HORMONES
Induction
AND BEHAVIOR
6, 259-276 (1975)
of Lordosis in Female Rats: Two Modes of Estrogen Action and the Effect of Adrenalectomy LEE-MING KOW and DONALD W. PFAFF Rockefeller University, New York, New York IOO2l
In ovariectomized female rats, progesterone treatment alone does not induce lordosis, but following estrogen treatment by an appropriate interval it greatly enhances the performance of lordosis compared to that with effect of progesterone is thought to act estrogen alone. This “facilitating” effect of estrogen. In the present synergistically with the initial “priming” experiments a second estrogen treatment given to estrogen-primed ovariectomized rats in place of progesterone was found to facilitate lordosis. Latency of the facilitation of lordosis following this second estrogen treatment was similar to that of progesterone and was much shorter than that required for the usual “priming” effect, but higher doses were needed for the “facilitatory” effect. Experiments with adrenalectomized-ovariectomized female rats showed that this short latency effect of second estrogen treatment need not be mediated by the adrenals. These results raise the possibility that estrogen acts on the central nervous system in more than one way to induce lordosis.
INTRODUCTION In the induction of sexual receptivity in female rats, progesterone is not merely facilitatory (Boling and Blandau, 1939; Beach, 1942; Lisk, 1960; Edwards, Whalen, and Nadler, 1968; Whalen and Gorzalka, 1972) but is essential under physiological conditions (Powers, 1970) and for ovariectomized rats primed with a small dosage of estrogen (Powers and Valenstein, 1972). From these observations evolves the concept that in female rats, and other the neural substrate for lordosis rodents as well, estrogen serves to “prime” and progesterone subsequently facilitates appearance of full sexual receptivity. However, there are indications that the effect of estrogen may not be confined to the priming. In ovariectomized rats, receptivity can be induced with estrogen alone (Davidson, Rodgers, Smith, and Bloch, 1968; Edwards, Whalen, and Nadler, 1968; Pfaff, 1970; Hendricks, 1972). If both “priming” and “facilitating” neural effects were required for the induction of receptivity, the above observation would imply that estrogen can serve to do both. This possibility is supported by the observation that implantation of estrogen into the midbrain 259 Copyright @ 1975 by Academic Press, Inc. AU rights of reproduction in any form reserved.
260
KOW AND PFAFF
reticular formation of estrogen-primed, ovariectomized rats can facilitate lordosis as similar implantation of progesterone does (Ross, Claybaugh, . Clemens, and Gorski, 1971). Another line of evidence, from hormone uptake studies, also supports indirectly the notion that estrogen may act in at least two different ways to induce lordosis. It has been shown that in rats (Eisenfeld and Axelrod, 1965, 1966; Kato and Villee, 1967a, 1967b; Pfaff, 1968; Pfaff and Keiner, 1973) as well as in guinea pigs (Wade and Feder, 1972a), there are two types of estrogen accumulation in the brain; one, which is saturable with estrogen, located in hypothalamic and limbic structures; the other, unsaturable system is widely spread throughout the entire brain. Wade and Feder (1972a, b) further showed that in addition to estrogen, the unsaturable system also took up progesterone, 20a-hydroxy-pregn-u-en-3-one (20~OHP) and corticosterone in essentially the same pattern. These three steroids are known to have a facilitatory effect on receptivity in guinea pigs (see Discussion by Wade and Feder, 1972b). These lines of evidence suggest the possibility that estrogen has, in addition to its priming effect, a facilitatory effect as progesterone does in the induction of sexual receptivity. Evidence for this possibility was found in Experiment 1, of which the preliminary results have been reported (Kow, Malsbury, and Pfaff, 1974). Whether the presence of adrenal gland is essential for estrogen to facilitate receptivity was investigated in Experiment 2.
EXPERIMENT 1 This experiment was designed to see if estrogen can substitute for progesterone to facilitate sexual receptivity in the conventionally estrogenprimed ovariectomized rat. Methods Subjects and Be-Experimental
Treatment
Ovariectomized, adult Sprague-Dawley rats (Hormone Assay Lab. Inc.), with body weights ranging from 222 to 274 gm, were used. Upon their arrival they were allowed to adapt to the new environment and the reversed light-dark cycle for at least 1 wk. To verify the ovariectomy, every female was tested by manual stimulation for lordosis every day for the last six days of the adaptation period. Any rat showing definite lordosis in response to manual test was excluded. The subjects were then divided randomly into eight groups, each consisting of eight or nine females. In order to “sensitize” the ovariectomized females (Gerall and Dunlap, 1973) each group was given subcutaneous
261
MODES OF ESTROGEN ACTION
injections of 10 pgrn estradiol benzoate (EB)/rat/day for two consecutive days and 500 pgrn progesterone (P)/rat on the third day. Five hours after P injection the females were subjected to a behavioral test, with the procedure that will be described below, to evaluate their receptiveness. All of the females used showed a high degree of receptivity, proving that they were capable of performing lordosis if treated with appropriate hormones. The results of these sensitization tests were not included in the data analyses. Two weeks after the sensitization treatment none of the females showed lordosis in response to manual test, indicating that the effect of the treatment had disappeared. The females were then subjected to experimental treatment and testing. Experimental Treatment Each group was given two injections two days apart, as shown in Table 1. Different groups received different combinations of treatments. All the injections were given subcutaneously in sesame oil. In all except group III, in which vehicle (oil) alone was given in the first injection, the first injection was intended to “prime” the animal for lordosis. For this purpose the dosage of estrogen, 3 ~.lgm EB/rat, was chosen to be sufficient for priming but small enough to exert no (or minimal) facilitatory effect, assuming that estrogen has this effect. Except for group II, in which oil was given in the second injection, the second injection was intended for facilitation. Tests for Lordosis Behavioral tests were carried our 4, 6, 8, 10 and 24 hr after the second injection for all except group II, which was tested only three times at 4, 8 and 24 hr after the second injection. A group of more than 10 sexually active male rats was used as “stud” males. To perform the test, each female was put into an observation cage (9 X 17 X 7 in.) with one of the stud males. Using more than one stud male to test each female kept the males at their best and TABLE I Hormonal Treatments of Ovariectomized Rats Group I II Ill IV V VI VII VIII
n
Day 1 3 pgm EB 3 wgm EB Oil
3 pgm EB 3 pgm EB 3 pgm EB 3 pgm EB 3 pgm EB
Day 3 400 pgrn P Oil
400 MgrnEB 400 pgrn EB 200 ccgmEB 100 CLgmEB 50 pgrn EB 10 ggrn EB
262
KOW AND PFAFF
shortened the testing time. The behavior of the female was scored for 15 mounts by the male or for 15 min, whichever came first. The degree of receptivity was evaluated with three measures of behavior: lordosis quotient (LQ), quality of lordosis (Q), and the percentage response (PR). Lordosis quotient was defined as (number of lordoses/number of mounts by male) X 100. The quality of lordosis in response to each mount by male was scored, based on the degree of the vertebral dorsiflexion, with a zero to five scale as follows: 0, no lordosis; 1, weak but definite; 2, moderate; 3, strong; 4, moderate and outlasting the mount; 5, strong and outlasting the mount. The Q for each test .was calculated as the average of the quality, including O’s, of responses to mounts by the male. The mean value of Q excluding all zeroes can easily be obtained by the formula (Q including zeroes) X lOO/mean LQ. The percentage response is defined as the percentage of the females in each group showing at least one definite lordosis in each test. For the females that did not show any lordosis in any of the behavior tests, manual testing was employed 3 to 4 days after the second injection. Statistics For comparisons between groups t-tests for independent samples (unpaired) were used. Results and Discussion The results of the behavior tests for each group are shown in Table 2 and illustrated in Fig. 1 and 2. In group I, which was treated with EB and P, all the females have shown lordosis (PR = 100) in the first test 4 hr after P injection. At this time the levels of both LQ and Q were already higher @< .OOl) than those of group II. This high level of receptivity was maintained until 8 hr after P injection. Thereafter all measures of receptivity declined, and at 24 hr the levels of LQ and Q were no longer significantly different from those of group II. The results from group I indicate that under the present conditions 3 pgrn EB/rat is sufficient to prime the ovariectomized rats for subsequent facilitation. In group II, although there is a small, gradual increase in PR in the subsequent tests, the levels of the other two measures, LQ and Q, remain very low and constant in each test. The results from group II therefore indicate that although 3 pgrn EB/rat is sufficient to prime the rats, it alone can not induce any significant degree of receptivity. In group III, which received no priming estrogen in the Day 1 injection but 400 pgrn EB/rat in the Day 3 injection, none of the females showed any lordosis in any of the tests up through 24 hr after the Day 3 injection. (On Day 6 they were again tested, manually, and at this time responded with strong lordoses.) This latency of more than 24 hr is longer than that (16-20 hr) reported by Green, Luttge, and Whalen (1970). However, the conditions they employed (IV
0.21 +.12
0.22 +.06
0.16 2.10
0.41 +.26
0.32 *.14
9.6 k4.7
11.8* k2.7
5.9 k3.2
14.3 29.0
15.3* k5.9
IVb
Vb
VIC
VII
VW
75
38
38
88
44
0
13
1oo
PR
16.9 59.0
15.1 +10.4
15.9* t6.3
12.4 +4.9
32.1*** ?7.6
0.0
-
51.6*** f 10.5
LQ
15.1* *6.3
63
0.35 r.21
13.5 k6.4
20.0* 63 +10.5
63
0.36 k.28
0.43 k.23
29.9 *** +I.6
63
0.20 *.08
0.0 +6.9 25.2***
0
y.7,; -. 100
0.00
3.4 +2.5
-
-
LQ 60.9*** k6.4
PR 1oo
Q 1.66 k.45
6 hr
0.39 +.19
0.55 A.31
0.38 +.22
0.50 A.14
k.20 0.68
0.00
0.06 +.04
1.88 t.33
Q
8hr
15.0* +_6.4
15.8* *5 .I
+6.2 15.6*
0.0
-
;;.r**
LQ
,5
12.5 *7.3
14.1 5. k-10.3
5.
