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Dose-response and time-response relationships between progesterone and the display of patterns of receptive and proceptive behavior in the female rat
HORMONES
AND BEHAVIOR
13, 40-48 (1979)
Dose-Response and Time-Response Relationships Between Progesterone and the Display of Patterns of Receptive and Proceptive Behavior in the Female Rat BARBARA H. FADEM, RONALD J. BARFIELD, AND RICHARD E. WHALEN* Department of Biology, Livingston College, Rutgers University, New Brunswick, New Jersey 08903, and *Department of Psychobiology, University of California, Irvine, California 92717
The relationship between administration of progesteroneand the display of patterns of receptive (responseto the male) and proceptive (female initiated) sexual behavior was examinedin ovariectomized,estrogen-primedfemalerats in a “restrained male” test situation. It was found that the degreeof receptivity and proceptivity displayed was directly proportional to progesteronedose and time from progesteroneinjection (up to 4.5 hr). Higher progesterone doses and longer period of time from progesterone injection (up to 4.5 hr) were both associated with shorter latencies to return to the male following intromission and ejaculation. Receptivity could be induced with estrogen alone but progesterone was required for the display of proceptivity and higher doses of progesterone were needed to effect increases in proceptivity relative to receptivity. Proceptive behavior also occurred in a narrower time range than did receptive behavior. Receptivity alone is characterized as the lowest degree, and receptivity plus proceptivity as the highest degree, of expression of the total behavior pattern of the estrous female rat. Receptivity and proceptivity together constitute a continuum of estrous responsiveness. Increasing the progesterone dose from 0 to 200 pg, and increasing the latency from progesterone injection from 0 to 4.5 hr, were associated with increasing degree of expression of the total behavioral continuum.
Receptive behavior in the female rat, that is, the likelihood that the female will display the lordosis posture when mounted by the male, is dependent both on the dose of estrogen (Davidson, Smith, Rodgers, and Block, 1968; Whalen and Hardy, 1970; Hardy and DeBold, 1971) and of progesterone (Lisk, 1960; Meyerson, 1972) and on the time since each
hormone is administered (Green Luttge, and Whalen, 1970; Lisk, 1960; Meyerson, 1972). Receptive behavior of estrous females occurs as a response to maleinitiated behavior during mating. Estrous females also exhibit “proceptive” behavior which involves female initiative in mating (Beach, 1976). In the female rat this includes darting, hopping, rapid ear vibration (ear 40 0018-506W79/040040-09$01.00/O Copyright @ 1979 by Academic Press. inc. All rights of reproduction in any form reserved.
RECEFIWITY,
PROCEFHVITY,
AND
PROGESTERONE
41
wiggling), and approach to and withdrawal from the male, all of which presumably serve to arouse the male sexually (Hlinak and Madlafousek, 1971; Madlafousek, Hlinak, and Beran, 1976). Although the time course of the display of proceptive behavior following exogenous hormone administration has not been examined, it differs from that of receptive behavior in naturally estrous female hamsters. Proceptivity begins later into estrus and terminates sooner (Beach, Stern, Carmichael, and Ranson, 1976). In the female rat, there is also evidence that the proceptive component of sexual behavior is primarily affected by progesterone rather than estrogen dose (Whalen, 1974; Fadem, 1977; Gilman and Hitt, 1978) and thus it might be expected that the time course for the display of receptivity and proceptivity is different following exogenous hormone administration as well. Ideally, feminine sexual behavior, especially its active “proceptive” component, should be assessed in a test situation in which the variability due to the activity of the male is minimized. This goal can be achieved by using a test apparatus in which the mobility of the male is restricted. Such an apparatus (“the tether apparatus”) has been previously employed (Krieger, Orr, and Perper, 1976; Fadem, 1977). The present study was designed to reexamine dose-response and time-response functions for progesterone for both receptive and proceptive behavior. To quantify these two aspects of feminine sexual behavior, “the tether apparatus” was used. GENERAL
METHODS
Test procedure. The animals were observed in an 89 x 18-cm plywood box with a Plexiglas front. The box was divided into two compartments by a 9-cm-high Plexiglas barrier placed 26 cm from one end (floor areas: 972 and 3 13 cm2). The stimulus male was tethered in the smaller compartment by attaching a wire loop to the nape of the neck with a wound clip and attaching the loop to a string secured to the wall of the chamber. To start a test, the experimental female was placed in the larger compartment. A test was over when the female returned to the male’s compartment following ejaculation or when 300 set had elapsed. Tests were conducted weekly for 5 weeks between 1 and 4 PM. Behavioral measures. For the male, the occurrence of mounts, intromissions, and ejaculation was recorded on an event recorder. Ejaculation latency (EL-the time in seconds from the first intromission to the ejaculation) was then obtained. For the female, the incidence of “solicits” (hops and darts) rapid ear vibration (ear wiggling), and entrances to and exits from the male’s compartment (visits) was recorded. The adoption of lordosis postures in response to mounts by males and the occurrence of rejection of males during mounting attempts, characterized by hind leg kicks, were recorded as well. [The kicking response is an index of sexual
