycle. The rats were ovariectomized under ether anesthesia during the course of one day. They were then returned to their respective cages and body weight, food and water consumption were monitored postoperatively in order to determine when nutrient intake and body weight had returned to pre-operative levels. At this point, the experiment was started.
Hormone Administration Equimolar solutions of estradiol-17fl (5/~g/0.1 ml arachis oil), ethynyl estradiol (5.44/~g) and mestranol (5.69/zg) were prepared. The hormones were kept in blackened bottles and maintained at room temperature (21°C) to avoid decomposition. Fresh solutions of the hormones were prepared before each treatment period.
1The authors wish to thank Professor F. V. Smith and Professor M. J. Morgan for laboratory facilities in the Department of Psychology, University of Durham; Dr. A. Still and Dr. D. Wastell for statistical advice, and Dr. K. Kendrick and D. Harper for technical assistance. 2Reprint requests should be sent to: Nello J. Spiteri, Department of Zoology, State University of Groningen, Kerklaan 30, 9751 NN HAREN, The Netherlands.
C o p y r i g h t © 1980 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/80/090409-07502.00/0
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Experimental Design All rats were treated with all 3 hormones. The treatment order for the 18 rats was counterbalanced to give three replications of the six possible orders. Three "treatment periods" were separated by ten "treatment-free" days, which allowed body weight, food and water intake to return to normal. Each treatment period consisted of four days during which the animals were given hormone injections. The animals were injected with one of the three hormones from Monday to Thursday of each treatment week. Food and water intake were measured from Monday to Friday of each week: the food hoppers and water bottles were filled and weighed daily. The animals were weighed daily and all measurements made between 09.00 hr and 10.00 hr, 2 hours before lights-out. On four days during the recovery period (from Tuesday to Friday) body weight, food and water intake were measured as for the treatment period. Thus records were kept for both treatment and recovery periods; the overall duration of the experiment was six weeks.
Statistical Analyses The results were analysed using analysis of variance (0between, two-within factors). The Least Significant Difference test [10] was used for post-hoc comparisons between means. RESULTS AND DISCUSSION
Food Intake Figure 1 shows the effect of estrogen treatment on daily food intake. As expected, estrogen treatment depressed food intake (when compared with pre-treatment levels). Although E caused a decrease in food intake, MEE caused a greater decrease and EE caused the greatest depression in food intake. The overall difference in food intake was significant, F(2,34)=54.24, p<0.001, and both individual comparisons were also significant, E vs MEE: F(1,34)=15.17, p<0.001; MEE vs EE: F(1,34)=41.30, p<0.001.
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Water Intake Figure 2 shows that the depression of water consumption by estrogen administration was similar to the depression in food intake, and the overall difference was significant, F(2,34)=26.36, p<0.01. Individual comparisons, however, showed that there was no difference in water consumption between E and MEE treated rats, F(1,34)=4.08, 0.06> p>0.05. However, comparisons between these two treatment groups (between means on the fourth day of treatment), using the appropriate error term, revealed that there was reliable difference in water intake, F(1,102)=7.63, p<0.01, on that day. Rats treated with EE drank significantly less than MEE treated rats, F(i,34)=25.26, p<0.001.
Body Weight The results were expressed as % change in body weight. Figure 3 shows that there was a significant change in weight with estrogen treatment, F(2,34)=20.23, p<0.001. Rats treated with MEE and E did not show any difference in percentage weight change, F(1,34)<1, p = n s , although by Day 5 a difference appeared, F(I,102)=5.23, p<0.05. Rats treated with EE significantly lost more weight than MEE treated rats, F(1,34)=25.26, p<0.001. These rats also showed a greater weight loss over days than either E or MEE treated rats, F(6,102)= 10.80, p<0.001. During the recovery period, the body weight of all animals approximated normal levels. The experiment reported here shows that the two synthetic estrogens used in oral contraceptive formulations, MEE and EE, depress food and water intake as well as body weight. Other estrogens are known to have similar effects |251. MEE and E had similar effects, but MEE treated rats showed a slower recovery to control levels of food intake, water intake and body weight. EE treatment produced the greatest depression of all three measures which were recorded during the experiment. These various effects may reflect differences in the pharmaco-kinetic properties of these synthetic estrogens.