88
78
0
25
1oo
PR
n.3; -.
+“$ -.
-. f’;;
0.00
-
Q
10 hr
25
50
67
0
-
PR
0.46 +.18
Ozi7 *.19
24.4t k8.0 24.3t ~7.8
0.75 *.20
32.4tt +7.3
y.;; -. 1.09 +.25
0.00
0.0 35.6ttt *6.2 A10.2 45.9tt
f*;; -.
o-31 +.13
Q
4.5 +2.7
17.4 +7.6
LQ
24 hr
88
15
88
88
100
0
38
50
PR
PP< .OOl; *>P< .05. Comparison between the mean LQ of each behavior test and mean LQ of group II (4 and 8 hr pooled). ttt 9 < ,001; tt ?P< .Ol; t,P< .05. Comparison of the mean LQ’s at 24 hr, between group II and other groups.
10 hr p$&ed).
alp < .OOl; b,P < .O1;crP< .05. Comparison between the pooled mean LQ’s of group II (4 and 8 hr pooled) and other groups (4,6,8, and
Statistical comparisons of mean LQ’s (t-test; p, two-tailed):
0.00
0.0
0.05 2.05
1.77 -1.45
Q
IIIC
+_3.5 3.4
50.8*** t9.3
IQ
II
LQ
Group
4 hr
TABLE 2
Results of Lordosis Tests of Ovariectomized Rats (j; f SE) Hours after second injection
264
KOW AND PFAFF
E
II: llI
70=
G+
HI-
Q, ‘Z 2
-
I
= Group1
In=
= GroupJI
(n r 8)
8)
0
= GrouplE
(n z 8)
0
= Grcup~
(n= 9)
u 40.!? s P ,o 20 0.
m 2
J .-----.---9 4
6
-_---------------. 1 6 10 Hours after
1 24
nd 2
injection
Fig. 1. Mean results of lordosis tests of overiectomized rats groups I through IV. injection of free estradiol in aqueous suspetision, and the subsequent administration of 500 pgrn P) were intended to minimize the latency of estrogen priming. Thus, although one can induce receptivity in ovariectomized rats with a single injection of 4OOpgm EB/rat, under the present conditions it required a latency of more than 24 hr.
MODES OF ESTROGEN
26.5
ACTION
50 3)$ .
&
40
.-
= Group~(n=9) = Groups
0
= Group~(n=8)
0
= Groupm(n=8)
(n=8)
:
,,.’
.
3 *.s
30
4 k 4
20
10
I!ziII szu x?I m 911 1
4
6
6
10
Hours after
24 2
nd
injection
Fig. 2. Mean results of lordosis tests of ovariectomized rats groups IV through VIII. Note the ordinates of this figure are different from those of Fig. 1; those for lordosis quotient and quality have been expanded.
266
KOW AND PFAFF
While neither 3 pgm EB (“priming”) nor 400 pgrn EB (on Day 3) individually induced significant receptivity within the testing period, the combined treatment with both can do so, as shown by the results from group IV (Table 2). Lordosis quotient was significantly higher than group II as soon as 6 hr after injection of the facilitating estrogen dose. However, up through 10 hr after injection, the facilitating effectiveness of estrogen was generally less than that of progesterone at the same dosage. This facilitatory effect of estrogen can be achieved with dosages smaller than 400 pgrn EB/rat, as shown by the results from groups V through VIII. From groups V to VIII the dosages of estrogen given in the Day 3 injection are progressively smaller (see Table 1). For convenience, to plot dose against response more clearly, the highest values of each measure of receptivity from the first four behavior tests were gathered from groups II and IV through VIII and plotted in Fig. 3. As shown in this figure, the level of each measure of receptivity tends to decline as the dosage of the second estrogen treatment was reduced from 400 to 10 pgrn EB/rat. From 10 to 0 pgrn EB/rat there is a
Fig. 3. Relationships between mean lordosis measures and doses of EB in the second injection. For each dose, and each measure of lordosis, the highest mean result from among the mean results for the first four tests (4-10 hi post 2nd injection) is plotted. Pooled data, averaging across the 6 and 8 hr tests or across 4, 6, 8 and 10 hr tests, show the same patterns of results.
MODES OF ESTROGEN
ACTION
267
big drop in each measure. Similar curves are seen in plots of data pooled from all 6 and 8 hr tests. The fact that all measures of receptivity from the group (II) that received no second estrogen are lower than those from groups (IV through VIII) that received second estrogen treatment indicates that the second estrogen can facilitate receptivity even at the dosage as low as 10 pgrn EB/rat. However, because of individual variability in each group the facilitation by lower doses of EB were not statistically significant in as many tests or at as high a level of significance as the higher doses (Table 2). The temporal patterns of facilitation for groups IV through VIII are basically the same. In every one of these five groups receptivity was increased in less than 10 hr by the second estrogen (Day 3) injection. In this respect, the temporal pattern of the estrogen facilitation is similar to that of the progesterone facilitation (Fig. 1). However, at 24 hr, whereas the LQ of each of the groups receiving second estrogen treatment was higher than that of group II (see Table 2) the high LQ after 24 hr was not seen in the group (I) treated with progesterone. This experiment has demonstrated that a second injection of estrogen can facilitate receptivity in estradiol-primed, ovariectomized rats. A comparison between the facilitatory effects of estrogen and progesterone reveals both similarities and differences. They are similar in (I) having a comparable short latency (4-8 hr), and (2) requiring priming with estrogen (in regard to P, see Experiment 3). On the other hand, they are different in effectiveness. At the dosage of 400 pgm the facilitatory effect of P was about twice as great as that of EB (Table 2). Work with lower doses also provides interesting comparisons between progesterone and the “second estrogen” effect. In ovariectomized rats primed with 1Opgm EB/kgm Powers and Valenstein (1972) found that the threshold dosage of progesterone for a statistically significant facilitation of lordosis was between 100 and 250 pgm, while in the present study some instances of statistically significant EB facilitation were found at 100 pgrn EB/rat and lower. Whalen (1974) saw a large increase in progesterone effect between 50 and 1001.lgm P, whereas in the present study, the first visible effect of a second estrogen treatment appeared at 1Opgm EB/rat. Moreover, in an unpublished study by Modianos (personal communication), the relationship of progesterone dose to LQ was obtained from groups of ovariectomized rats primed with 1Opgm EB/rat. If the dose-response curve of his study and the dose-response curve for the facilitatory EB effect obtained in the present study are plotted together, the two curves, one for P and the other for EB, are not parallel, and cross at 200 pgm. Above 200 pgm, LQ’s induced by EB t P are higher than those by EB + EB, while below 200 pgrn, the opposite is the case, suggesting that at dosage smaller than 200kgm EB is more effective than P in facilitating lordosis in estrogen-primed, ovariectomized rats. The second injection of EB is also different from progesterone in that it, but not progesterone, can induce a prolonged (24 hr) increase in lordosis.