42
FADEM, BARFIELD,
AND WHALEN
refractoriness and has been used to distinguish receptive from nonreceptive females (Whalen and Nadler, 1%5).] Receptivity was measured in terms of lordosis quotient (LQ-the number of lordoses/number of mounts x 100) and acceptance (AC-absence of kicking responses during a test). Proceptivity was measured in terms of (1) solicitation rate (SRnumber of solicits/EL x 1000); (2) visit rate (VR-number of times the female crossed the barrier to the male’s compartment/EL x 1000); (3) ear wiggling (EW-presence in a test); and (4) intromission and ejaculation return latencies (IRL and ERL-the time, in seconds, between intromission or ejaculation and the female’s next return to the male’s compartment). In the few instances in which the female did not leave the male’s compartment following an intromission, no return latency was calculated. Females invariably left the male’s compartment following an ejaculation. Maximum scores of 200 and 300 set were assigned to IRL and ERL, respectively, in tests where the female failed to return to the male’s compartment during a test. Hormone treatment. Approximately 1 week after ovariectomy, each female received 10 pg estradiol benzoate in oil (peanut oil in Experiment I and sesame oil in Experiment II) followed 2 days later by 500 pg progesterone in oil. One week later, each female was given the same hormone treatment and was tested with a male in the tether apparatus to familiarize her with the apparatus. Hormone treatments as described under Methods of Experiments I and II were given weekly for each of the 5 test weeks. Statistical tests. Differences between group means for IRL, ERL, SR, and VR were tested using an analysis of variance (ANOVA). The Scheffe test was used to test for significant differences between individual doses and times. This procedure was also used for LQ in Experiment I. For data analyses, raw scores were used for IRL and ERL in Experiment I and for ERL in Experiment II. For scores that were not normally distributed, the data were transformed. Fifth-root transformation of the data was used for analysis of IRL in Experiment II. Log transformation was used for SR and square-root transformation for VR in both experiments. Nonparametric statistics were used to test for significant dose and time effects on EW and AC (Cochran’s Q) in Experiments I and II, and Friedman’s two-way ANOVA on ranks for LQ in Experiment II. EXPERIMENT
I
The objective of this experiment was to examine the effect of different doses of progesterone on measures of receptivity and proceptivity. Methods The subjects were 20, sexually inexperienced, ovariectomized, Sprague-Dawley female rats weighing approximately 200 g. They were housed in groups of 6 or 7 and maintained on a 12- 12 reversed light-dark
RECEPIIVITY,
PROCEPTIVITY,
43
AND PROGESTERONE
cycle with lights off at 31 AM. Ten intact Long-Evans males were used as stimulus animals. The dose of estradiol benzoate in all tests was 8 pg per animal. This was followed 2 days later by one of the following progesterone doses: (a) 0 pg, (b) 50 pg, (c) 75 pg, (d) 100 pg, or (e) 200 pg. Behavioral testing was conducted from a minimum of 3.5 to a maximum of 6 hr following progesterone injection. Each animal received each dose (a-e) and test order was varied randomly. In all tests, the stimulus male was removed following ejaculation and a new male was introduced. This change in males eliminates the effect of the male’s behavior during his postejaculatory refractory period on the female’s latency to return following ejaculation (Fadem, 1977). Results
At the higher progesterone doses, average lordosis quotients (LQs) were higher and fewer females rejected the male when mounted. Mean intromission and ejaculation return latencies (IRLs and ERLs) were shorter, solicitation rate (SR) was higher, and more females demonstrated ear TABLE 1 The Relationship between Progesterone Dose and Measures of Receptive and Proceptive Behavior in the Estrogen-Primed Female Rat Dose Lug) Behavioral measure
0
50
75
100
200
PC
Mean intromission return latency (set)
168
154 <
118 *
84 >
67
0.0001
Mean ejaculation return latency (sec)a
268
258
230 c
205 *
149 >
0.0001
Mean solicitation ratea
0.30
1.50
6.60 ‘
*
13.55
23.25 >
0.0001
Mean visit rate’
21.10
22.45
21.15
19.58
18.40
Mean lordosis quotienta
16 <
28
50 ,
74
90
0.0001
*
*
t
Ear Wiggling (No. of animals)b Acceptance (No. of animal+ (1ANOVA. * Cochran’s Q. * Scheffe test significance.
