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The effects of estrogen and progesterone on female rat proceptive behavior
HORMONES
AND
BEHAVIOR
14, 65-75 (1980)
The Effects of Estrogen and Progesterone on Female Rat Proceptive Behavior BARBARA J. TENNENT, ERLA R. SMITH, AND JULIAN M. DAVIDSON Department of Physiology, Stanford University, School of Medicine, Stanford, California p4305 The relative importance of estrogen (EB) and progesterone (P) in stimulating proceptivity in ovariectomized female rats was studied. Proceptive behavior was measured quantitatively, providing a clear measure of response to experimental manipulation. When rats were tested biweekly after daily treatment with 0.4 cLB/lOOg body wt EB for 4 days, they showed maximal lordosis but low levels of proceptive behavior by the second test. Additional EB (3.0 cLB/loO g body wt daily) failed to stimulate additional proceptivity. A graded increase in proceptive behavior resulted from administration of increasing doses of P (50, 100,500 pg and 1.0 mg) to animals receiving EB priming as described above. The level of “soliciting” was significantly higher than EB-only-treated rats when 500 pg or 1.0 mg P was given. Ovariectomized, adrenalectomized rats, primed with 2.5 &IO0 g body wt EB daily for 7 days and tested on Day 8, were significantly less proceptive than ovariectomized, sham-adrenalectomized rats with the same hormone treatment. Four hours after injection of 1.0 mg P, there was no difference in proceptive or receptive behavior between sham- and adrenalectomized rats. It was concluded that if an EB dose is sufficient to induce maximal receptivity, additional estrogen does not stimulate proceptivity; unlike previous studies, the present data are not consistent with a global effect of ovarian steroids on both components of female behavior. Progesterone is more effective than estrogen in stimulating proceptive behavior, although proceptivity is not absolutely dependent on progesterone for expression. Proceptivity in EB-only-treated rats appears to be facilitated by adrenal P.
The role of the female rat in mating consists of three major components (Beach, 1976). Attractivity reflects the intensity of the female’s sexual stimulus properties for the male. Rroceptivity is expressed by the female’s appetitive activities, such as stereotyped hopping, darting, and ear wiggling movements, that are directed at the male. Receptivity, or lordosis behavior, is defined as the female’s reactions to the male that are necessary for successful copulation. Proceptivity and receptivity, the two active components of female sexual behavior, are both dependent on ovarian hormones for expression (Boling and Blandau, 1939; Beach, 1942). Lordosis can be elicited by estrogen alone in ovariectomized, adrenalec65 0018-506x/80/010065-1 1$01.00/O Copyright @ 1980 by Academic Press. Inc. All rights of reproduction in any form reserved.
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tomized rats, although progesterone facilitates its expression at lower estrogen doses (Davidson, Rodgers, Smith, and Bloch, 1968; Pfaff, 1970). The hormonal regulation of proceptive behavior has not been systematically explored. Zemlan and Adler (1978) observed both lordosis and proceptive behavior in ovariectomized, adrenalectomized rats treated with very large doses of estrogen, although the behavior was not measured quantitatively. These authors suggest that both behavioral components are primarily regulated by estrogen, but that proceptivity is much less sensitive to the hormone and requires higher levels for expression. Progesterone, while not essential, was presumed merely to amplify the effect of low estrogen doses on proceptive and receptive behavior. It is also possible that full proceptive behavior, comparable to that shown by a naturally estrous female, may require progesterone in addition to estrogen. Supporting this second alternative, recent studies investigating hormonal regulation of proceptivity (Hardy and DeBold, 1971; Hlinak and Madlafousek, 1971; Whalen, 1974) have shown that progesterone stimulates proceptive behavior in estrogen-primed animals. In none of these studies, however, was proceptivity quantitatively assessed, nor were the differential effects of estrogen and progesterone on receptivity and proceptivity systematically explored. Gilman and Hill (1978) showed that increasing progesterone but not estrogen doses shortened the latency for a female’s return to a male following intromission in an apparatus allowing the female to control timing of the copulatory pattern. In this experiment, the percentage of females soliciting increased with both increasing estrogen (EB) and progesterone (P) doses, but again, the rare of soliciting was not measured. While this paper was in preparation, Fadem, Barfield, and Whalen (1980), using a single low EB priming dose, also reported that increased P shortens return latencies and increases solicitation rate. The role of estrogen in proceptivity was not, however, clearly demonstrated vis-a-vis that of progesterone. This study was designed to distinguish between the two alternatives of a global effect of ovarian hormones on proceptive and receptive behavior and specific effects of estrogen and progesterone. By first administering sufficient estrogen to stimulate full receptive behavior, the effects of additional estrogen and varying amounts of progesterone on proceptive behavior could be assessed. Further, since the effects of estrogen alone could be mediated or at least augmented, by adrenal progesterone, adrenalectomized rats treated with estrogen were also studied. Proceptive behavior was measured quantitatively, since simple calculation of percentage of animals “soliciting” might obscure possible differences in the inrensity of behavior expressed. METHODS
This study was conducted in two parts. Experiment I established a dose-response curve for progesterone using high and low doses of estro-
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adrenalectomized and shamgen priming. In Experiment II, adrenalectomized estrogen-primed rats showing high LQ (lordosis quotients) were tested for proceptivity before and following progesterone treatments. Experiment 1 The subjects were forty 45day-old Long-Evans female rats provided with ad lib. food and water and maintained on a reversed 14:lO hr light-dark schedule. One-half of the animals were ovariectomized (Ovx) under ether anesthesia 1 week after their arrival and the remainder after 2 weeks. Two weeks after ovariectomy all animals were divided into four experimental groups of 10 animals each. Groups I and II received SCinjections of 0.4 pg/lOO g body wt estradiol benzoate (EB) in 0.1 cc sesame oil and groups III and IV received 3.0 &lo0 g body wt EB. These treatments were continued daily for 4 days, and on the fifth morning groups II and IV received a dose of progesterone (P) in oil, while the remaining animals received vehicle injections. Four to six hours later a behavioral test was given. In subsequent biweekly tests, all animals received the same EB doses and groups II and IV received progressively higher doses of P (50, 100, 500, and 1000 pg on successive tests). Behavioral testing began 3 hr after light offset. Each animal was placed in a semicircular Plexiglas mating arena for a 3-min adaptation period. The arena was 76 cm in diameter, sufficient to observe the components of solicitation behavior. A male which had shown one intromission with a highly estrous, nonexperimental female in a separate arena was placed with the experimental female immediately following his intromission. The test continued until the female received 10 mounts. If the male ejaculated, a new male was substituted as quickly as possible. Both lordosis and proceptive behavior were recorded. A lordosis was scored only if the rump and head were both elevated and the back arched; mere flattening of the back was not counted. Three elements of proceptive behavior were recorded, using the classifications of Madlafousek and Hlinak (1978). Hopping was defined as a short leap with the animal landing on all four paws, followed by the assumption of a crouching posture in which the animal was braced for accepting the male’s weight. Darting was defined as a run consisting of several steps, abruptly terminated by the adoption of the crouching position. Ear wiggling was recorded when the ears vibrated rapidly, usually accompanied by an upward toss of the head. The occurrence of these behaviors was recorded on an event recorder. The behavior of the male (mounts, intromissions, and ejaculations) was also noted on the event recorder, and the duration of each test was measured from that record, Following the final test, each female was immediately removed from the
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arena and a l.O-cc blood sample was drawn by cardiac puncture ether anesthesia. The blood was later assayed for progesterone.