268
KOW AND PFAFF
EXPERIMENT 2 When a facilitatory effect of the second estrogen treatment had been demonstrated in Experiment 1, a second experiment was designed to investigate whether the facilitatory effect of the second estrogen treatment demonstrated in Experiment 1 was due to the release of adrenal progesterone. This possibility was suggested by the findings that: (1) adrenal cortex stores a significant quantity of progesterone (Holzbauer and Newport, 1967); (2) in estrogen-primed, ovariectomized rats and guinea pigs ACTH administration can facilitate receptivity, presumably by causing the release of adrenal progesterone (Feder and Ruf, 1969); (3) adrenal progesterone probably is involved in the timing of the heat in normal rats (Nequin and Schwartz, 1971; Barfield and Lisk, 1974); and, (4) estrogen can stimulate adrenocortical. activity in rats (Kitay, Coyne, Newsom, and Nelson, 1965). The absence of the adrenal gland does not necessarily prevent the receptivity induced in ovariectomized rats with daily estrogen administration (Davidson, Rodgers, Smith, and Bloch, 1968). But, since a new paradigm of estrogen administration was used in Experiment 1 to demonstrate a “facilitatory” estrogen effect, the possibility of adrenal involvement in this effect still remained to be investigated. Methods Subjects and Pre-Experimental Treatment Adult, ovariectomized and adrenalectomized Sprague-Dawley rats, with body weights ranging from 230 to 311 gm were used. They were handled and treated, including sensitization and pretests, in the same way as those in Experiment 1, except that in addition to the tap water they were also supplied with an aqueous drinking solution containing NaCl (2%) sucrose (2%) and Terramycin (3.3%). No mineralocorticoid or other “maintenance” hormone treatment was given. Experimental Treatment The subjects were divided randomly into four groups, IX through XII, each consisting of 7 to 11 rats. As shown in Table 3, each group received two to three subcutaneous injections of EB with the dosage of the last injection, which was intended for facilitation, being the same, 400 pgm/rat, for all groups. Group IX received exactly the same paradigm as did group IV in Experiment 1, i.e., a “priming” estrogen injection 3 /..tgmEB/rat, followed by a “facilitating” estrogen injection, 4OOpgm EB/rat, after a “priming period” of 2 days. In group X, both the priming estrogen dosage (doubled by two 3 pgrn EB/rat injections) and the period of priming (extended from 2 to 6 days) were increased. In group XI, the “priming” dosage remained the same
269
MODESOF ESTROGENACTION TABLE 3 EstrogenTreatment (pgm EB/rat/injection) of Adrenalectomized~vaiectomized Rats Day 5
Day 7
-
3
400
10
3
3
400
XI
11
-
3
-
400
XII
7
-
-
6
400
Group
n
Day 1
IX
10
X
Day 3
but the priming period extended, while in group XII the priming dosage doubled but the priming period remained the same, as compared to group IX. Tests for Lordosis Lordosis was evaluated with the same behavioral testing and scoring procedures used in Experiment 1, except that now subjects were tested only twice: first just before (“pre”), and then 6 hr after (“post”) the last estrogen injection. The “pre” tests were carried out l-2 hr after the onset of the dark period. Autopsy At the completion of the experiment, each rat was given an overdose of anesthesia and autopsied to verify adrenalectomy. Only the data of the rats with verified complete adrenalectomies are included in the Results. Statistics For comparisons of the means between “pre” and “post” tests within the same group, t-tests for matched pairs were used. Results and Discussion The results of the behavioral tests for this experiment are shown in Table 4. The mean values of LQ and Q between the “pre” and the “post” tests were compared statistically for each group. In group IX, all measures of receptivity increased from “pre” to “post” test, but the increases were not statistically significant 0, > .05). To explore the possibility that in the absence of adrenal gland the subjects may require more priming, both the priming dosage and the priming period were increased in group X. All measures of receptivity in this group were quite high in the “pre” test, and the subsequent
270
KOW AND PFAFF TABLE 4 Results of LordosisTestsof Adrenalectomized-QvaiectomizedRats Before C.&e-)and 6 hr after (Post-)Final EstrogenTreatment (j; + SE)
Lordosisquotient __ PrePostP
Pre- Post-
IX
3.8 *2.2
18.7 ~8.9
N.S.
X
25.3 54.8
68.0 k5.7
10.0 k3.9 8.1 t3.8
Group
XI XII
Quality
% Response p
Pre-
Post-
.05 .41 N.S. +.03 c.23
30
70
< .OOl
.47 2.09 +.ll +.16 < ’ool
90
100
24.9 k7.8
< .025
.16 .61 2.06 +.22 < .025
45
73
28.6 t6.6
< .OlO
.09 t.16 A’ k.04
43
86
< .025
administration of the “facilitating” final dose of estrogen greatly enhanced lordosis (Table 4). Experiments were done on group XI, with an extended period of priming, and group XII, with doubled priming dose of estrogen to see if one factor was responsible for the great facilitation observed in group X. Under either condition, injection of 400 pgrn EB/rat significantly facilitated receptivity (Table 4). Both factors, increased priming dosage of estrogen and increased “priming period”, apparently were important for estrogen to show a facilitatory effect on lordosis in the absence of the adrenal glands. Results from this experiment have shown that secretions of the adrenal gland are not essential for estrogen to facilitate sexual receptivity in the estrogen-primed, ovariectomized rat. But, in order for the estrogen to show a significant facilitatory effect in the absence of the adrenal glands either the dosage of the priming estrogen or the duration of priming have to be increased.
EXPERIMENT 3 In Experiment 1 it was found that estrogen priming is essential for the second estrogen treatment to facilitate lordosis. In the course of comparing the facilitating actions of the second estrogen treatment and progesterone we noticed that the necessity of estrogen priming for progesterone to show facilitation has not been well documented in female rats. The following experiment was performed to confirm the necessity of estrogen priming.