E T H Y N Y L E S T R O G E N S A N D F E M A L E RAT B E H A V I O R
411 either estradiol or ethynyl estradiol [2,3], could account for the slower recovery to normal levels of feeding, drinking and body weight in mestranol treated rats.
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Apart from the metabolism of estradiol to estrone leading to catecholestrogen formation, competitive hydroxylation also occurs at the C-16 position giving rise to estriol which is highly uterotrophic [12,19]. On the other hand, hydroxylation at the C-2 position, and therefore catecholestrogen formation, is the major metabolic process for ethynyl estradiol. In the rat, this process is in fact 51)% higher than for estradiol [4]. Recently it has been observed that catecholestrogens significantly reduced food intake in rats (13 cited in [11]). It is possible, therefore, that an increase in catecholestrogen formation may have caused partly, the greater decrease in food intake shown by rats treated with ethynyl estradiol. A second possibility is that ethynyl estradiol may have peripheral metabolic effects, for example by inducing changes in triglyceride clearance and fat metabolism, leading to reduced food intake. This has been recently suggested for the naturally occurring ovarian estrogens [27]. The observed depression in water intake by ethynyl estradiol treated rats may also be due to a reduction in /3-adrenergic responsiveness. It has been shown that chronic treatment with ethynyl estradiol plus norethynodrel (a progestogen with estrogenic properties) attenuates the responses to both hypovolemic and osmotic thirst mechanisms; and that these responses are mediated by EE alone [24]. The changes in food and water intake observed in rats treated with mestranol are probably due to the action of its major metabolite ethynyl estradioi, since mestranol itself is of very low "estrogenicity". Further, since mestranol is capable of binding to estrogen receptors in a form with low estrogenic activity [15], the number of receptors available for the uptake of its active metabolite (EE) would be reduced. The " a c t i v a t i o n " of such receptors would depend, to a large extent, on firstly the high lipophilicity of mestranol leading to its greater storage (and slower release rate), by adipose, adrenal and brain tissue [2,3], and secondly the metabolism Of mestranol to ethynyl estradiol. These properties, associated with the different pattern of excretion of mestranoi from
Treatment of ovariectomized rats with estradiol benzoate induces high levels of sexual behavior within five days of treatment [6]. Nearly all contraceptive steroids of the combination-type contain synthetic estrogens, either ethynyl estradiol or mestranol. We have therefore investigated the effects of ethynyl estrogens on sexual behavior of female rats. There is some evidence that ethynyl estradiol increases the receptivity of intact female rats [28]; but no detail of measures of sexual receptivity or proceptivity is reported. This experiment, therefore, investigates the effects of ethynyl estradioi and mestranol in combination with progesterone on the receptivity and proceptivity of ovariectomized female rats. Experiment 3 examines the effect of daily administration of ethynyl estrogens in the absence of exogenous progesterone. Estradiol-17/3 was used for comparison. METHOD
Experimental Animals and Housing Twenty-four female Wistar rats, 150 days of age, were housed in large group cages, in batches of 8 animals per cage, under a 12 hr light-dark reversed cycle (lights out at 12.00 hr). F o o d and water were available ad lib. The temperature was thermostatically controlled at 21°C. All the females were ovariectomized under ether anaesthesia 3 weeks before the start of any experimental procedures. Estrus was reinstated with injections of estradiol benzoate (5/xg in 0.1 ml arachis oil, 48 and 24 hr before the test) and progesterone (500/zg in 0.1 ml arachis oil, 6 hr before testing), and all females were given sexual experience by allowing them ten mounts with intromissions, by active males on three separate occasions in a testing arena. The females were randomly divided into three groups, consisting of eight rats per group. Twenty sexually experienced Wistar males (proven copulators) were used in the tests for sexual behavior. The testing arena was 90 cm in diameter and had a circular blackened wall 30 cm high. Experimental testing was carried out during the dark period of the animals' light-dark cycle in a room lit by a 60 W red light-bulb. Animals were habituated to the arena before the test. A female was placed with a sexually active male until the male performed 10 mounts with pelvic thrusting. Lordosis was recorded when a female showed pronounced concave arching of the back in response to mounting by a male. In addition to this measure, a general measure of proceptivity (or soliciting behavior, see [1]) was obtained by counting the number of inter-mount intervals during which soliciting behaviors such as darting and hopping, ear wiggling or nipping the male's face occurred [17].