under
Experiment II The subjects were forty 60-day-old Ovx Long-Evans female rats, maintained as described in Expt. I. The animals were given sexual experience in two “shaping” tests. For these tests, they were primed with 0.5 pg EB/lOO g body wt for 4 days and given 1.0 mg P, 4 to 6 hr before testing. Two days after the second “shaping” test, 22 of these animals were adrenalectomized (adx) under ether anesthesia, while 17 others were sham-adrenalectomized (sham-adx). Sham operations included making an incision in the body wall and probing with a cotton swab. On the following day, daily treatment was begun with 2.5 pg EB/lOO g body wt and continued through the day of testing. On Day 4, the animals were anesthetized under ether, to stimulate the adrenal cortex. Fifteen minutes later they were reanesthetized and a l.O-cc blood sample was collected by jugular venipuncture. The plasma was stored frozen and later assayed for P. On Day 8, the first postoperative behavior test was conducted 1 hr after lights-off. The females were then given 1.0 mg P immediately following this EB-only test and were retested 4 to 5 hr after the injection. After 1 week with no hormonal treatment, the hormone injections and testing procedures were repeated. The radioimmunoassay for progesterone was a modification of the method of Gibori, Antczak, and Rothchild (1977). Samples were diluted in 300 ~1 distilled water, and 2 ml of hexane was added. The samples remained at room temperature for 2 hr and were then centrifuged at 2000 rpm for 10 min. Recovery of [SH]progesterone from serum pools averaged 74.3% so individual recoveries were routinely performed. The intraassay coefficient of variation was 10.7% and the interassay coefficient was 15.4%. All samples from each experiment were included in one assay, and all samples were assayed in triplicate. RESULTS
For analysis, all hopping, darting, and ear wiggling during a test were totaled and divided by the length of the test to establish a standard measure of proceptive behavior, solicitations per minute (sol/min). Experiment I Estrogen dose had no significant effect on either proceptive or receptive behavior in any test (Mann-Whitney U test). Table 1 shows LQ, solicitations per minute, and percentage of animals responding for each group. Concordance between the high and low EB treatment groups was very high in both the P-treated [intergroup concordance with Jack knife estimate (Kendall, 1%2); proceptivity 1.0251, P < 0.002; receptivity, 0.9424, P < 0.0021 and the EB-only group (proceptivity, 0.9964, P < 0.001;
57.5 2 7.W 90
Combined EB + P (II and IV) % Responding
0.4 +- 0.1 60
0.4 k 0.1 45
0.6 2 0.2 70
0.3 2 0.2 50
0.3 f 0.1 50
0.6 + 0.3 40
sol/min’
85.0 + 6.8 95
84.5 2 5.0 95
91.0 ? 6.9 100
82.0 2 8.1 100
63.6 k 13.7 90
81.0 f 10.5 90
LQ
1.8 + 0.3d 80
1.2 f 0.2 75
1.8 2 0.4 80
1.5 2 0.4 80
1.8 + 0.4 80
0.8 2 0.3 70
sol/min
94.0 ” 2.6 100
88.5 + 3.8 95
94.0 ” 3.4 100
85.6 f 7.3 100
94.0 + 4.0 100
71.0 2 11.4 90
LQ
2.9 + 0.4d** 95
1.4 2 0.3 80
2.8 f 0.5 100
1.5 + 0.5 80
3.0 f 0.6 90
1.1 2 0.3 80
sol/min
Test 3 (500 pg P)
98.5 ?z 1.1 100
94.5 2 2.6 100
99.0 + 1.0 100
90.0 + 4.7 100
97.8 5 2.2 100
99.0 f 1.0 100
LQ
3.8 2 0.5’,’ 100
1.5 + 0.3 80
3.8 + 0.7 100
1.5 + 0.3 80
3.5 + 0.8 100
1.5 + 0.5 80
soYmin
Test 4 (1000 pg P)
“Solicitations per minute. “Standard error of the mean. cSignificantly lower than subsequent tests P < 0.01. dSignificantly higher than previous test P < 0.01. eSignificantly higher than previous test P < 0.02. ‘Significantly higher than controls P < 0.02. #Significantly higher than controls P < 0.002.