ns
>
1
3
8
8
19
0.001
10
11
16
14
19
0.001
44
FADEM, BARFIELD,
AND WHALEN
wiggling (EW) at higher progesterone doses (Table 1). The highest progesterone dose used in this study (200 pg) was associated with maximum values for all measures. The effect of progesterone dose was significant for all measures (p < 0.001) except visit rate (VR). Although some receptivity (lordosis) was shown without progesterone, almost no proceptivity (solicitation or ear wiggling) occurred in its absence. Significant changes in LQ occurred between 0 and 75 pg, 50 and 100 pg, and 75 and 200 pg, while significant changes in SR only occurred between 75 and 2OOpgof progesterone (Table 1). Thus, as shown in Fig. la, progesterone was required for the display of proceptive behavior; and furthermore, higher progesterone doses were necessary to effect a significant increase in a measure of proceptivity (SR) relative to a measure of receptivity (LQ). EXPERIMENT
II
The purpose of this experiment was to examine the time course of the expression of receptive and proceptive behavior following progesterone injection. Methods The subjects were 18, sexually inexperienced, ovariectomized, LongEvans female rats weighing approximately 200 g. They were housed in pairs and maintained on a 12-12 reversed light-dark cycle with lights off at 9:30 AM. Eight intact Long-Evans males were used as stimulus animals. Forty-eight hours prior to testing, each female received 8 pg estradiol benzoate and 200 pg progesterone at 0, 0.5, 1.5, 4.5, or 13.5 hr prior to testing. Test order was varied randomly and the male was changed following ejaculation as in Experiment I. Results As the time interval from progesterone injection increased, measures of both receptivity and proceptivity increased to a maximum at 4.5 hr and all measures had decreased by 13.5 hr (Table 2). The effect of time from progesterone injection on behavior was significant for all measures (p < 0.009) except VR. In the absence of progesterone (at 0 hr), receptivity was shown (LQ = 56%), but little proceptivity was observed. High levels of receptivity were observed sooner after progesterone injection than high levels of proceptivity (LQ reached 71% of maximum by 0.5 hr while SR only reached 11% of maximum by 0.5 hr). Proceptivity decreased sooner than receptivity (LQ was 77% of maximum at 13.5 hr and SR was only 27% of maximum at 13.5 hr) (Fig. lb). Thus, the time course following progesterone injection for the display of proceptivity differs from that of receptivity, viz., proceptivity peaks more sharply in time.
RECEPTIVITY,
PROCEPTIVITY,
45
AND PROGESTERONE
a 0
so
75
100
200
DOSE lug)
/ / I
/
/
//
\
\
. \
\
\ Sl?\‘\
I I I I I
\
\
\
\
\
\
\
TIME klrs.) The effect of progesterone on receptivity and proceptivity in ovariectomized, estrogen-primed female rats. (a) Dose-response relationship and (b) the time-response relationship between progesterone nand both lordosis quotient (LQ-a behavioral measure of receptivity) and solicitation rate (SR-a behavioral measure of proceptivity). Dose of progesterone and time from progesterone injection are shown on the abscissa. Using 100% as the maximum level of behavior observed, the percentage of this maximum level at other test doses and times from injection are shown on the ordinate. FIG.
I.
46
FADEM, BARFIELD,
AND WHALEN
TABLE 2 The Relationship between Time from Progesterone Injection and Measures of Receptive and Proceptive Behavior in the Estrogen-Primed Female Rat Time of test (hr) Behavioral measure
0
0.5
Mean intromission return latency (set)
102
70
Mean ejaculation return latency (set)
173 ‘
148
Mean solicitation rate”
2.22
1.5
4.5
73
*
2.61 L
143
13.5
P <
39
68
0.005
89
161
0.009
, 30.33
62.72
8.55
0.8001
A A Mean visit rate”
24.22
29.39
20.78.
Mean lordosis quotienP
25.56
20.12
0.06
56
71
92
100
77
0.001
Ear Wiggling (No. of animals)c
1
1
9
17
5
0.001
Acceptance (No. of animals)
8
5
10
17
9
0.05
a ANOVA. b Friedman’s two-way ANOVA. c Cochran’s Q. * Scheffe test significance.
DISCUSSION
The present study demonstrates the dose- and time-dependent relationships between exogenous progesterone and the display of receptive and proceptive behavior in the ovariectomized, estrogen-primed female rat. The main findings are as follows: (1) Progesterone affects the female’s latency to initiate contact with the male following copulatory events (IRL and ERL). This effect has been previously observed (Fadem, 1977; Gilman and Hitt, 1978). (2) The degree of receptivity and proceptivity displayed is directly related to the progesterone dose and time from progesterone injection (up to 4.5 hr). (3) Although the female may show partial receptivity, i.e., exhibit lordosis, without progesterone, proceptivity (hopping, darting and ear wiggling) rarely if ever occurs in the absence of progesterone. (4) Higher doses of progesterone are necessary to effect increases in proceptivity relative to receptivity. And (5), proceptivity occurs in a narrower time range than receptivity.