MODES OF ESTROGEN
ACTION
271
Methods Subjects and Pre-Experimental Treatment Eleven adult, ovariectomized Sprague-Dawley rats (Hormone Assay Lab., Inc.) with body weights from 299 to 339 gm were used. They were housed individually under a reversed light-dark cycle. As in Experiment 1, the females were subjected to two consecutive weekly “sensitization” treatments. Each treatment consisted of administration of 1Oygm EB/rat and then 500 pgrn P/rat two days later. All the hormones were administered by subcutaneous injection in 0.1 ml sesame oil in the pre-experimental as well as the experimental treatments. Each sensitization treatment was followed by the behavioral test described below. All of the subjects showed a high degree of receptivity following the second sensitization treatment. Two weeks after this treatment the subjects were no longer receptive when tested, indicating that residual depots of steroids, if any, were not sufficient to induce lordosis. Experimental Treatment Starting more than 2 wk after the last sensitization injection, all the females were subjected to seven weekly hormone treatments. On the Day 1 of each of the first 6 wk the females were injected subcutaneously with 0 (oil alone), 1, 3, 5, 10 or 20 pgrn EB/rat, in that order. Each Day 1 injection was followed by an injection of 5001.tgrn P/rat on Day 3. In the seventh week, each female was given seven daily injections of 10 pgm EB/rat (10 X 7), and on the last day an injection of 500/.4gm P was also given. Test for Lordosis Approximately 6 hr after each P injection, several hours after the beginning of dark phase of light cycle, the females were tested for lordosis. The procedure of the test is the same as that described in Experiment 1, except that the quality of lordosis in response to mounts by male was not scored. Lordosis quotient and percentage response were defined and calculated in the same ways as in Experiment 1. Results and Discussion The results of the behavioral tests are summarized in Table 5. When not preceded with estrogen priming (0 ggrn EB/rat), progesterone treatment at a dosage of 500 pgm/rat did not induce lordosis in any of the 11 females. Lordosis was observed only when the female was primed with estrogen. As the dose of the priming estrogen was increased while the dose of progesterone was kept constant, both LQ and PR increased monotonically.
272
KOW AND PFAFF TABLE 5 Lordosis Quotient (LQ) and Percentage Response (PR) of Ovariectomized Female Rats to Mounts by Male 6 hr after Progesterone, Following Various Doses of Estrogen Priming (?it SE;n = 11) Estradiol benzoate priming dose (clpm/rat)
LQ PR (%)
-__-
0
1
3
5
10
20
10x7
0 +O
10.8 25.4
36.0 +8.6
68.6 k11.4
80.6 +8.5
86.0 *8.1
95.7 t1.6
45
82
91
100
100
0
91
The results of this experiment show that estrogen priming is essential for progesterone to facilitate lordosis in ovariectomized female rats. In this respect progesterone is similar to the second estrogen treatment.
GENERAL DISCUSSION Experiment 1 demonstrated that estrogen can facilitate sexual receptivity in female rats in the type of paradigm used to show the facilitatory effect of progesterone. This facilitatory action of estrogen may be exerted directly on the central nervous system, rather than indirectly through stimulating the release of adrenal progesterone or other adrenal hormones (Experiment 2). Thus, in the induction of sexual receptivity in female rats estrogen can act in two modes: priming and facilitation. From the results of Experiment 1, one can see that there are differences between these two modes of estrogen action. Priming action requires small dosage (< 3 @gm EB/rat) and long latency (Z 24 hr), but not previous estrogen treatment; whereas facilitating action tends to require larger dosage (see Table 2 for significance levels), short latency (4 to 8 hr), and estrogen priming. Such a short latency for facilitation has never been found for the priming action of estrogen. Even under conditions intended to minimize latency of the priming action, lordosis was not observed until more than 16 hr after the injection of estradiol (Green et al., 1970). These differences suggest that the two modes of actions may be achieved by estrogen acting on neural substrates with two different mechanisms. This suggestion, together with the findings that there are two types of estrogen accumulation in the brains of rats, as well as guinea pigs (see Introduction), makes the following proposal theoretically feasible. To a female rat, given a single small dose (3 pgm EB or less) of estrogen, the hormone may be taken
MODES OF ESTROGEN
ACTION
273
up mainly by the saturable hypothalamic and limbic estrogen-concentrating neurons (see references in Introduction) and act to prime the animal. If large or repeated doses of estrogen were given, estrogen accumulated by unsaturable, extrahypothalamic sites might act to facilitate receptivity. This proposal may serve as an explanation for how estrogen alone, with large, or repeated doses, but not a single, small dose, induces sexual receptivity. Together with the prolonged (24 hr) increase in receptivity observed in Experiment 1, following the second estrogen injection, the above proposal can explain the ambiguity of progesterone’s biphasic effect observed in female rats. In this species, the biphasic effect of progesterone can be observed when the dosage of the priming estrogen is low (Nadler, 1970) but not when it is high (Zucker, 1967; Edwards, Whalen, and Nadler, 1968). It is possible that in this species progesterone does have a biphasic effect, and when a small dosage of priming estrogen was used, the facilitating effect of progesterone alone, and hence the biphasic effect, was observed. But, when large dosages of priming estrogen were used, receptivity may be facilitated by both progesterone and estrogen, and since estrogen can induce a secondary, prolonged increase of receptivity the second (inhibitory) phase of the progesterone effect might be masked. In this respect it is interesting to note that in estrogen-primed, ovariectomized guinea pigs the heat duration induced by progesterone can be prolonged if a supplemental dose of estrogen is given simultaneously with progesterone (Collins, Boling, Dempsey, and Young, 1938; Joslyn and Feder, 1971). With the demonstration that estrogen can facilitate receptivity using a paradigm similar to that for progesterone, it is important to examine whether the same mechanisms are employed by the two ovarian hormones. As shown in Experiment 1, the receptivity-facilitating effect of the second estrogen treatment is similar to that of progesterone in having a comparable short latency and in requiring estrogen priming. With intracerebral implantation technique it has been demonstrated that both hormones can achieve this facilitatory effect by acting at the same neural loci in the midbrain reticular formation (Ross, Claybaugh, Clemens, and Gorski, 1971). Moreover, the lordosis-facilitating effect is not the only action through which estrogen can mimic progesterone. In the estrogen-primed, ovariectomized rats a sharp increase of plasma luteinizing hormone (LH) not only can be induced by injection of progesterone (Caligaris, Astrada, and Taleisnik, 1968; Kalra, Krulich, Quijada, Kalra, Fawcett, and McCann, 1970; Kalra, Kalra, Krulich, Fawcett, and McCann, 1972), but can also be induced by an injection of estrogen (Cahgaris, Astrada, and Taleisnik, 1971; Kalra and McCann, 1973). The LH release effects of estrogen and progesterone seem to be similar in mechanism (Kalra and McCann, 1973) and latency. The LH release in female rats induced by cerebral cortex stimulation has been shown to be blocked not only by an injection of progesterone but by estrogen as well (Taleisnik, Caligaris, and DeOlmos, 1962).
274
KOW AND PFAFF
There are differences between the effects of repeated estrogen treatment and progesterone. As discussed in Experiment 1, the second estrogen treatment, but not progesterone, can induce a secondary increase in receptivity observable 24 hr after the treatment. In this connection, it is interesting to note that in estrogen-primed, ovariectomized rats estrogen injection induced LH release not only at 6 hr but also at around 29 hr (Caligaris, Astrada, and Taleisnik, 1971; Kalra and McCann, 1973). Another difference also discussed in Experiment 1 is the dose-response relationship. Whereas at high dosages the second estrogen treatment is less effective than progesterone in facilitating receptivity, at low dosages the situation seems to be reversed. Finally, there are indirect pieces of evidence suggesting that the second estrogen treatment and progesterone employ different mechanisms in facilitating sexual receptivity. In male rats castrated as adults, feminine mating behavior can be induced with high dose of estrogen alone in repeated treatments, but can not be facilitated with progesterone, not even with a very high dosage (see review by Kow, Malsbury, and Pfaff, 1974). Female hamsters and guinea pigs are behaviorally more sensitive to progesterone than are rats (see Introduction in Wade, Harding, and Feder, 1973) but it is very difficult to induce receptivity with estrogen alone (hamsters: Ciaccio and Lisk, 1967; guinea pigs: Boling, Young, and Dempsey, 1938). Thus, while some evidence suggests that progesterone and repeated estrogen treatments employ the same mechanisms in facilitating receptivity, other evidence suggests otherwise. The best explanation at present seems to be that estrogen employs more than one mechanism in facilitating sexual receptivity, and one of these mechanisms is also employed by progesterone. In summary, it was found that, in the induction of lordosis, estrogen can have two kinds of action: priming and facilitatory. For estrogen to show a facilitatory action the presence of the adrenal gland is not essential. To the extent that this facilitatory action of estrogen requires large dosages, its physiological significance has not been evaluated. However, in the study of estrogen action on the central nervous system one must be aware of the fact that estrogen could act in more than one way. ACKNOWLEDGMENTS The authors express their appreciation to Mr. M. Montgomery for his technical assistance and preparation of illustrations. This study was supported by NIH Fellowship No. NS.53743 (L.-M. Kow). REFERENCES Barfield, M. A., and Lisk, R D. (1974). Relative contributions of ovarian and adrenal progesterone to the timing of heat in the 4day cyclic rat. Endocrinology 94, 571-575.