50.5 + 7.lC 95
Combined EB (I and III) % Responding
61.0 k 10.2 90
43.0 2 9.4 80
39.0 * 1l.P 100
72.0 + 8.4 100
3.0 pg EB % Responding
0.4 pg EB + P % Responding
0.4 pg EB % Responding
LQ
3.0 pg EB + P % Responding
IV
III
II
I
Group
Test 2 (100 pg P)
1. Effects of Estrogen and Progesterone on Receptive and Proceptive Behavior in the Rat
Test 1 (50 pg P)
TABLE
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receptivity, 1.0234, P < 0.002). Data from these EB treatment groups were therefore combined for subsequent analysis. In both EB treatment groups, proceptive behavior significantly increased in response to increasing progesterone doses (Friedman’s twoway analysis of variance; x2 = 36.92 P < 0.002). A graded dose-response relationship was apparent; in successive tests each increase in progesterone dose resulted in a correspondingly increased frequency of solicitations (Wilcoxon matched pairs, P < 0.01 for 50-, loo-, and 500-pg tests, P < 0.02 for l.O-mg test). Animals treated with EB alone showed no such change in proceptive behavior during successive tests (Friedman’s twoway analysis of variance; x2 = 8.565) although the first test tended to be lower than subsequent tests. Differences in proceptivity between P-treated and EB-only-treated animals were not significant in the 50-pg or lOO-pg tests, but became significant in the 500~pg (P < 0.02) and 1.O-mg (P < 0.002) tests (Mann-Whitney U). Receptive behavior also increased in successive tests in both the EBonly and EB + P groups (Friedman’s). In both groups, LQ in the first test was significantly lower (P < 0.01, Wilcoxon matched pairs) than in subsequent tests, in which LQ was maximal for both groups. In every test, LQ in the EB-only-treated groups was not significantly different from that shown by the EB + P-treated animals. The different elements of proceptive behavior-hopping, darting, and ear wiggling-were scored separately to see if there were differences in their sensitivity to hormonal treatment (Fig. 1). In those rats treated with EB + 500 pug P, hopping was the most consistently exhibited behavior. When animals received 1.0 mg P, however, hopping was less consistent, and the decrease in hopping may have been related to an increase in darting. Ear wiggling showed a clear progesterone dose effect. Statistical tests were performed only for combined “solicitation scores” since individual animals tended either to hop or to dart, and the particular behavior occurring appeared partially dependent on the action of the male. Females seemed to hop more frequently when the males were quick to mount and they darted more when the males were slower to respond. Hops
Darts
Ear wiggles
EE+P ,’ 3’
TEST #
1
2
3
4
FIG. 1. The effects of estrogen (EB) and progesterone (P) on the different elements of proceptive behavior: hops, darts, and ear wiggles.
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The mean plasma progesterone level in blood drawn 4-5 hr following injection of 1.O mg P (and shortly after mating) was 23.2 k 1.4 rig/ml, while the mean level in EB-treated mated control animals was 10.7 + 0.8 nglml. Experiment ZZ
test of this experiment, mean LQ for all In the second “shaping” animals was 92.1 f 3.3 and mean solicitations per minute was 7.0 + 0.9. LQ and proceptivity levels for both postadrenalectomy tests are shown in Table 2. Analysis of pre-P tests vs post-P tests was by the Wilcoxon matched pairs test, while experimental and control groups were compared by Mann-Whitney U. Completeness of adrenalectomy was verified by measurement of plasma P in ether-stressed animals. Mean P level in sham-adx rats was 5.7 t 1.0 r&ml, while there was no detectable P in any adx animal. In the first postadrenalectomy test with EB alone, sham-adx animals showed significantly (P < 0.05) more proceptive behavior than adx rats. Before P treatment, solicitation behavior was present in 32% of the adx group and 71.4% of the sham-adx controls. Differences in proceptivity between the groups were significant both when all animals were included and when only those that showed any proceptive behavior were considered. Before P treatment, there was no significant difference in LQ between groups, although the control animals tended to be more receptive. Following P treatment, solicitations frequency increased significantly in both groups, while there was no significant increase in LQ. Progesterone TABLE 2 Lordosis and Proceptive Behavior before and following Progesterone Treatment in EB-Primed Ovariectomized and Ovariectomized-Adrenalectomized Female Rats Lordosis Test 1
Ovx-adx ovx
Test 2
Preprogesterone
Postprogesterone
66.4 * 8.3 81.3 2 7.0
81.3 f 4.8 89.2 f 1.4
Preprogesterone
Postprogesterone
76.4 k 7.4 86.3 + 6.6
91.5 f 3.6b 93.8 f 4.5
Solicitations per minute Test 1 Preprogesterone Ovx-adx ovx
0.2 t 0.09e 1.3 k 0.5
Test 2
Postprogesterone 3.1 k 0.7” 4.6 2 1.1”
Preprogesterone 0.2 -c 0.2c 1.1 k 0.4
u Significant difference from preprogesterone test p < 0.01. b Significant difference from preprogesterone test p < 0.05. c Significant difference from controls (Ovx) p < 0.05.
Postprogesterone 3.7 + 1.2” 4.6 in 0.9”
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treatment to adx animals restored both proceptive and receptive behavior to sham-adx levels. In the second pre-P test, adx animals still showed significantly less proceptive behavior than did control animals. In the adx group, only 14.3% showed any proceptive behavior, while 68.8% of sham-adx animals solicited. As in the first test, there was no significant difference in LQ between the groups. Following progesterone treatment, proceptive behavior increased in both groups. LQ showed a significant increase ti-om the pre-P level in adx but not control animals, which were already showing high LQ. The two groups did not show significantly different lordosis behavior level before and after adrenalectomy. After surgery, proceptive behavior tended to be lower in both groups, but the difference was significant (P < 0.02) only in the adx group. DISCUSSION
The first experiment distinguished the relative effects of EB and P on receptive and proceptive behavior and examined the relationship between these two components of female sexual behavior. It was found that treatment of spayed rats with EB alone does facilitate a low level of proceptive behavior, but proceptivity did not respond to EB in a doseresponse fashion. Hardy and DeBold (1971) using animals treated with varying EB doses and 500 pg P, showed that the percentage of animals soliciting did not increase with EB dose, once a certain level of EB stimulation was reached. The present study confirms and extends that observation; when the EB dose is sufficient to induce maximal receptivity, additional EB, with or without P, has no effect on the percentage of animals soliciting or on the level of proceptivity displayed by an animal. This finding contrasts with the conclusion that receptive and then proceptive behavior can be sequentially elicited by increasing estrogen doses (Zemlan and Adler, 1978). In contrast, progesterone did stimulate dose-related increases in proceptive behavior. These results show the importance of quantitatively assessing proceptive behavior. The percentage of animals showing proceptivity after administration of 500 pg and 1 .O mg P does not reflect the observed graded changes in level of behavior. Thus, recording of each observed behavior provides a more sensitive measure of the effects of experimental manipulation. Although responses increased significantly with each increase in P dose, differences between EB-only- and P-treated groups became significant only when 500 pg or 1.O mg P was given. That these data are relevant to natural conditions of the estrous cycle is suggested by the fact that plasma P levels 4 hr after injection of 1 .O mg P resembled that found on the evening of proestrus in our colony (Gray, Smith, Tennent, and Davidson, in press). Again, there was no difference between EB treatment groups in any test, nor was the rate of