RECEFI-IVITY,
PROCEF’TIVITY,
AND
PROGESTERONE
47
In the intact female rat, progesterone secretion increases following the surge of luteinizing hormone (LH) which triggers ovulation after a latency of about 12 hr. High levels of plasma progesterone are associated with high levels of estrous behavior (Feder, Goy, and Resko, 1967), and the increasing levels of progesterone which occur following the LH surge may act to increase the degree of expression of the total estrous behavior pattern, i.e., receptivity alone followed by receptivity plus a full display of proceptivity, when progesterone levels are highest. The complete pattern would thus occur some hours before ovulation and might serve to increase the probability that mating would occur at the physiologically optimal time for impregnation. Also, because the female’s male-seeking, solicitation behavior (proceptivity) is active, it involves considerable energy expenditure, and it can be assumed that in the natural situation such behavior would occur during a short time span relative to passive receptivity, i.e., only at the optimal time for mating to lead to impregnation. Thus, the finding in the present study that proceptivity occurs in a narrower time range than receptivity following exogenous hormone treatment is not unexpected. The total pattern of female rat sexual behavior involves both receptive and proceptive components. It has been suggested that these two components may represent two separate behavioral systems which are controlled to some extent by different brain regions (Modianos, Hitt, and Popolow, 1975; Ward, Crowley, Zemlan, and Margules, 1975) and may depend on different gonadal hormones, lordosis upon estrogen alone and ear wiggling and hopping upon progesterone following estrogen priming (Hardy and DeBold, 1971; Whalen, 1974). Alternatively, proceptivity and receptivity may represent the graded expression of a single system controlling estrous behavior. Regardless of whether receptivity and proceptivity are controlled by the same or by different physiological systems, the results of this study show that receptivity and proceptivity represent varying levels in a continuum of estrous responsiveness. Lowest levels of the continuum are characterized by irregular displays of lordosis alone and highest levels are characterized by reliable displays of lordosis plus hopping, darting, and ear wiggling. The notion that, in the female rat, lordosis plus hopping and darting represents a higher level of sexual responsiveness than lordosis alone has been previously advanced and has been used in quantifying feminine sexual behavior (Ball, 1937; Gerall, Hendricks, Johnson, and Bounds, 1967). In the present study, the degree of expression of the total behavioral continuum appeared to depend on progesterone dose and latency from progesterone injection. The results of this study then, show a critical role for progesterone in proceptive behavior in the female rat and also indicate that receptive and proceptive behavior together constitute a continuum of estrous responsiveness.
48
FADEM, BARFIELD,
AND WHALEN
ACKNOWLEDGMENTS This research was supported by Grant HD-04484 to R.J.B. from the National Institutes of Health and by a Sigma Xi National Research Council of North America, Grant-in-Aid of Research to B.H.F.
REFERENCES Ball, J. (1937). A test for measuring sexual excitability in the female rat. Camp. Psychof. Monogr.
14, l-37.
Beach, F. A. (1976). Sexual attractivity, proceptivity and receptivity in female mammals. Horm. Behav. 7, 105-138. Beach, F. A., Stern, B., Carmichael, M., and Ranson, E. (1976). Comparisons of sexual receptivity and proceptivity in female hamsters. Behav. Biol. 18,473-487. Davidson, J. M., Smith, E. R., Rodgers, C. H., and Bloch, G. J. (1%8). Relative thresholds of behavioral and somatic responses to estrogen. Physiol. Behav. 3, 227-229. Fadem, B. H. (1977). Copulatory Behavior in the Female Rat: The Effect of Local Stimulation, Progesterone Level, the Stimulus Qualities of the Male and Individual Differences on Proceptivity. Unpublished Master’s thesis, Department of Zoology, Rutgers Univer-
sity, New Brunswick, N.J. Feder, H. H., Goy, R. W., and Resko, J. A. (1967). Progesterone concentrations in the peripheral plasma of cyclic rats. J. Physiol. 191, 136-137. Gerall, A. A., Hendricks, S. E., Johnson, L. L., and Bounds, T. W. (1%7). Effects of early castration in male rats on adult sexual behavior. J. Comp. Physiol. 64, 206-212. Gilman, D. P., and Hitt, J. C. (1978). Effects of gonadal hormones on pacing of sexual contacts by female rats. Behav. Biol. 24 (1) 77-87. Green, R., Luttge, W. G., and Whalen, R. E. (1970). Induction of receptivity in ovariectomized female rats by a single intravenous injection of estradiol-17b Physiol. Behav. 5, 137-141. 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. Hlinak, Z., and Madlafousek, J. (1971). The dependence of sexual behavior of inexperienced males on the precopulatory behaviour of female albino rats. Proc. Czek. Physiol. Sot. 21, 83.
Krieger, M. S., Orr, D., and Perper, T. (1976). Temporal patterning of sexual behavior in the female rat. Behav. Biol. 18, 379-386. Lisk, R. D. (1960). A comparison of the effectiveness of intravenous as opposed to subcutaneous injection of progesterone for the induction of estrous behavior in the rat. Canad. J. Biochem. Physiol. 38, 1381-1383. Madlafousek, J., Hlinak, Z., and Beran, J. (1976). Decline of sexual behavior in castrated male rats: Effects of female precopulatory behavior. Harm. Behav. 7, 245-252. Meyerson, B. J. (1972). Latency between intravenous injection of progestins and the appearance of estrous behavior in estrogen-treated ovarectomized rats. Harm. Behav. 3, l-9.