MODES OF ESTROGEN ACTION
275
Beach, F. A. (1942). Importance of progesterone to induction of sexual receptivity in spayed female rats. Proc. Sot. Expt. Biol. Med. 51, 361-371. Boling, J. L., and Blandau, R. J. (1939). The estrogen-progesterone induction of mating responses in the spayed female rat. Endocrinology 25, 359-364. Boling, J. L., Young, W. C., and Dempsey, E. W. (1938). Miscellaneous experiments on the estrogen-progesterone induction of heat in the spayed guinea pig. Endocrinology 23, 182-187. Caligaris, L., Astrada, J. J., and Taleisnik, S. (1968). Stimulating and inhibiting effects of progesterone on the release of luteinizing hormone. Acta Endocrinol. 59, 177-185. Caligaris, L., Astrada, J. J., and Taleisnik, S. (1971). Release of luteinizing hormone induced by estrogen injection into ovariectomized rats. Endocrinology 88, 810-815. Ciaccio, L, A., and Lisk, R. D. (1967). Facilitation and inhibition of estrous behavior in the spayed female golden hamster (Mesocricetus auratus). Amer. Zool. 7, 712 (abstr). Collins, V. J., Baling, J. L., Dempsey, E. W., and Young, W. C. (1938). Some quantitative studies of experimentally induced sexual receptivity in spayed guinea pigs. Endocrinology 23, 188-196. Davidson, J. M., Rodgers, C. R, Smith, E. R., and Bloch, G. J. (1968). Stimulation of female sex behavior in adrenalectomized rats with estrogen alone. Endocrinology 82, 193-195. Edwards, D. A., Whalen, R. E., and Nadler, R. D. (1968). induction of estrus: estrogen-progesterone interactions. Physiol. Behav. 3, 29-33. Eisenfeld, A. J., and Axelrod, J. (1965). Selectivity of estrogen distribution in tissues. J. Pharm Exp. Ther. 150, 469-475. Eisenfeld, A. J., and Axehod, J. (1966). Effect of steroid hormones, ovariectomy, estrogen pretreatment, sex, and immaturity on the distribution of 3H-estradiol. Endocrinology
79, 38-42.
Feder, H. H., and Ruf, K. B. (1969). Stimulation of progesterone release and estrous behavior by ACTH in ovariectomized rodents. Endocrinology 84, 171-174. Gerall, A. A., and Dunlap, J. L (1973). The effects of experience and hormones on the initial receptivity in female and male rats. Physiol. Behav. 10, 851-854. Green, R, Luttge, W. G., and Whalen, R. E. (1970). Induction of receptivity in ovariectomized female rats by a single intravenous injection of estradiol-176 Physiol. Behav. 5, 137-141. Hendricks, S. E. (1972). Androgen modification of behavioral responsiveness to estrogen in the male rat. Horm. Behav. 3, 47-54. Holzbauer, M., and Newport, M. (1967). The effect of stress on the concentration of 3@-hydroxypregn-5en-20-one (pregnenolone) and pregn4ene-3, 20dione (progesterone) in the adrenal gland of the rat. J. Physiol. 193, 131-140. Joslyn, W. D., and Feder, H. H. (1971). Facihtatory and inhibitory effects of supplementary estradiol benzoate given to ovariectomized, estrogen-primed guinea pigs. Horm
Behav. 2, 307-314.
Kaba, P. S., and McCann, S. M. (1973). Involvement of catecholamines in feedback mechanisms. Prog. Brain Res. 39, 185-197. Kalra, P. S., Kaba, S. P., Krulich, L., Fawcett, C. P., and McCann, S. M. (1972). Involvement of norepinephrine in transmission of the stimulatory influence of progesterone on gonadotropin release. Endocrinology 90, 1168-1176. Kalra, P. S., Krulich, L., Quijada, M., Kalra, S. P., Fawcett, C. P., and McCann, S. M. (1970). Feedback of gonadal steroids on gonadotropins and prolactin in the rat, Excerpta.
Med. Int. Congr. Ser. 219, 708-715.
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Kato, J., and Villee, C. A. (1967a). Preferential uptake of estradiol by the anterior hypothalamus of the rat. Endocrinology 80, 567-575. Kato, J., and Villee, C. A. (1967b). Factors affecting uptake of estradiol-6, 7-3H by the hypophysis and hypothalamus. Endocrinology 80, 1133-1138. Kitay, J. I., Coyne, M. D., Newsom, W,, and Nelson, R. (1965). Relation of the ovary to adrenal corticosterone production and adrenal enzyme activity in the rat. Endocrinology
77, 902-908.
Kow, L.-M., Malsbury, C. W., and Pfaff, D. W. (1974). Effects of progesterone on female reproductive behavior in rats: possible modes of action and role in behavioral sex differences. In W. Montagna and W. A. Sadler (Eds.), Reproductive Behavior. Adv. Behav. Biol. 11, 179-210. Plenum Press, New York. 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. Can. J. Biochem. 38, 1381-1383. Nadler, R. D. (1970). A biphasic influence of progesterone on sexual receptivity of spayed female rats. Physiol. Behav. 5, 95-97. Nequin, L. Cl., and Schwartz, N. B. (1971). Adrenal participation in the timing of mating and LH release in the cyclic rat. Endocrinology 88, 325-331. Pfaff, D. W. (1968). Autoradiographic localization of radioactivity in rat brain after injection of tritiated sex hormones. Science 161, 1355-1356. Pfaff, D. W. (1970). Nature of sex hormone effects on rat sex behavior: specificity of effects and individual patterns of response. J. Comp. Phvsiol. Psychol. 73, 349-358.
Pfaff, D. W., and Keiner, M. (1973). Atlas of estradiol-concentrating cells in the central nervous system of the female rat. J. Camp. Ncurol. 151, 121-158. Powers, J. B. (1970). Hormonal control of sexual receptivity during the estrous cycle of the rat. Physiol. Behav. 5, 831-835. Powers, J. B., and Valenstein, E. S. (1972). Individual differences in sexual responsiveness to estrogen and progesterone in ovariectomized rats. Physiol. Behav. 8, 673-676. Ross, J., Claybaugh, C., Clemens, L G., and Gorski, R. A. (1971). Short latency induction of estrous behavior with intracerebral gonadal hormones in ovariectomized rats. Endocrinology 89, 32-38. Taleisnik, S., Caligaris, L., and De Olmos, J. (1962). Luteinizing hormone release by cerebral cortex stimulation. Amer. J. Physiol. 203, 1109-l 112. Wade, G. N., and Feder, H. H. (1972a). [ 1,2-3H] Progesterone uptake by guinea pig brain and uterus: differential localization, time-course of uptake and metabolism, and effects of age, sex, estrogen-priming and competing steroids. Brain Res. 45, 525-543. Wade, G. N., and Feder, H. H. (1972b). Uptake of (1,2-‘H) 20a-hydroxypregn4cn-3one, (1,2 -3H) corticosterone, and (6,7-3H) estradiol-17p by guinea pig brain and uterus: comparison of uptake of (1,2-3H) progesterone. Brain Res. 45, 545-554. Wade, G. N., Harding C. I;., and Fcder, H. H. (1973). Neural uptake of (1,2-“H) progesterone in ovariectomized rats, guinea pigs, and hamsters: correlation with species differences in behavioral responsiveness. Brain Res. 61, 357-367. Whalen, R. E. and Gorzalka, B. B. (1972). The effects of progesterone and its metabolites on the induction of sexual receptivity in rats. Horm. Behav. 3, 221-226. Whalen, R. E. (1974). Estrogen-progesterone induction of mating in female rats. Harm. Behav. 5, 157-162. Zucker, I. (1967). Actions of progesterone in the control of sexual receptivity of the spayed female rat. J. Comp. Physiol. Psychol. 63, 313-316.