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increase in response to increasing P different in high and low EB groups. In several earlier studies of proceptivity (Hardy and DeBold, 1971; Hlinak and Madlafousek, 1971; Whalen, 1974) proceptive behavior varied with both EB and P doses; it was stated (Hlinak and Madlafousek, 1971) that less P was needed with increasing EB to achieve the same level of proceptivity, Only percentage of animals showing proceptivity and not solicitation rate was reported. Exposure to EB increases subsequent responses to steroid administration (Beach, 1974; Damassa and Davidson, 1973), and repeated testing also facilitates lordosis (Hardy and DeBold, 1973; Henrik and Gerall, 1976), and soliciting (Hlinak, 1975). In the EB-alone-treated groups, proceptivity tended to increase after the first test, although the level was not significant, and remained the same on all subsequent tests. P-Treated animals showed significant increases after each test, and differences were greatest after the second test, while EB-only animals showed relatively constant behavior. These increases could not, therefore, have been caused by successive testing and re eated EB administration alone. LQ did increase significantly after the Krst test in all groups; this difference may have resulted from successive testing and steroid administration. The adrenal gland can release substantial amounts of P. Plasma P levels measured in EB-treated Ovx rats following mating even exceeded that found following ether stress, presumably due to considerable matinginduced adrenal P secretion. A number of studies (Davidson ef al., 1968; Pfaff, 1970; Larsson et al., 1974) have assessed the role of adrenal P in facilitating lordosis, but none has specifically studied its effect on proceptivity. The second experiment demonstrated the importance of the adrenal in maintaining this behavior. After adrenalectomy, both the proportion of animals showing proceptive behavior and the amount of proceptivity in response to EB alone, were considerably reduced. Because Zemlan and Adler (1978) did not measure proceptive behavior quantitatively, it is difficult to compare these results with their report on proceptivity in adrenalectomized rats. Some proceptivity was observed in some adrenalectomized rats in this experiment, but the level was well below ovariectomized EB-treated levels, and far below that shown by progesterone-treated animals. Zemlan and Adler also used a different EB treatment paradigm, injecting 500 &kg 46 hr before testing, as compared to the present use of lower doses injected for several days. Decrements in proceptive behavior observed in adx EB-only-treated animals were probably not caused by ill effects of the surgery. Exogenous P restored the level of behavior to that shown by sham-adx animals. In post-adx tests, proceptive behavior tended to be lower than in the shaping tests in both groups, but the difference was significant only in the adx animals. While this may have indicated postsurgical illness, this is unlikely since lordosis behavior in both groups was restored to pread-
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renalectomy levels by administration of 1.O mg P. The lowered proceptivity of adx animals postoperatively more likely reflected the loss of supplementary adrenal progesterone. These experiments have shown that proceptive behavior, while not absolutely dependent on progesterone, responds in a graded manner to increasing P doses. Adrenal P also appears to stimulate proceptivity. If EB is sufficient to stimulate maximal receptivity, additional EB has no effect on the low level of proceptivity expressed. ACKNOWLEDGMENTS This work was supported by NIMH Grant MH-21178. B. Tennent was supported by NIMH Bioscience Training Program MH 8304 and NIH predoctoral fellowship No. 1 F31 MH 07645. The able technical assistance of D. TaIlentire is gratefully acknowledged. Progesterone antiserum was kindly supplied by Dr. G. D. Niswender.
REFERENCES Beach, F. A. (1942). Importance of progesterone to induction of sexual receptivity in spayed female rats. Proc. Sot. Exp. Biol. Med. 51, 369-371. Beach, F. A. (1976). Sexual attractivity, proceptivity, and receptivity in female mammals. Horm.
Behav.
7, 105-138.
Baling, J. L., and Blandau, R. J. (1939). The estrogen-progesterone induction of mating response in the spayed female rat. Endocrinology 25, 359-364. Damassa, D., and Davidson, J. M. (1973). Effects of ovariectomy and constant light on responsiveness to estrogen in the rat. Horm. Behav. 4, 269-279. Davidson, J. M., Rodgers, C. H., Smith, E. R., and Bloch, G. J. (1968). Stimulation of female behavior in adrenalectomized rats with estrogen alone. Endocrinology 82, 193195. Fadem, B. H., Bartield, R. J., and Whalen, R. E. (1980). Dose response and time-response relationships between progesterone and the display of patterns of receptive and proceptive behavior in the female rat. Horm. Behav. 13, 40-48. Gibori, G., Antczak, E., and Rothchild, I. (1977). The role of estrogen in the regulation of luteal progesterone secretion in the rat after day 12 of pregnancy. Endocrinology 100, 1483- 1495. Gilman, D. P., and Hitt, J. C. (1978). Effects of gonadal hormones on pacing of sexual contacts by female rats. Behav. Biol. 24, 77-87. Gray, G. D., Tennent, B., Smith, E. R., and Davidson, J. M. (1980). LH regulation and sexual behavior in middle-aged female rats. Endocrinology, in press. Hardy, D. F., and DeBold, J. F. (1971). The relationship between levels of exogenous hormones and the display of lordosis by the female rat. Harm. Behav. 2, 287-297. Hardy, D. F., and DeBold, J. F. (1973). Effects of repeated testing on sexual behavior of the female rat. J. Camp. Physiol. Psycho/. 85, 195-202. Henrik, E., and Gerall, A. A. (1976). Facilitation of receptivity in estrogen-primed rats during successive mating tests with progestins and methysergide. J. Camp. Physiol. Psychol.
90, 590-600.
Hlinak, Z., and Madlafousek, J. (1971). Positive and negative effects of progesterone on the precopulatory behavior of ovariectomized rats. Activ. Nerv. Super. 14, 3. Hlinak, Z. (1975). Strain-associated differences in the action of oestradiol and progesterone in inducing pre-copulatory behaviour in ovariectomized rats. Physiol. Bohemoslov. 24, 381-383. Kendall, M. G. (1962). Rank Correlation Methods. Hafner, New York.
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Larsson, F., Feder, H. H., and Komisaruk, B. R. (1974). Role of the adrenal glands, repeated matings, and monamines in lordosis behavior of rats. Pharmacol. Biochem. Behav. 2, 685-692. Madlafousek, J., and Hlinak, Z. (1978). Sexual behaviour of the female laboratory rat: Inventory, patterning, and measurement. Behaviour 63, 129-173. Pfaff, D. W. (1970). Nature of sex hormone effect on rat sex behavior; specificity of effects and individual patterns of response. J. Comp. Physiol. Psychol. 73, 349-358. Whalen, R. E. (1974). Estrogen, progesterone induction of mating in female rats. Horm. Behav. 5, 157-162. Zemlan, F., and Adler, N. T. (1978). Hormonal control of female sexual behavior in the rat. Horm. Behav. 9. 345-357.