Modianos, D. T., Hitt, J. C., and Popolow, H. B. (1975). Habenular lesions and feminine sexual behavior of ovariectomized female rats: diminished responsiveness to the synergistic effects of estrogen and progesterone. J. Comp. Physiol. Psychol. 89, 231-237. Ward, I. L., Crowley, W. R., Zemlan, F. P., and Margules, D. L. (1975). Monoaminergic mediation of female sexual behavior. J. Camp. Physiol. Psychol. 88, 53-61. Whalen, R. E. (1974). Estrogen-progesterone induction of mating in female rats. Harm. Behav.
5, 157-162.
Whalen, R. E., and Hardy, D. F. (1970). Induction of receptivity in female rats and cats with estrogen and testosterone. Physiol. Behav. 5, 529-533. Whalen, R. E., and Nadler, R. D. (1965). Modification of spontaneous and hormone induced sexual behavior by estrogen administered to neonatal female rats. J. Camp. Physiol. Psychol. 60, (1) 150-152.
AND BEHAVIOR
13, 40-48 (1979)
Dose-Response and Time-Response Relationships Between Progesterone and the Display of Patterns of Receptive and Proceptive Behavior in the Female Rat BARBARA H. FADEM, RONALD J. BARFIELD, AND RICHARD E. WHALEN* Department of Biology, Livingston College, Rutgers University, New Brunswick, New Jersey 08903, and *Department of Psychobiology, University of California, Irvine, California 92717
The relationship between administration of progesteroneand the display of patterns of receptive (responseto the male) and proceptive (female initiated) sexual behavior was examinedin ovariectomized,estrogen-primedfemalerats in a “restrained male” test situation. It was found that the degreeof receptivity and proceptivity displayed was directly proportional to progesteronedose and time from progesteroneinjection (up to 4.5 hr). Higher progesterone doses and longer period of time from progesterone injection (up to 4.5 hr) were both associated with shorter latencies to return to the male following intromission and ejaculation. Receptivity could be induced with estrogen alone but progesterone was required for the display of proceptivity and higher doses of progesterone were needed to effect increases in proceptivity relative to receptivity. Proceptive behavior also occurred in a narrower time range than did receptive behavior. Receptivity alone is characterized as the lowest degree, and receptivity plus proceptivity as the highest degree, of expression of the total behavior pattern of the estrous female rat. Receptivity and proceptivity together constitute a continuum of estrous responsiveness. Increasing the progesterone dose from 0 to 200 pg, and increasing the latency from progesterone injection from 0 to 4.5 hr, were associated with increasing degree of expression of the total behavioral continuum.
Receptive behavior in the female rat, that is, the likelihood that the female will display the lordosis posture when mounted by the male, is dependent both on the dose of estrogen (Davidson, Smith, Rodgers, and Block, 1968; Whalen and Hardy, 1970; Hardy and DeBold, 1971) and of progesterone (Lisk, 1960; Meyerson, 1972) and on the time since each
hormone is administered (Green Luttge, and Whalen, 1970; Lisk, 1960; Meyerson, 1972). Receptive behavior of estrous females occurs as a response to maleinitiated behavior during mating. Estrous females also exhibit “proceptive” behavior which involves female initiative in mating (Beach, 1976). In the female rat this includes darting, hopping, rapid ear vibration (ear 40 0018-506W79/040040-09$01.00/O Copyright @ 1979 by Academic Press. inc. All rights of reproduction in any form reserved.
RECEFIWITY,
PROCEFHVITY,
AND
PROGESTERONE
41
wiggling), and approach to and withdrawal from the male, all of which presumably serve to arouse the male sexually (Hlinak and Madlafousek, 1971; Madlafousek, Hlinak, and Beran, 1976). Although the time course of the display of proceptive behavior following exogenous hormone administration has not been examined, it differs from that of receptive behavior in naturally estrous female hamsters. Proceptivity begins later into estrus and terminates sooner (Beach, Stern, Carmichael, and Ranson, 1976). In the female rat, there is also evidence that the proceptive component of sexual behavior is primarily affected by progesterone rather than estrogen dose (Whalen, 1974; Fadem, 1977; Gilman and Hitt, 1978) and thus it might be expected that the time course for the display of receptivity and proceptivity is different following exogenous hormone administration as well. Ideally, feminine sexual behavior, especially its active “proceptive” component, should be assessed in a test situation in which the variability due to the activity of the male is minimized. This goal can be achieved by using a test apparatus in which the mobility of the male is restricted. Such an apparatus (“the tether apparatus”) has been previously employed (Krieger, Orr, and Perper, 1976; Fadem, 1977). The present study was designed to reexamine dose-response and time-response functions for progesterone for both receptive and proceptive behavior. To quantify these two aspects of feminine sexual behavior, “the tether apparatus” was used. GENERAL
METHODS