Induction
AND BEHAVIOR
6, 259-276 (1975)
of Lordosis in Female Rats: Two Modes of Estrogen Action and the Effect of Adrenalectomy LEE-MING KOW and DONALD W. PFAFF Rockefeller University, New York, New York IOO2l
In ovariectomized female rats, progesterone treatment alone does not induce lordosis, but following estrogen treatment by an appropriate interval it greatly enhances the performance of lordosis compared to that with effect of progesterone is thought to act estrogen alone. This “facilitating” effect of estrogen. In the present synergistically with the initial “priming” experiments a second estrogen treatment given to estrogen-primed ovariectomized rats in place of progesterone was found to facilitate lordosis. Latency of the facilitation of lordosis following this second estrogen treatment was similar to that of progesterone and was much shorter than that required for the usual “priming” effect, but higher doses were needed for the “facilitatory” effect. Experiments with adrenalectomized-ovariectomized female rats showed that this short latency effect of second estrogen treatment need not be mediated by the adrenals. These results raise the possibility that estrogen acts on the central nervous system in more than one way to induce lordosis.
INTRODUCTION In the induction of sexual receptivity in female rats, progesterone is not merely facilitatory (Boling and Blandau, 1939; Beach, 1942; Lisk, 1960; Edwards, Whalen, and Nadler, 1968; Whalen and Gorzalka, 1972) but is essential under physiological conditions (Powers, 1970) and for ovariectomized rats primed with a small dosage of estrogen (Powers and Valenstein, 1972). From these observations evolves the concept that in female rats, and other the neural substrate for lordosis rodents as well, estrogen serves to “prime” and progesterone subsequently facilitates appearance of full sexual receptivity. However, there are indications that the effect of estrogen may not be confined to the priming. In ovariectomized rats, receptivity can be induced with estrogen alone (Davidson, Rodgers, Smith, and Bloch, 1968; Edwards, Whalen, and Nadler, 1968; Pfaff, 1970; Hendricks, 1972). If both “priming” and “facilitating” neural effects were required for the induction of receptivity, the above observation would imply that estrogen can serve to do both. This possibility is supported by the observation that implantation of estrogen into the midbrain 259 Copyright @ 1975 by Academic Press, Inc. AU rights of reproduction in any form reserved.
260
KOW AND PFAFF
reticular formation of estrogen-primed, ovariectomized rats can facilitate lordosis as similar implantation of progesterone does (Ross, Claybaugh, . Clemens, and Gorski, 1971). Another line of evidence, from hormone uptake studies, also supports indirectly the notion that estrogen may act in at least two different ways to induce lordosis. It has been shown that in rats (Eisenfeld and Axelrod, 1965, 1966; Kato and Villee, 1967a, 1967b; Pfaff, 1968; Pfaff and Keiner, 1973) as well as in guinea pigs (Wade and Feder, 1972a), there are two types of estrogen accumulation in the brain; one, which is saturable with estrogen, located in hypothalamic and limbic structures; the other, unsaturable system is widely spread throughout the entire brain. Wade and Feder (1972a, b) further showed that in addition to estrogen, the unsaturable system also took up progesterone, 20a-hydroxy-pregn-u-en-3-one (20~OHP) and corticosterone in essentially the same pattern. These three steroids are known to have a facilitatory effect on receptivity in guinea pigs (see Discussion by Wade and Feder, 1972b). These lines of evidence suggest the possibility that estrogen has, in addition to its priming effect, a facilitatory effect as progesterone does in the induction of sexual receptivity. Evidence for this possibility was found in Experiment 1, of which the preliminary results have been reported (Kow, Malsbury, and Pfaff, 1974). Whether the presence of adrenal gland is essential for estrogen to facilitate receptivity was investigated in Experiment 2.
EXPERIMENT 1 This experiment was designed to see if estrogen can substitute for progesterone to facilitate sexual receptivity in the conventionally estrogenprimed ovariectomized rat. Methods Subjects and Be-Experimental
Treatment
Ovariectomized, adult Sprague-Dawley rats (Hormone Assay Lab. Inc.), with body weights ranging from 222 to 274 gm, were used. Upon their arrival they were allowed to adapt to the new environment and the reversed light-dark cycle for at least 1 wk. To verify the ovariectomy, every female was tested by manual stimulation for lordosis every day for the last six days of the adaptation period. Any rat showing definite lordosis in response to manual test was excluded. The subjects were then divided randomly into eight groups, each consisting of eight or nine females. In order to “sensitize” the ovariectomized females (Gerall and Dunlap, 1973) each group was given subcutaneous
261
MODES OF ESTROGEN ACTION
injections of 10 pgrn estradiol benzoate (EB)/rat/day for two consecutive days and 500 pgrn progesterone (P)/rat on the third day. Five hours after P injection the females were subjected to a behavioral test, with the procedure that will be described below, to evaluate their receptiveness. All of the females used showed a high degree of receptivity, proving that they were capable of performing lordosis if treated with appropriate hormones. The results of these sensitization tests were not included in the data analyses. Two weeks after the sensitization treatment none of the females showed lordosis in response to manual test, indicating that the effect of the treatment had disappeared. The females were then subjected to experimental treatment and testing. Experimental Treatment Each group was given two injections two days apart, as shown in Table 1. Different groups received different combinations of treatments. All the injections were given subcutaneously in sesame oil. In all except group III, in which vehicle (oil) alone was given in the first injection, the first injection was intended to “prime” the animal for lordosis. For this purpose the dosage of estrogen, 3 ~.lgm EB/rat, was chosen to be sufficient for priming but small enough to exert no (or minimal) facilitatory effect, assuming that estrogen has this effect. Except for group II, in which oil was given in the second injection, the second injection was intended for facilitation. Tests for Lordosis Behavioral tests were carried our 4, 6, 8, 10 and 24 hr after the second injection for all except group II, which was tested only three times at 4, 8 and 24 hr after the second injection. A group of more than 10 sexually active male rats was used as “stud” males. To perform the test, each female was put into an observation cage (9 X 17 X 7 in.) with one of the stud males. Using more than one stud male to test each female kept the males at their best and TABLE I Hormonal Treatments of Ovariectomized Rats Group I II Ill IV V VI VII VIII
n
Day 1 3 pgm EB 3 wgm EB Oil