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The Effects of Estrogen and Progesterone on Female Rat Proceptive Behavior BARBARA J. TENNENT, ERLA R. SMITH, AND JULIAN M. DAVIDSON Department of Physiology, Stanford University, School of Medicine, Stanford, California p4305 The relative importance of estrogen (EB) and progesterone (P) in stimulating proceptivity in ovariectomized female rats was studied. Proceptive behavior was measured quantitatively, providing a clear measure of response to experimental manipulation. When rats were tested biweekly after daily treatment with 0.4 cLB/lOOg body wt EB for 4 days, they showed maximal lordosis but low levels of proceptive behavior by the second test. Additional EB (3.0 cLB/loO g body wt daily) failed to stimulate additional proceptivity. A graded increase in proceptive behavior resulted from administration of increasing doses of P (50, 100,500 pg and 1.0 mg) to animals receiving EB priming as described above. The level of “soliciting” was significantly higher than EB-only-treated rats when 500 pg or 1.0 mg P was given. Ovariectomized, adrenalectomized rats, primed with 2.5 &IO0 g body wt EB daily for 7 days and tested on Day 8, were significantly less proceptive than ovariectomized, sham-adrenalectomized rats with the same hormone treatment. Four hours after injection of 1.0 mg P, there was no difference in proceptive or receptive behavior between sham- and adrenalectomized rats. It was concluded that if an EB dose is sufficient to induce maximal receptivity, additional estrogen does not stimulate proceptivity; unlike previous studies, the present data are not consistent with a global effect of ovarian steroids on both components of female behavior. Progesterone is more effective than estrogen in stimulating proceptive behavior, although proceptivity is not absolutely dependent on progesterone for expression. Proceptivity in EB-only-treated rats appears to be facilitated by adrenal P.
The role of the female rat in mating consists of three major components (Beach, 1976). Attractivity reflects the intensity of the female’s sexual stimulus properties for the male. Rroceptivity is expressed by the female’s appetitive activities, such as stereotyped hopping, darting, and ear wiggling movements, that are directed at the male. Receptivity, or lordosis behavior, is defined as the female’s reactions to the male that are necessary for successful copulation. Proceptivity and receptivity, the two active components of female sexual behavior, are both dependent on ovarian hormones for expression (Boling and Blandau, 1939; Beach, 1942). Lordosis can be elicited by estrogen alone in ovariectomized, adrenalec65 0018-506x/80/010065-1 1$01.00/O Copyright @ 1980 by Academic Press. Inc. All rights of reproduction in any form reserved.
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tomized rats, although progesterone facilitates its expression at lower estrogen doses (Davidson, Rodgers, Smith, and Bloch, 1968; Pfaff, 1970). The hormonal regulation of proceptive behavior has not been systematically explored. Zemlan and Adler (1978) observed both lordosis and proceptive behavior in ovariectomized, adrenalectomized rats treated with very large doses of estrogen, although the behavior was not measured quantitatively. These authors suggest that both behavioral components are primarily regulated by estrogen, but that proceptivity is much less sensitive to the hormone and requires higher levels for expression. Progesterone, while not essential, was presumed merely to amplify the effect of low estrogen doses on proceptive and receptive behavior. It is also possible that full proceptive behavior, comparable to that shown by a naturally estrous female, may require progesterone in addition to estrogen. Supporting this second alternative, recent studies investigating hormonal regulation of proceptivity (Hardy and DeBold, 1971; Hlinak and Madlafousek, 1971; Whalen, 1974) have shown that progesterone stimulates proceptive behavior in estrogen-primed animals. In none of these studies, however, was proceptivity quantitatively assessed, nor were the differential effects of estrogen and progesterone on receptivity and proceptivity systematically explored. Gilman and Hill (1978) showed that increasing progesterone but not estrogen doses shortened the latency for a female’s return to a male following intromission in an apparatus allowing the female to control timing of the copulatory pattern. In this experiment, the percentage of females soliciting increased with both increasing estrogen (EB) and progesterone (P) doses, but again, the rare of soliciting was not measured. While this paper was in preparation, Fadem, Barfield, and Whalen (1980), using a single low EB priming dose, also reported that increased P shortens return latencies and increases solicitation rate. The role of estrogen in proceptivity was not, however, clearly demonstrated vis-a-vis that of progesterone. This study was designed to distinguish between the two alternatives of a global effect of ovarian hormones on proceptive and receptive behavior and specific effects of estrogen and progesterone. By first administering sufficient estrogen to stimulate full receptive behavior, the effects of additional estrogen and varying amounts of progesterone on proceptive behavior could be assessed. Further, since the effects of estrogen alone could be mediated or at least augmented, by adrenal progesterone, adrenalectomized rats treated with estrogen were also studied. Proceptive behavior was measured quantitatively, since simple calculation of percentage of animals “soliciting” might obscure possible differences in the inrensity of behavior expressed. METHODS
This study was conducted in two parts. Experiment I established a dose-response curve for progesterone using high and low doses of estro-
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adrenalectomized and shamgen priming. In Experiment II, adrenalectomized estrogen-primed rats showing high LQ (lordosis quotients) were tested for proceptivity before and following progesterone treatments. Experiment 1 The subjects were forty 45day-old Long-Evans female rats provided with ad lib. food and water and maintained on a reversed 14:lO hr light-dark schedule. One-half of the animals were ovariectomized (Ovx) under ether anesthesia 1 week after their arrival and the remainder after 2 weeks. Two weeks after ovariectomy all animals were divided into four experimental groups of 10 animals each. Groups I and II received SCinjections of 0.4 pg/lOO g body wt estradiol benzoate (EB) in 0.1 cc sesame oil and groups III and IV received 3.0 &lo0 g body wt EB. These treatments were continued daily for 4 days, and on the fifth morning groups II and IV received a dose of progesterone (P) in oil, while the remaining animals received vehicle injections. Four to six hours later a behavioral test was given. In subsequent biweekly tests, all animals received the same EB doses and groups II and IV received progressively higher doses of P (50, 100, 500, and 1000 pg on successive tests). Behavioral testing began 3 hr after light offset. Each animal was placed in a semicircular Plexiglas mating arena for a 3-min adaptation period. The arena was 76 cm in diameter, sufficient to observe the components of solicitation behavior. A male which had shown one intromission with a highly estrous, nonexperimental female in a separate arena was placed with the experimental female immediately following his intromission. The test continued until the female received 10 mounts. If the male ejaculated, a new male was substituted as quickly as possible. Both lordosis and proceptive behavior were recorded. A lordosis was scored only if the rump and head were both elevated and the back arched; mere flattening of the back was not counted. Three elements of proceptive behavior were recorded, using the classifications of Madlafousek and Hlinak (1978). Hopping was defined as a short leap with the animal landing on all four paws, followed by the assumption of a crouching posture in which the animal was braced for accepting the male’s weight. Darting was defined as a run consisting of several steps, abruptly terminated by the adoption of the crouching position. Ear wiggling was recorded when the ears vibrated rapidly, usually accompanied by an upward toss of the head. The occurrence of these behaviors was recorded on an event recorder. The behavior of the male (mounts, intromissions, and ejaculations) was also noted on the event recorder, and the duration of each test was measured from that record, Following the final test, each female was immediately removed from the
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TENNENT,
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arena and a l.O-cc blood sample was drawn by cardiac puncture ether anesthesia. The blood was later assayed for progesterone.