Experimental Design Each group of females received all three hormone treatments (3×3 latin square design). The three experimental periods were separated by ten days during which no treatment was given. This period minimized any possible interaction between estrogens. The rats were given four daily subcutaneous injections of either estradiol (5 p.g/0.1 ml arachis oil), or ethynyl estradiol (5.44/xg), or mestranol (5.69 ptg).
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Twenty-four hours after the 4th injection and 6 hours before testing began at 15.00 hr, all the rats were injected with 500 tzg of progesterone in 0.1 ml arachis oil per rat. All testing during the experiment was run "blind".
Statistical Analyses Data were analysed with a Friedman two-way analysis of variance, and the Wilcoxon matched-pairs signed-ranks test with a z-transformation was used for post-hoc comparisons between groups [9]. All tests were two-tailed.
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Number of inter-mount intervals with soliciting behavior FIG. 5. The number of inter-mount intervals during which ovariectomized rats, injected with equimolar doses of either E or EE or MEE (for 4 days before testing), and progesterone (6 hr before testing), showed soliciting behavior. also shown by sexually receptive females, but were remarkably passive during mounts with pelvic thrusting by the males. Some females sniffed or chewed the sawdust, and continued doing so during mounts. Others investigated the males' anogenital region quite thoroughly, but did not show any soliciting behavior. This passivity is uncharacteristic of diestrous females, which show active rejection of the male [12].
RESULTS
The overall difference in lordosis behavior was significant (~(2) =37,02, p <0.001). The difference in the number of lordosis to ten mounts by a male, between the E and EE treated females and the MEE treated females is evident from Fig. 4. Females treated with EE, however, showed a higher number of lordosis responses than females treated with E (z=2.551, p<0.01). Proceptivity was also different between the groups (X2(2)=36.02; p<0.001); and the rats treated with EE showed significantly more soliciting behavior than females treated with E (z=2.229, p<0.05). Rats treated with MEE showed no soliciting behavior (Fig. 5). However, these rats did not actively reject the males' attempts to mount. They all showed a noticeable stiffening of the tail, a characteristic
EXPERIMENT 3 METHOD
Experimental Animals Forty-two female Wistar rats, 150 to 200 days of age, were ovariectomized at least two weeks before the start of any experimental procedures.
Experimental Design Estrus was reinstated in all the rats and they were given sexual experience as in Experiment 2. Each rat was randomly allocated to one of three equal groups: one group received 5/.tg of E in 0.1 ml arachis oil per
E T H Y N Y L E S T R O G E N S A N D F E M A L E RAT B E H A V I O R
413 100
rat per day. A second group received 5.44/xg of EE in 0.1 ml oil; and a third group received 5.69/zg of M E E in 0.1 ml oil (n= 14 per group). All injections were administered subcutaneously, at 09.00 hr every morning. The room, testing arena and testing procedure were the same as those used in Experiment 2. In addition to measures of lordosis and proceptivity, a rejection measure was also obtained. This was done by counting the number of intermount intervals during which either kicking away the male, or fighting or " b o x i n g " [22] were observed to occur. These behaviors are also shown by diestrous females in response to mounts or attempted mounts by a male. The lordosis quotient was derived by applying the following formula:
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Statistical Analyses
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The lordosis, proceptivity and rejection quotients for each day were analysed using a Kruskal-Wallis one-way analysis of variance. Mann-Whitney U-tests, with a z-transformation were used in conducting post-hoc comparisons between groups [9]. All tests were two-tailed. RESULTS
The results are presented in Fig. 6. Lordosis quotients for Day 1 were the same for all three groups (0%), and were therefore excluded from statistical analysis. However, there was an overall significant difference in the performance of lordosis between groups from Day 3 to Day 9 inclusive (p<0.01). The difference in lordosis responses between EE and E treated females over M E E treated females was obvious and large (Fig. 6(A)). Females treated with E and EE did not show a difference in lordosis behavior up to and including Day 3; but EE treated rats showed significantly (p<0.05) more lordosis in response to mounts by a male thereafter, with the exception of Day 8 (z= 1.86; p~<0.06). These results followed a similar trend to the one observed in Experiment 2. There appeared a significant change in soliciting behavior (Fig. 6(B)) after Day 3 of testing (p<0.001). The M E E treated females showed significantly less soliciting behavior than the two other estrogen groups (p<0.01). A difference in proceptivity between E and EE females did not appear until the 5th day of testing and lasted until the 7th day (p<0.05). Females from both these groups showed a similar amount of soliciting behavior on Days 8 and 9. The rejection of a male by a female rat was frequent and similar for all three groups during the first three days of treatment (Fig. 6(C)). During the next 6 days, however, there
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FIG. 6. (A) Lordosis quotients, (B) proceptivity quotients and (C) rejection quotients of ovariectomized rats treated with equimolar doses of either estradiol (5 /zg standard), or ethynyl estradiol or mestranol daily, for 9 days; n= 14 per group.