3 pgm EB 3 pgm EB 3 pgm EB 3 pgm EB 3 pgm EB
Day 3 400 pgrn P Oil
400 MgrnEB 400 pgrn EB 200 ccgmEB 100 CLgmEB 50 pgrn EB 10 ggrn EB
262
KOW AND PFAFF
shortened the testing time. The behavior of the female was scored for 15 mounts by the male or for 15 min, whichever came first. The degree of receptivity was evaluated with three measures of behavior: lordosis quotient (LQ), quality of lordosis (Q), and the percentage response (PR). Lordosis quotient was defined as (number of lordoses/number of mounts by male) X 100. The quality of lordosis in response to each mount by male was scored, based on the degree of the vertebral dorsiflexion, with a zero to five scale as follows: 0, no lordosis; 1, weak but definite; 2, moderate; 3, strong; 4, moderate and outlasting the mount; 5, strong and outlasting the mount. The Q for each test .was calculated as the average of the quality, including O’s, of responses to mounts by the male. The mean value of Q excluding all zeroes can easily be obtained by the formula (Q including zeroes) X lOO/mean LQ. The percentage response is defined as the percentage of the females in each group showing at least one definite lordosis in each test. For the females that did not show any lordosis in any of the behavior tests, manual testing was employed 3 to 4 days after the second injection. Statistics For comparisons between groups t-tests for independent samples (unpaired) were used. Results and Discussion The results of the behavior tests for each group are shown in Table 2 and illustrated in Fig. 1 and 2. In group I, which was treated with EB and P, all the females have shown lordosis (PR = 100) in the first test 4 hr after P injection. At this time the levels of both LQ and Q were already higher @< .OOl) than those of group II. This high level of receptivity was maintained until 8 hr after P injection. Thereafter all measures of receptivity declined, and at 24 hr the levels of LQ and Q were no longer significantly different from those of group II. The results from group I indicate that under the present conditions 3 pgrn EB/rat is sufficient to prime the ovariectomized rats for subsequent facilitation. In group II, although there is a small, gradual increase in PR in the subsequent tests, the levels of the other two measures, LQ and Q, remain very low and constant in each test. The results from group II therefore indicate that although 3 pgrn EB/rat is sufficient to prime the rats, it alone can not induce any significant degree of receptivity. In group III, which received no priming estrogen in the Day 1 injection but 400 pgrn EB/rat in the Day 3 injection, none of the females showed any lordosis in any of the tests up through 24 hr after the Day 3 injection. (On Day 6 they were again tested, manually, and at this time responded with strong lordoses.) This latency of more than 24 hr is longer than that (16-20 hr) reported by Green, Luttge, and Whalen (1970). However, the conditions they employed (IV
0.21 +.12
0.22 +.06
0.16 2.10
0.41 +.26
0.32 *.14
9.6 k4.7
11.8* k2.7
5.9 k3.2
14.3 29.0
15.3* k5.9
IVb
Vb
VIC
VII
VW
75
38
38
88
44
0
13
1oo
PR
16.9 59.0
15.1 +10.4
15.9* t6.3
12.4 +4.9
32.1*** ?7.6
0.0
-
51.6*** f 10.5
LQ
15.1* *6.3
63
0.35 r.21
13.5 k6.4
20.0* 63 +10.5
63
0.36 k.28
0.43 k.23
29.9 *** +I.6
63
0.20 *.08
0.0 +6.9 25.2***
0
y.7,; -. 100
0.00
3.4 +2.5
-
-
LQ 60.9*** k6.4
PR 1oo
Q 1.66 k.45
6 hr
0.39 +.19
0.55 A.31
0.38 +.22
0.50 A.14
k.20 0.68
0.00
0.06 +.04
1.88 t.33
Q
8hr
15.0* +_6.4
15.8* *5 .I
+6.2 15.6*
0.0
-
;;.r**
LQ
,5
12.5 *7.3
14.1 5. k-10.3
5.
88
78
0
25
1oo
PR
n.3; -.
+“$ -.
-. f’;;
0.00
-
Q
10 hr
25
50
67
0
-
PR
0.46 +.18
Ozi7 *.19
24.4t k8.0 24.3t ~7.8
0.75 *.20
32.4tt +7.3
y.;; -. 1.09 +.25
0.00
0.0 35.6ttt *6.2 A10.2 45.9tt
f*;; -.
o-31 +.13
Q
4.5 +2.7
17.4 +7.6
LQ
24 hr
88
15
88
88
100
0
38
50
PR
PP< .OOl; *>P< .05. Comparison between the mean LQ of each behavior test and mean LQ of group II (4 and 8 hr pooled). ttt 9 < ,001; tt ?P< .Ol; t,P< .05. Comparison of the mean LQ’s at 24 hr, between group II and other groups.
10 hr p$&ed).
alp < .OOl; b,P < .O1;crP< .05. Comparison between the pooled mean LQ’s of group II (4 and 8 hr pooled) and other groups (4,6,8, and
Statistical comparisons of mean LQ’s (t-test; p, two-tailed):
0.00
0.0
0.05 2.05
1.77 -1.45
Q
IIIC
+_3.5 3.4
50.8*** t9.3
IQ
II
LQ
Group
4 hr
TABLE 2
Results of Lordosis Tests of Ovariectomized Rats (j; f SE) Hours after second injection
264
KOW AND PFAFF
E
II: llI
70=
G+
HI-
Q, ‘Z 2
-
I
= Group1
In=
= GroupJI
(n r 8)
8)
0
= GrouplE
(n z 8)
0
= Grcup~
(n= 9)
u 40.!? s P ,o 20 0.
m 2
J .-----.---9 4
6
-_---------------. 1 6 10 Hours after
1 24
nd 2
injection
Fig. 1. Mean results of lordosis tests of overiectomized rats groups I through IV. injection of free estradiol in aqueous suspetision, and the subsequent administration of 500 pgrn P) were intended to minimize the latency of estrogen priming. Thus, although one can induce receptivity in ovariectomized rats with a single injection of 4OOpgm EB/rat, under the present conditions it required a latency of more than 24 hr.
MODES OF ESTROGEN
26.5
ACTION
50 3)$ .
&
40
.-
= Group~(n=9) = Groups
0
= Group~(n=8)
0
= Groupm(n=8)
(n=8)
:
,,.’
.
3 *.s
30
4 k 4
20
10
I!ziII szu x?I m 911 1
4
6
6
10
Hours after
24 2
nd
injection
Fig. 2. Mean results of lordosis tests of ovariectomized rats groups IV through VIII. Note the ordinates of this figure are different from those of Fig. 1; those for lordosis quotient and quality have been expanded.
266
KOW AND PFAFF
While neither 3 pgm EB (“priming”) nor 400 pgrn EB (on Day 3) individually induced significant receptivity within the testing period, the combined treatment with both can do so, as shown by the results from group IV (Table 2). Lordosis quotient was significantly higher than group II as soon as 6 hr after injection of the facilitating estrogen dose. However, up through 10 hr after injection, the facilitating effectiveness of estrogen was generally less than that of progesterone at the same dosage. This facilitatory effect of estrogen can be achieved with dosages smaller than 400 pgrn EB/rat, as shown by the results from groups V through VIII. From groups V to VIII the dosages of estrogen given in the Day 3 injection are progressively smaller (see Table 1). For convenience, to plot dose against response more clearly, the highest values of each measure of receptivity from the first four behavior tests were gathered from groups II and IV through VIII and plotted in Fig. 3. As shown in this figure, the level of each measure of receptivity tends to decline as the dosage of the second estrogen treatment was reduced from 400 to 10 pgrn EB/rat. From 10 to 0 pgrn EB/rat there is a
Fig. 3. Relationships between mean lordosis measures and doses of EB in the second injection. For each dose, and each measure of lordosis, the highest mean result from among the mean results for the first four tests (4-10 hi post 2nd injection) is plotted. Pooled data, averaging across the 6 and 8 hr tests or across 4, 6, 8 and 10 hr tests, show the same patterns of results.