Test procedure. The animals were observed in an 89 x 18-cm plywood box with a Plexiglas front. The box was divided into two compartments by a 9-cm-high Plexiglas barrier placed 26 cm from one end (floor areas: 972 and 3 13 cm2). The stimulus male was tethered in the smaller compartment by attaching a wire loop to the nape of the neck with a wound clip and attaching the loop to a string secured to the wall of the chamber. To start a test, the experimental female was placed in the larger compartment. A test was over when the female returned to the male’s compartment following ejaculation or when 300 set had elapsed. Tests were conducted weekly for 5 weeks between 1 and 4 PM. Behavioral measures. For the male, the occurrence of mounts, intromissions, and ejaculation was recorded on an event recorder. Ejaculation latency (EL-the time in seconds from the first intromission to the ejaculation) was then obtained. For the female, the incidence of “solicits” (hops and darts) rapid ear vibration (ear wiggling), and entrances to and exits from the male’s compartment (visits) was recorded. The adoption of lordosis postures in response to mounts by males and the occurrence of rejection of males during mounting attempts, characterized by hind leg kicks, were recorded as well. [The kicking response is an index of sexual
42
FADEM, BARFIELD,
AND WHALEN
refractoriness and has been used to distinguish receptive from nonreceptive females (Whalen and Nadler, 1%5).] Receptivity was measured in terms of lordosis quotient (LQ-the number of lordoses/number of mounts x 100) and acceptance (AC-absence of kicking responses during a test). Proceptivity was measured in terms of (1) solicitation rate (SRnumber of solicits/EL x 1000); (2) visit rate (VR-number of times the female crossed the barrier to the male’s compartment/EL x 1000); (3) ear wiggling (EW-presence in a test); and (4) intromission and ejaculation return latencies (IRL and ERL-the time, in seconds, between intromission or ejaculation and the female’s next return to the male’s compartment). In the few instances in which the female did not leave the male’s compartment following an intromission, no return latency was calculated. Females invariably left the male’s compartment following an ejaculation. Maximum scores of 200 and 300 set were assigned to IRL and ERL, respectively, in tests where the female failed to return to the male’s compartment during a test. Hormone treatment. Approximately 1 week after ovariectomy, each female received 10 pg estradiol benzoate in oil (peanut oil in Experiment I and sesame oil in Experiment II) followed 2 days later by 500 pg progesterone in oil. One week later, each female was given the same hormone treatment and was tested with a male in the tether apparatus to familiarize her with the apparatus. Hormone treatments as described under Methods of Experiments I and II were given weekly for each of the 5 test weeks. Statistical tests. Differences between group means for IRL, ERL, SR, and VR were tested using an analysis of variance (ANOVA). The Scheffe test was used to test for significant differences between individual doses and times. This procedure was also used for LQ in Experiment I. For data analyses, raw scores were used for IRL and ERL in Experiment I and for ERL in Experiment II. For scores that were not normally distributed, the data were transformed. Fifth-root transformation of the data was used for analysis of IRL in Experiment II. Log transformation was used for SR and square-root transformation for VR in both experiments. Nonparametric statistics were used to test for significant dose and time effects on EW and AC (Cochran’s Q) in Experiments I and II, and Friedman’s two-way ANOVA on ranks for LQ in Experiment II. EXPERIMENT
I
The objective of this experiment was to examine the effect of different doses of progesterone on measures of receptivity and proceptivity. Methods The subjects were 20, sexually inexperienced, ovariectomized, Sprague-Dawley female rats weighing approximately 200 g. They were housed in groups of 6 or 7 and maintained on a 12- 12 reversed light-dark
RECEPIIVITY,
PROCEPTIVITY,
43
AND PROGESTERONE
cycle with lights off at 31 AM. Ten intact Long-Evans males were used as stimulus animals. The dose of estradiol benzoate in all tests was 8 pg per animal. This was followed 2 days later by one of the following progesterone doses: (a) 0 pg, (b) 50 pg, (c) 75 pg, (d) 100 pg, or (e) 200 pg. Behavioral testing was conducted from a minimum of 3.5 to a maximum of 6 hr following progesterone injection. Each animal received each dose (a-e) and test order was varied randomly. In all tests, the stimulus male was removed following ejaculation and a new male was introduced. This change in males eliminates the effect of the male’s behavior during his postejaculatory refractory period on the female’s latency to return following ejaculation (Fadem, 1977). Results
At the higher progesterone doses, average lordosis quotients (LQs) were higher and fewer females rejected the male when mounted. Mean intromission and ejaculation return latencies (IRLs and ERLs) were shorter, solicitation rate (SR) was higher, and more females demonstrated ear TABLE 1 The Relationship between Progesterone Dose and Measures of Receptive and Proceptive Behavior in the Estrogen-Primed Female Rat Dose Lug) Behavioral measure
0
50
75
100
200
PC
Mean intromission return latency (set)
168
154 <
118 *
84 >
67
0.0001
Mean ejaculation return latency (sec)a
268
258
230 c
205 *
149 >
0.0001
Mean solicitation ratea
0.30
1.50
6.60 ‘
*
13.55
23.25 >
0.0001
Mean visit rate’
21.10
22.45
21.15
19.58
18.40
Mean lordosis quotienta
16 <
28
50 ,
74
90
0.0001
*
*
t
Ear Wiggling (No. of animals)b Acceptance (No. of animal+ (1ANOVA. * Cochran’s Q. * Scheffe test significance.