was an overall significant change in rejection behavior (p<0.001). Females treated with MEE rejected the males' attempts to mount more often than E treated females (range of z=2.41 to 4.61, p=0.05 to 0.001); but female rats treated with EE rejected males on a fewer number of occasions than E treated females. This difference proved significant between Days 4 and 7 of hormone treatment (p<0.01). DISCUSSION Two main points have emerged from the present experiments. First, the quality of female sexual behavior is differentially affected by the estrogens. Second, daily treatment with these estrogens leads to the differential appearance of pre-copulatory and copulatory components of sexual behavior. The most prominent female sexual behaviors seen in response to daily administration of estradiol or ethynyl estradiol were (1) a decrease in male rejection, (2) soliciting behavior and (3) lordosis behavior.
414
SPITERI E T A L .
While female aggression towards a male was obvious during the initial days of hormone treatment, females gradually showed soliciting behavior and then lordosis behavior in response to a male's mount (with the exception of mestranol treated rats). This type of lordosis behavior seemed similar to " f o r c e d " lordosis, the female sometimes moving away as the male dismounted [30]. With continued hormone treatment, the elicited lordoses were more intense, with deeper arching of the back, and the posture was kept after the male dismounted. Females which demonstrated intense lordosis showed this behavior when a male bumped into the back of a female prior to mounting. The degree of concavity of the back during lordosis is more pronounced as the estrous period progresses [21], and there is a similar effect with increasing, exogenously administered estrogen doses [30]. In this study, high proceptivity quotients were associated with high lordosis levels. The spontaneous occurrence of dart-hop movements, ear wiggling and lordosis is indicative of the very highest levels of receptivity at estrus [17]. Spontaneous dart-hop movements and ear wiggling were seen most frequently in females treated with ethynyl estradiol. Ethynyl estradiol enhanced lordosis behavior and reduced rejection behavior in females to a greater extent than estradiol. The transient difference in soliciting behavior between the two female groups appeared more gradually and, by the final day, all the females treated with estradiol or ethynyl estradiol showed high and similar levels of soliciting behavior. One point that is of interest is that during the development of sexual behavior in females which were treated with either estradiol or ethynyl estradiol, females showed lordosis and soliciting behavior as well as rejecting the males. This suggests that although a female rat showed lor-
dosis to mounts by a male, she did not readily accept all his mounting attempts. Lordosis and soliciting behavior are consistent with enhanced receptivity while rejection behavior suggests the opposite. Mestranol treated rats, however, did not exhibit any signs of receptivity. They did show some passivity to attempted mounts by males, particularly during the last two days of treatment; but generally they were not as passive as the mestranol + progesterone treated females in Experiment 2, frequently rejecting and fighting with the males. Although this comparison is somewhat subjective, it supports findings which suggest that progesterone has sedative or depressant effects on central nervous system activity [14]. Differences in the uptake and metabolism of estradiol17/3, ethynyl estradiol and mestranol may have also led to the differential appearance of appetitive and consummatory components of sexual behavior. The finding that mestranol suppressed food intake reliably more than estradiol (Experiment 1), but failed to stimulate sexual behavior (Experiment 2 and 3) may be attributable, in part, to the different "response thresholds" of these two behavioral systems to the different estrogens. For example, it has been demonstrated that doses of estradiol which reliably depressed food intake [8] were considerably lower than those which stimulated either running activity [10,29] or sexual behavior [30] in the female rat. Clearly, animal studies, particularly those involving subprimate species are of limited relevance to any studies of the effects of synthetic estrogens on human behavior. They could, however, provide useful guidelines for further work in this field.