under
Experiment II The subjects were forty 60-day-old Ovx Long-Evans female rats, maintained as described in Expt. I. The animals were given sexual experience in two “shaping” tests. For these tests, they were primed with 0.5 pg EB/lOO g body wt for 4 days and given 1.0 mg P, 4 to 6 hr before testing. Two days after the second “shaping” test, 22 of these animals were adrenalectomized (adx) under ether anesthesia, while 17 others were sham-adrenalectomized (sham-adx). Sham operations included making an incision in the body wall and probing with a cotton swab. On the following day, daily treatment was begun with 2.5 pg EB/lOO g body wt and continued through the day of testing. On Day 4, the animals were anesthetized under ether, to stimulate the adrenal cortex. Fifteen minutes later they were reanesthetized and a l.O-cc blood sample was collected by jugular venipuncture. The plasma was stored frozen and later assayed for P. On Day 8, the first postoperative behavior test was conducted 1 hr after lights-off. The females were then given 1.0 mg P immediately following this EB-only test and were retested 4 to 5 hr after the injection. After 1 week with no hormonal treatment, the hormone injections and testing procedures were repeated. The radioimmunoassay for progesterone was a modification of the method of Gibori, Antczak, and Rothchild (1977). Samples were diluted in 300 ~1 distilled water, and 2 ml of hexane was added. The samples remained at room temperature for 2 hr and were then centrifuged at 2000 rpm for 10 min. Recovery of [SH]progesterone from serum pools averaged 74.3% so individual recoveries were routinely performed. The intraassay coefficient of variation was 10.7% and the interassay coefficient was 15.4%. All samples from each experiment were included in one assay, and all samples were assayed in triplicate. RESULTS
For analysis, all hopping, darting, and ear wiggling during a test were totaled and divided by the length of the test to establish a standard measure of proceptive behavior, solicitations per minute (sol/min). Experiment I Estrogen dose had no significant effect on either proceptive or receptive behavior in any test (Mann-Whitney U test). Table 1 shows LQ, solicitations per minute, and percentage of animals responding for each group. Concordance between the high and low EB treatment groups was very high in both the P-treated [intergroup concordance with Jack knife estimate (Kendall, 1%2); proceptivity 1.0251, P < 0.002; receptivity, 0.9424, P < 0.0021 and the EB-only group (proceptivity, 0.9964, P < 0.001;
57.5 2 7.W 90
Combined EB + P (II and IV) % Responding
0.4 +- 0.1 60
0.4 k 0.1 45
0.6 2 0.2 70
0.3 2 0.2 50
0.3 f 0.1 50
0.6 + 0.3 40
sol/min’
85.0 + 6.8 95
84.5 2 5.0 95
91.0 ? 6.9 100
82.0 2 8.1 100
63.6 k 13.7 90
81.0 f 10.5 90
LQ
1.8 + 0.3d 80
1.2 f 0.2 75
1.8 2 0.4 80
1.5 2 0.4 80
1.8 + 0.4 80
0.8 2 0.3 70
sol/min
94.0 ” 2.6 100
88.5 + 3.8 95
94.0 ” 3.4 100
85.6 f 7.3 100
94.0 + 4.0 100
71.0 2 11.4 90
LQ
2.9 + 0.4d** 95
1.4 2 0.3 80
2.8 f 0.5 100
1.5 + 0.5 80
3.0 f 0.6 90
1.1 2 0.3 80
sol/min
Test 3 (500 pg P)
98.5 ?z 1.1 100
94.5 2 2.6 100
99.0 + 1.0 100
90.0 + 4.7 100
97.8 5 2.2 100
99.0 f 1.0 100
LQ
3.8 2 0.5’,’ 100
1.5 + 0.3 80
3.8 + 0.7 100
1.5 + 0.3 80
3.5 + 0.8 100
1.5 + 0.5 80
soYmin
Test 4 (1000 pg P)
“Solicitations per minute. “Standard error of the mean. cSignificantly lower than subsequent tests P < 0.01. dSignificantly higher than previous test P < 0.01. eSignificantly higher than previous test P < 0.02. ‘Significantly higher than controls P < 0.02. #Significantly higher than controls P < 0.002.
50.5 + 7.lC 95
Combined EB (I and III) % Responding
61.0 k 10.2 90
43.0 2 9.4 80
39.0 * 1l.P 100
72.0 + 8.4 100
3.0 pg EB % Responding
0.4 pg EB + P % Responding
0.4 pg EB % Responding
LQ
3.0 pg EB + P % Responding
IV
III
II
I
Group
Test 2 (100 pg P)
1. Effects of Estrogen and Progesterone on Receptive and Proceptive Behavior in the Rat
Test 1 (50 pg P)
TABLE
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TENNENT,
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receptivity, 1.0234, P < 0.002). Data from these EB treatment groups were therefore combined for subsequent analysis. In both EB treatment groups, proceptive behavior significantly increased in response to increasing progesterone doses (Friedman’s twoway analysis of variance; x2 = 36.92 P < 0.002). A graded dose-response relationship was apparent; in successive tests each increase in progesterone dose resulted in a correspondingly increased frequency of solicitations (Wilcoxon matched pairs, P < 0.01 for 50-, loo-, and 500-pg tests, P < 0.02 for l.O-mg test). Animals treated with EB alone showed no such change in proceptive behavior during successive tests (Friedman’s twoway analysis of variance; x2 = 8.565) although the first test tended to be lower than subsequent tests. Differences in proceptivity between P-treated and EB-only-treated animals were not significant in the 50-pg or lOO-pg tests, but became significant in the 500~pg (P < 0.02) and 1.O-mg (P < 0.002) tests (Mann-Whitney U). Receptive behavior also increased in successive tests in both the EBonly and EB + P groups (Friedman’s). In both groups, LQ in the first test was significantly lower (P < 0.01, Wilcoxon matched pairs) than in subsequent tests, in which LQ was maximal for both groups. In every test, LQ in the EB-only-treated groups was not significantly different from that shown by the EB + P-treated animals. The different elements of proceptive behavior-hopping, darting, and ear wiggling-were scored separately to see if there were differences in their sensitivity to hormonal treatment (Fig. 1). In those rats treated with EB + 500 pug P, hopping was the most consistently exhibited behavior. When animals received 1.0 mg P, however, hopping was less consistent, and the decrease in hopping may have been related to an increase in darting. Ear wiggling showed a clear progesterone dose effect. Statistical tests were performed only for combined “solicitation scores” since individual animals tended either to hop or to dart, and the particular behavior occurring appeared partially dependent on the action of the male. Females seemed to hop more frequently when the males were quick to mount and they darted more when the males were slower to respond. Hops
Darts
Ear wiggles
EE+P ,’ 3’
TEST #
1
2
3
4
FIG. 1. The effects of estrogen (EB) and progesterone (P) on the different elements of proceptive behavior: hops, darts, and ear wiggles.