ns
>
1
3
8
8
19
0.001
10
11
16
14
19
0.001
44
FADEM, BARFIELD,
AND WHALEN
wiggling (EW) at higher progesterone doses (Table 1). The highest progesterone dose used in this study (200 pg) was associated with maximum values for all measures. The effect of progesterone dose was significant for all measures (p < 0.001) except visit rate (VR). Although some receptivity (lordosis) was shown without progesterone, almost no proceptivity (solicitation or ear wiggling) occurred in its absence. Significant changes in LQ occurred between 0 and 75 pg, 50 and 100 pg, and 75 and 200 pg, while significant changes in SR only occurred between 75 and 2OOpgof progesterone (Table 1). Thus, as shown in Fig. la, progesterone was required for the display of proceptive behavior; and furthermore, higher progesterone doses were necessary to effect a significant increase in a measure of proceptivity (SR) relative to a measure of receptivity (LQ). EXPERIMENT
II
The purpose of this experiment was to examine the time course of the expression of receptive and proceptive behavior following progesterone injection. Methods The subjects were 18, sexually inexperienced, ovariectomized, LongEvans female rats weighing approximately 200 g. They were housed in pairs and maintained on a 12-12 reversed light-dark cycle with lights off at 9:30 AM. Eight intact Long-Evans males were used as stimulus animals. Forty-eight hours prior to testing, each female received 8 pg estradiol benzoate and 200 pg progesterone at 0, 0.5, 1.5, 4.5, or 13.5 hr prior to testing. Test order was varied randomly and the male was changed following ejaculation as in Experiment I. Results As the time interval from progesterone injection increased, measures of both receptivity and proceptivity increased to a maximum at 4.5 hr and all measures had decreased by 13.5 hr (Table 2). The effect of time from progesterone injection on behavior was significant for all measures (p < 0.009) except VR. In the absence of progesterone (at 0 hr), receptivity was shown (LQ = 56%), but little proceptivity was observed. High levels of receptivity were observed sooner after progesterone injection than high levels of proceptivity (LQ reached 71% of maximum by 0.5 hr while SR only reached 11% of maximum by 0.5 hr). Proceptivity decreased sooner than receptivity (LQ was 77% of maximum at 13.5 hr and SR was only 27% of maximum at 13.5 hr) (Fig. lb). Thus, the time course following progesterone injection for the display of proceptivity differs from that of receptivity, viz., proceptivity peaks more sharply in time.
RECEPTIVITY,
PROCEPTIVITY,
45
AND PROGESTERONE
a 0
so
75
100
200
DOSE lug)
/ / I
/
/
//
\
\
. \
\
\ Sl?\‘\
I I I I I
\
\
\
\
\
\
\
TIME klrs.) The effect of progesterone on receptivity and proceptivity in ovariectomized, estrogen-primed female rats. (a) Dose-response relationship and (b) the time-response relationship between progesterone nand both lordosis quotient (LQ-a behavioral measure of receptivity) and solicitation rate (SR-a behavioral measure of proceptivity). Dose of progesterone and time from progesterone injection are shown on the abscissa. Using 100% as the maximum level of behavior observed, the percentage of this maximum level at other test doses and times from injection are shown on the ordinate. FIG.
I.
46
FADEM, BARFIELD,
AND WHALEN
TABLE 2 The Relationship between Time from Progesterone Injection and Measures of Receptive and Proceptive Behavior in the Estrogen-Primed Female Rat Time of test (hr) Behavioral measure
0
0.5
Mean intromission return latency (set)
102
70
Mean ejaculation return latency (set)
173 ‘
148
Mean solicitation rate”
2.22
1.5
4.5
73
*
2.61 L
143
13.5
P <
39
68
0.005
89
161
0.009
, 30.33
62.72
8.55
0.8001
A A Mean visit rate”
24.22
29.39
20.78.
Mean lordosis quotienP
25.56
20.12
0.06
56
71
92
100
77
0.001
Ear Wiggling (No. of animals)c
1
1
9
17
5
0.001
Acceptance (No. of animals)
8
5
10
17
9
0.05
a ANOVA. b Friedman’s two-way ANOVA. c Cochran’s Q. * Scheffe test significance.
DISCUSSION
The present study demonstrates the dose- and time-dependent relationships between exogenous progesterone and the display of receptive and proceptive behavior in the ovariectomized, estrogen-primed female rat. The main findings are as follows: (1) Progesterone affects the female’s latency to initiate contact with the male following copulatory events (IRL and ERL). This effect has been previously observed (Fadem, 1977; Gilman and Hitt, 1978). (2) The degree of receptivity and proceptivity displayed is directly related to the progesterone dose and time from progesterone injection (up to 4.5 hr). (3) Although the female may show partial receptivity, i.e., exhibit lordosis, without progesterone, proceptivity (hopping, darting and ear wiggling) rarely if ever occurs in the absence of progesterone. (4) Higher doses of progesterone are necessary to effect increases in proceptivity relative to receptivity. And (5), proceptivity occurs in a narrower time range than receptivity.