REFERENCES
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9. Ferguson, G. A. Statistical Analysis in Psychology and Education. Int. student edition. Tokyo: McGraw Hill Kogakusha, 1976. 10. Finger, F. W. Estrus and general activity in the rat. J. comp. physiol. Psychol. 68: 461-466, 1969. 11. Fishman, J. Appetite and sex hormones. In: Appetite and Food Intake, edited by T. Silverstone. Berlin: Dahlem Konferenzen. Life Sciences Research Report, No. 2,207-218, 1976. 12. Fishman, J., H. Bradlow and T. F. Gallagher. Oxidative metabolism of estradiol. J. biol. Chem. 235: 3104-3107, 1%0.
13. Garattini, S. and J. Fishman. 2-Hydroxyestrone-induced reduction of food intake by rats. Unpublished observation, 1975, cited in [11]. 14. Gyermek, G., G. Genther and N. Fleming. Some effects of progesterone and related steroids on the central nervous system. Int. J. Neuropharmae. 6: 191-198, 1967. 15. Hahn, D. W., J. L. McGuire, F. C. Greenslade and G. D. Turner. Molecular parameters involved in the estrogenicity of mestranol and ethynylestradiol. Proe. Soc. exp. Biol. Med. 137: 1180-1185, 1971. 16. Helton, E. D. and J. W. Goldzieher. The pharmacokinetics of ethynyl estrogens: A review. Contraception 15: 255-284, 1977. 17. Hemmingsen, A. M. Studies on the oestrus-producing hormone (Oestrin). Skand. Arch Physiol. 65: 97-250, 1933. 18. Kappus, H., H. M. Bolt and H. Remmer. Demethylation of mestranol to ethynylestradiol in vitro and in vivo. Acta Endocr. 71: 374-384, 1972. 19. Kappus, H., H. M. Bolt and H. Remmer. Affinity of ethynylestradiol and mestranol for the uterine estrogen receptor and the microsomal mixed function oxidase of the liver. J. Steroid Biochem. 4: 121-128, 1973. 20. Keppel, G. Design and Analysis: A Researcher's Handbook. Englewood Cliffs, New Jersey: Prentice-Hall Inc., 1973. 21. Komisaruk, B. R. and C. Diakow. Lordosis reflex intensity in rats in relation to the estrous cycle, ovariectomy, estrogen administration and mating behavior. Endocrinology 93: 548-557, 1973. 22. Pfaff, D. W. and C. Lewis. Film analysis of lordosis in female rats. Hormones Behav. 5: 317-335, 1974. 23. Tarttelin, M. F. and R. A. Gorski. Varitions in food and water intake in the normal and acyclic female rat. Physiol. Behav. 7: 847-852, 1971.
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24. Thrasher, T. N. and M. J. Fregly. Effect of chronic treatment with an estrogen-progestogen combination on beta-adrenergic induced thirst. Pharmac. Biochem. Behav. 8: 177-183, 1978. 25. Wade, G. N. Some effects of ovarian hormones on food intake and body weight in female rats. J. comp. physiol. Psychol. 88: 183-193, 1975. 26. Wade, G. N. Sex hormones, regulatory behaviors, and body weight. In: Advances in the Study o f Behavior, Vol. 6, edited by J. S. Rosenblatt, R. A. Hinde, E. Shaw and C. G. Beer. New York: Academic Press, 1976, pp. 201-279.
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27. Wade, G. N. and J. M. Gray. Gonadal effects of food intake and adiposity: A metabolic hypothesis. Physiol. Behav. 22: 583-593, 1979. 28. Watnick, A. S., J. Gibson, M. Vinegra and S. Talksdorf. Ethinyl estradiol: A potent orally active contraceptive in rats. Proc. Soc. exp. Biol. Med. 116: 343-347, 1964. 29. Young, W. and W. R. Fish: The ovarian hormones and spontaneous running activity in the female rat. Endocrinology 36: 181-189, 1945. 30. Zemlan, F. P. and N. T. Adler. Hormonal control of female sexual behavior in the rat. Hormones Behav. 9: 345-357, 1977.