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AND PROCEPTIVITY
The mean plasma progesterone level in blood drawn 4-5 hr following injection of 1.O mg P (and shortly after mating) was 23.2 k 1.4 rig/ml, while the mean level in EB-treated mated control animals was 10.7 + 0.8 nglml. Experiment ZZ
test of this experiment, mean LQ for all In the second “shaping” animals was 92.1 f 3.3 and mean solicitations per minute was 7.0 + 0.9. LQ and proceptivity levels for both postadrenalectomy tests are shown in Table 2. Analysis of pre-P tests vs post-P tests was by the Wilcoxon matched pairs test, while experimental and control groups were compared by Mann-Whitney U. Completeness of adrenalectomy was verified by measurement of plasma P in ether-stressed animals. Mean P level in sham-adx rats was 5.7 t 1.0 r&ml, while there was no detectable P in any adx animal. In the first postadrenalectomy test with EB alone, sham-adx animals showed significantly (P < 0.05) more proceptive behavior than adx rats. Before P treatment, solicitation behavior was present in 32% of the adx group and 71.4% of the sham-adx controls. Differences in proceptivity between the groups were significant both when all animals were included and when only those that showed any proceptive behavior were considered. Before P treatment, there was no significant difference in LQ between groups, although the control animals tended to be more receptive. Following P treatment, solicitations frequency increased significantly in both groups, while there was no significant increase in LQ. Progesterone TABLE 2 Lordosis and Proceptive Behavior before and following Progesterone Treatment in EB-Primed Ovariectomized and Ovariectomized-Adrenalectomized Female Rats Lordosis Test 1
Ovx-adx ovx
Test 2
Preprogesterone
Postprogesterone
66.4 * 8.3 81.3 2 7.0
81.3 f 4.8 89.2 f 1.4
Preprogesterone
Postprogesterone
76.4 k 7.4 86.3 + 6.6
91.5 f 3.6b 93.8 f 4.5
Solicitations per minute Test 1 Preprogesterone Ovx-adx ovx
0.2 t 0.09e 1.3 k 0.5
Test 2
Postprogesterone 3.1 k 0.7” 4.6 2 1.1”
Preprogesterone 0.2 -c 0.2c 1.1 k 0.4
u Significant difference from preprogesterone test p < 0.01. b Significant difference from preprogesterone test p < 0.05. c Significant difference from controls (Ovx) p < 0.05.
Postprogesterone 3.7 + 1.2” 4.6 in 0.9”
72
TENNENT,
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DAVIDSON
treatment to adx animals restored both proceptive and receptive behavior to sham-adx levels. In the second pre-P test, adx animals still showed significantly less proceptive behavior than did control animals. In the adx group, only 14.3% showed any proceptive behavior, while 68.8% of sham-adx animals solicited. As in the first test, there was no significant difference in LQ between the groups. Following progesterone treatment, proceptive behavior increased in both groups. LQ showed a significant increase ti-om the pre-P level in adx but not control animals, which were already showing high LQ. The two groups did not show significantly different lordosis behavior level before and after adrenalectomy. After surgery, proceptive behavior tended to be lower in both groups, but the difference was significant (P < 0.02) only in the adx group. DISCUSSION
The first experiment distinguished the relative effects of EB and P on receptive and proceptive behavior and examined the relationship between these two components of female sexual behavior. It was found that treatment of spayed rats with EB alone does facilitate a low level of proceptive behavior, but proceptivity did not respond to EB in a doseresponse fashion. Hardy and DeBold (1971) using animals treated with varying EB doses and 500 pg P, showed that the percentage of animals soliciting did not increase with EB dose, once a certain level of EB stimulation was reached. The present study confirms and extends that observation; when the EB dose is sufficient to induce maximal receptivity, additional EB, with or without P, has no effect on the percentage of animals soliciting or on the level of proceptivity displayed by an animal. This finding contrasts with the conclusion that receptive and then proceptive behavior can be sequentially elicited by increasing estrogen doses (Zemlan and Adler, 1978). In contrast, progesterone did stimulate dose-related increases in proceptive behavior. These results show the importance of quantitatively assessing proceptive behavior. The percentage of animals showing proceptivity after administration of 500 pg and 1 .O mg P does not reflect the observed graded changes in level of behavior. Thus, recording of each observed behavior provides a more sensitive measure of the effects of experimental manipulation. Although responses increased significantly with each increase in P dose, differences between EB-only- and P-treated groups became significant only when 500 pg or 1.O mg P was given. That these data are relevant to natural conditions of the estrous cycle is suggested by the fact that plasma P levels 4 hr after injection of 1 .O mg P resembled that found on the evening of proestrus in our colony (Gray, Smith, Tennent, and Davidson, in press). Again, there was no difference between EB treatment groups in any test, nor was the rate of
PROGESTERONE
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73