RECEFI-IVITY,
PROCEF’TIVITY,
AND
PROGESTERONE
47
In the intact female rat, progesterone secretion increases following the surge of luteinizing hormone (LH) which triggers ovulation after a latency of about 12 hr. High levels of plasma progesterone are associated with high levels of estrous behavior (Feder, Goy, and Resko, 1967), and the increasing levels of progesterone which occur following the LH surge may act to increase the degree of expression of the total estrous behavior pattern, i.e., receptivity alone followed by receptivity plus a full display of proceptivity, when progesterone levels are highest. The complete pattern would thus occur some hours before ovulation and might serve to increase the probability that mating would occur at the physiologically optimal time for impregnation. Also, because the female’s male-seeking, solicitation behavior (proceptivity) is active, it involves considerable energy expenditure, and it can be assumed that in the natural situation such behavior would occur during a short time span relative to passive receptivity, i.e., only at the optimal time for mating to lead to impregnation. Thus, the finding in the present study that proceptivity occurs in a narrower time range than receptivity following exogenous hormone treatment is not unexpected. The total pattern of female rat sexual behavior involves both receptive and proceptive components. It has been suggested that these two components may represent two separate behavioral systems which are controlled to some extent by different brain regions (Modianos, Hitt, and Popolow, 1975; Ward, Crowley, Zemlan, and Margules, 1975) and may depend on different gonadal hormones, lordosis upon estrogen alone and ear wiggling and hopping upon progesterone following estrogen priming (Hardy and DeBold, 1971; Whalen, 1974). Alternatively, proceptivity and receptivity may represent the graded expression of a single system controlling estrous behavior. Regardless of whether receptivity and proceptivity are controlled by the same or by different physiological systems, the results of this study show that receptivity and proceptivity represent varying levels in a continuum of estrous responsiveness. Lowest levels of the continuum are characterized by irregular displays of lordosis alone and highest levels are characterized by reliable displays of lordosis plus hopping, darting, and ear wiggling. The notion that, in the female rat, lordosis plus hopping and darting represents a higher level of sexual responsiveness than lordosis alone has been previously advanced and has been used in quantifying feminine sexual behavior (Ball, 1937; Gerall, Hendricks, Johnson, and Bounds, 1967). In the present study, the degree of expression of the total behavioral continuum appeared to depend on progesterone dose and latency from progesterone injection. The results of this study then, show a critical role for progesterone in proceptive behavior in the female rat and also indicate that receptive and proceptive behavior together constitute a continuum of estrous responsiveness.
48
FADEM, BARFIELD,
AND WHALEN
ACKNOWLEDGMENTS This research was supported by Grant HD-04484 to R.J.B. from the National Institutes of Health and by a Sigma Xi National Research Council of North America, Grant-in-Aid of Research to B.H.F.
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14, l-37.
Beach, F. A. (1976). Sexual attractivity, proceptivity and receptivity in female mammals. Horm. Behav. 7, 105-138. Beach, F. A., Stern, B., Carmichael, M., and Ranson, E. (1976). Comparisons of sexual receptivity and proceptivity in female hamsters. Behav. Biol. 18,473-487. Davidson, J. M., Smith, E. R., Rodgers, C. H., and Bloch, G. J. (1%8). Relative thresholds of behavioral and somatic responses to estrogen. Physiol. Behav. 3, 227-229. Fadem, B. H. (1977). Copulatory Behavior in the Female Rat: The Effect of Local Stimulation, Progesterone Level, the Stimulus Qualities of the Male and Individual Differences on Proceptivity. Unpublished Master’s thesis, Department of Zoology, Rutgers Univer-
sity, New Brunswick, N.J. Feder, H. H., Goy, R. W., and Resko, J. A. (1967). Progesterone concentrations in the peripheral plasma of cyclic rats. J. Physiol. 191, 136-137. Gerall, A. A., Hendricks, S. E., Johnson, L. L., and Bounds, T. W. (1%7). Effects of early castration in male rats on adult sexual behavior. J. Comp. Physiol. 64, 206-212. Gilman, D. P., and Hitt, J. C. (1978). Effects of gonadal hormones on pacing of sexual contacts by female rats. Behav. Biol. 24 (1) 77-87. Green, R., Luttge, W. G., and Whalen, R. E. (1970). Induction of receptivity in ovariectomized female rats by a single intravenous injection of estradiol-17b Physiol. Behav. 5, 137-141. 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. Hlinak, Z., and Madlafousek, J. (1971). The dependence of sexual behavior of inexperienced males on the precopulatory behaviour of female albino rats. Proc. Czek. Physiol. Sot. 21, 83.
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Whalen, R. E., and Hardy, D. F. (1970). Induction of receptivity in female rats and cats with estrogen and testosterone. Physiol. Behav. 5, 529-533. Whalen, R. E., and Nadler, R. D. (1965). Modification of spontaneous and hormone induced sexual behavior by estrogen administered to neonatal female rats. J. Camp. Physiol. Psychol. 60, (1) 150-152.