increase in response to increasing P different in high and low EB groups. In several earlier studies of proceptivity (Hardy and DeBold, 1971; Hlinak and Madlafousek, 1971; Whalen, 1974) proceptive behavior varied with both EB and P doses; it was stated (Hlinak and Madlafousek, 1971) that less P was needed with increasing EB to achieve the same level of proceptivity, Only percentage of animals showing proceptivity and not solicitation rate was reported. Exposure to EB increases subsequent responses to steroid administration (Beach, 1974; Damassa and Davidson, 1973), and repeated testing also facilitates lordosis (Hardy and DeBold, 1973; Henrik and Gerall, 1976), and soliciting (Hlinak, 1975). In the EB-alone-treated groups, proceptivity tended to increase after the first test, although the level was not significant, and remained the same on all subsequent tests. P-Treated animals showed significant increases after each test, and differences were greatest after the second test, while EB-only animals showed relatively constant behavior. These increases could not, therefore, have been caused by successive testing and re eated EB administration alone. LQ did increase significantly after the Krst test in all groups; this difference may have resulted from successive testing and steroid administration. The adrenal gland can release substantial amounts of P. Plasma P levels measured in EB-treated Ovx rats following mating even exceeded that found following ether stress, presumably due to considerable matinginduced adrenal P secretion. A number of studies (Davidson ef al., 1968; Pfaff, 1970; Larsson et al., 1974) have assessed the role of adrenal P in facilitating lordosis, but none has specifically studied its effect on proceptivity. The second experiment demonstrated the importance of the adrenal in maintaining this behavior. After adrenalectomy, both the proportion of animals showing proceptive behavior and the amount of proceptivity in response to EB alone, were considerably reduced. Because Zemlan and Adler (1978) did not measure proceptive behavior quantitatively, it is difficult to compare these results with their report on proceptivity in adrenalectomized rats. Some proceptivity was observed in some adrenalectomized rats in this experiment, but the level was well below ovariectomized EB-treated levels, and far below that shown by progesterone-treated animals. Zemlan and Adler also used a different EB treatment paradigm, injecting 500 &kg 46 hr before testing, as compared to the present use of lower doses injected for several days. Decrements in proceptive behavior observed in adx EB-only-treated animals were probably not caused by ill effects of the surgery. Exogenous P restored the level of behavior to that shown by sham-adx animals. In post-adx tests, proceptive behavior tended to be lower than in the shaping tests in both groups, but the difference was significant only in the adx animals. While this may have indicated postsurgical illness, this is unlikely since lordosis behavior in both groups was restored to pread-
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AND DAVIDSON
renalectomy levels by administration of 1.O mg P. The lowered proceptivity of adx animals postoperatively more likely reflected the loss of supplementary adrenal progesterone. These experiments have shown that proceptive behavior, while not absolutely dependent on progesterone, responds in a graded manner to increasing P doses. Adrenal P also appears to stimulate proceptivity. If EB is sufficient to stimulate maximal receptivity, additional EB has no effect on the low level of proceptivity expressed. ACKNOWLEDGMENTS This work was supported by NIMH Grant MH-21178. B. Tennent was supported by NIMH Bioscience Training Program MH 8304 and NIH predoctoral fellowship No. 1 F31 MH 07645. The able technical assistance of D. TaIlentire is gratefully acknowledged. Progesterone antiserum was kindly supplied by Dr. G. D. Niswender.
REFERENCES Beach, F. A. (1942). Importance of progesterone to induction of sexual receptivity in spayed female rats. Proc. Sot. Exp. Biol. Med. 51, 369-371. Beach, F. A. (1976). Sexual attractivity, proceptivity, and receptivity in female mammals. Horm.
Behav.
7, 105-138.
Baling, J. L., and Blandau, R. J. (1939). The estrogen-progesterone induction of mating response in the spayed female rat. Endocrinology 25, 359-364. Damassa, D., and Davidson, J. M. (1973). Effects of ovariectomy and constant light on responsiveness to estrogen in the rat. Horm. Behav. 4, 269-279. Davidson, J. M., Rodgers, C. H., Smith, E. R., and Bloch, G. J. (1968). Stimulation of female behavior in adrenalectomized rats with estrogen alone. Endocrinology 82, 193195. Fadem, B. H., Bartield, R. J., and Whalen, R. E. (1980). Dose response and time-response relationships between progesterone and the display of patterns of receptive and proceptive behavior in the female rat. Horm. Behav. 13, 40-48. Gibori, G., Antczak, E., and Rothchild, I. (1977). The role of estrogen in the regulation of luteal progesterone secretion in the rat after day 12 of pregnancy. Endocrinology 100, 1483- 1495. Gilman, D. P., and Hitt, J. C. (1978). Effects of gonadal hormones on pacing of sexual contacts by female rats. Behav. Biol. 24, 77-87. Gray, G. D., Tennent, B., Smith, E. R., and Davidson, J. M. (1980). LH regulation and sexual behavior in middle-aged female rats. Endocrinology, in press. Hardy, D. F., and DeBold, J. F. (1971). The relationship between levels of exogenous hormones and the display of lordosis by the female rat. Harm. Behav. 2, 287-297. Hardy, D. F., and DeBold, J. F. (1973). Effects of repeated testing on sexual behavior of the female rat. J. Camp. Physiol. Psycho/. 85, 195-202. Henrik, E., and Gerall, A. A. (1976). Facilitation of receptivity in estrogen-primed rats during successive mating tests with progestins and methysergide. J. Camp. Physiol. Psychol.
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Hlinak, Z., and Madlafousek, J. (1971). Positive and negative effects of progesterone on the precopulatory behavior of ovariectomized rats. Activ. Nerv. Super. 14, 3. Hlinak, Z. (1975). Strain-associated differences in the action of oestradiol and progesterone in inducing pre-copulatory behaviour in ovariectomized rats. Physiol. Bohemoslov. 24, 381-383. Kendall, M. G. (1962). Rank Correlation Methods. Hafner, New York.
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Larsson, F., Feder, H. H., and Komisaruk, B. R. (1974). Role of the adrenal glands, repeated matings, and monamines in lordosis behavior of rats. Pharmacol. Biochem. Behav. 2, 685-692. Madlafousek, J., and Hlinak, Z. (1978). Sexual behaviour of the female laboratory rat: Inventory, patterning, and measurement. Behaviour 63, 129-173. Pfaff, D. W. (1970). Nature of sex hormone effect on rat sex behavior; specificity of effects and individual patterns of response. J. Comp. Physiol. Psychol. 73, 349-358. Whalen, R. E. (1974). Estrogen, progesterone induction of mating in female rats. Horm. Behav. 5, 157-162. Zemlan, F., and Adler, N. T. (1978). Hormonal control of female sexual behavior in the rat. Horm. Behav. 9. 345-357.