MODES OF ESTROGEN
ACTION
267
big drop in each measure. Similar curves are seen in plots of data pooled from all 6 and 8 hr tests. The fact that all measures of receptivity from the group (II) that received no second estrogen are lower than those from groups (IV through VIII) that received second estrogen treatment indicates that the second estrogen can facilitate receptivity even at the dosage as low as 10 pgrn EB/rat. However, because of individual variability in each group the facilitation by lower doses of EB were not statistically significant in as many tests or at as high a level of significance as the higher doses (Table 2). The temporal patterns of facilitation for groups IV through VIII are basically the same. In every one of these five groups receptivity was increased in less than 10 hr by the second estrogen (Day 3) injection. In this respect, the temporal pattern of the estrogen facilitation is similar to that of the progesterone facilitation (Fig. 1). However, at 24 hr, whereas the LQ of each of the groups receiving second estrogen treatment was higher than that of group II (see Table 2) the high LQ after 24 hr was not seen in the group (I) treated with progesterone. This experiment has demonstrated that a second injection of estrogen can facilitate receptivity in estradiol-primed, ovariectomized rats. A comparison between the facilitatory effects of estrogen and progesterone reveals both similarities and differences. They are similar in (I) having a comparable short latency (4-8 hr), and (2) requiring priming with estrogen (in regard to P, see Experiment 3). On the other hand, they are different in effectiveness. At the dosage of 400 pgm the facilitatory effect of P was about twice as great as that of EB (Table 2). Work with lower doses also provides interesting comparisons between progesterone and the “second estrogen” effect. In ovariectomized rats primed with 1Opgm EB/kgm Powers and Valenstein (1972) found that the threshold dosage of progesterone for a statistically significant facilitation of lordosis was between 100 and 250 pgm, while in the present study some instances of statistically significant EB facilitation were found at 100 pgrn EB/rat and lower. Whalen (1974) saw a large increase in progesterone effect between 50 and 1001.lgm P, whereas in the present study, the first visible effect of a second estrogen treatment appeared at 1Opgm EB/rat. Moreover, in an unpublished study by Modianos (personal communication), the relationship of progesterone dose to LQ was obtained from groups of ovariectomized rats primed with 1Opgm EB/rat. If the dose-response curve of his study and the dose-response curve for the facilitatory EB effect obtained in the present study are plotted together, the two curves, one for P and the other for EB, are not parallel, and cross at 200 pgm. Above 200 pgm, LQ’s induced by EB t P are higher than those by EB + EB, while below 200 pgrn, the opposite is the case, suggesting that at dosage smaller than 200kgm EB is more effective than P in facilitating lordosis in estrogen-primed, ovariectomized rats. The second injection of EB is also different from progesterone in that it, but not progesterone, can induce a prolonged (24 hr) increase in lordosis.
268
KOW AND PFAFF
EXPERIMENT 2 When a facilitatory effect of the second estrogen treatment had been demonstrated in Experiment 1, a second experiment was designed to investigate whether the facilitatory effect of the second estrogen treatment demonstrated in Experiment 1 was due to the release of adrenal progesterone. This possibility was suggested by the findings that: (1) adrenal cortex stores a significant quantity of progesterone (Holzbauer and Newport, 1967); (2) in estrogen-primed, ovariectomized rats and guinea pigs ACTH administration can facilitate receptivity, presumably by causing the release of adrenal progesterone (Feder and Ruf, 1969); (3) adrenal progesterone probably is involved in the timing of the heat in normal rats (Nequin and Schwartz, 1971; Barfield and Lisk, 1974); and, (4) estrogen can stimulate adrenocortical. activity in rats (Kitay, Coyne, Newsom, and Nelson, 1965). The absence of the adrenal gland does not necessarily prevent the receptivity induced in ovariectomized rats with daily estrogen administration (Davidson, Rodgers, Smith, and Bloch, 1968). But, since a new paradigm of estrogen administration was used in Experiment 1 to demonstrate a “facilitatory” estrogen effect, the possibility of adrenal involvement in this effect still remained to be investigated. Methods Subjects and Pre-Experimental Treatment Adult, ovariectomized and adrenalectomized Sprague-Dawley rats, with body weights ranging from 230 to 311 gm were used. They were handled and treated, including sensitization and pretests, in the same way as those in Experiment 1, except that in addition to the tap water they were also supplied with an aqueous drinking solution containing NaCl (2%) sucrose (2%) and Terramycin (3.3%). No mineralocorticoid or other “maintenance” hormone treatment was given. Experimental Treatment The subjects were divided randomly into four groups, IX through XII, each consisting of 7 to 11 rats. As shown in Table 3, each group received two to three subcutaneous injections of EB with the dosage of the last injection, which was intended for facilitation, being the same, 400 pgm/rat, for all groups. Group IX received exactly the same paradigm as did group IV in Experiment 1, i.e., a “priming” estrogen injection 3 /..tgmEB/rat, followed by a “facilitating” estrogen injection, 4OOpgm EB/rat, after a “priming period” of 2 days. In group X, both the priming estrogen dosage (doubled by two 3 pgrn EB/rat injections) and the period of priming (extended from 2 to 6 days) were increased. In group XI, the “priming” dosage remained the same
269
MODESOF ESTROGENACTION TABLE 3 EstrogenTreatment (pgm EB/rat/injection) of Adrenalectomized~vaiectomized Rats Day 5
Day 7
-
3
400
10
3
3
400
XI
11
-
3
-
400
XII
7
-
-
6
400
Group
n
Day 1
IX
10
X
Day 3
but the priming period extended, while in group XII the priming dosage doubled but the priming period remained the same, as compared to group IX. Tests for Lordosis Lordosis was evaluated with the same behavioral testing and scoring procedures used in Experiment 1, except that now subjects were tested only twice: first just before (“pre”), and then 6 hr after (“post”) the last estrogen injection. The “pre” tests were carried out l-2 hr after the onset of the dark period. Autopsy At the completion of the experiment, each rat was given an overdose of anesthesia and autopsied to verify adrenalectomy. Only the data of the rats with verified complete adrenalectomies are included in the Results. Statistics For comparisons of the means between “pre” and “post” tests within the same group, t-tests for matched pairs were used. Results and Discussion The results of the behavioral tests for this experiment are shown in Table 4. The mean values of LQ and Q between the “pre” and the “post” tests were compared statistically for each group. In group IX, all measures of receptivity increased from “pre” to “post” test, but the increases were not statistically significant 0, > .05). To explore the possibility that in the absence of adrenal gland the subjects may require more priming, both the priming dosage and the priming period were increased in group X. All measures of receptivity in this group were quite high in the “pre” test, and the subsequent
270
KOW AND PFAFF TABLE 4 Results of LordosisTestsof Adrenalectomized-QvaiectomizedRats Before C.&e-)and 6 hr after (Post-)Final EstrogenTreatment (j; + SE)
Lordosisquotient __ PrePostP
Pre- Post-
IX
3.8 *2.2
18.7 ~8.9
N.S.
X
25.3 54.8
68.0 k5.7
10.0 k3.9 8.1 t3.8
Group
XI XII
Quality
% Response p
Pre-
Post-
.05 .41 N.S. +.03 c.23
30
70
< .OOl
.47 2.09 +.ll +.16 < ’ool
90
100
24.9 k7.8
< .025
.16 .61 2.06 +.22 < .025
45
73
28.6 t6.6
< .OlO
.09 t.16 A’ k.04
43
86
< .025
administration of the “facilitating” final dose of estrogen greatly enhanced lordosis (Table 4). Experiments were done on group XI, with an extended period of priming, and group XII, with doubled priming dose of estrogen to see if one factor was responsible for the great facilitation observed in group X. Under either condition, injection of 400 pgrn EB/rat significantly facilitated receptivity (Table 4). Both factors, increased priming dosage of estrogen and increased “priming period”, apparently were important for estrogen to show a facilitatory effect on lordosis in the absence of the adrenal glands. Results from this experiment have shown that secretions of the adrenal gland are not essential for estrogen to facilitate sexual receptivity in the estrogen-primed, ovariectomized rat. But, in order for the estrogen to show a significant facilitatory effect in the absence of the adrenal glands either the dosage of the priming estrogen or the duration of priming have to be increased.
EXPERIMENT 3 In Experiment 1 it was found that estrogen priming is essential for the second estrogen treatment to facilitate lordosis. In the course of comparing the facilitating actions of the second estrogen treatment and progesterone we noticed that the necessity of estrogen priming for progesterone to show facilitation has not been well documented in female rats. The following experiment was performed to confirm the necessity of estrogen priming.
MODES OF ESTROGEN
ACTION
271
Methods Subjects and Pre-Experimental Treatment Eleven adult, ovariectomized Sprague-Dawley rats (Hormone Assay Lab., Inc.) with body weights from 299 to 339 gm were used. They were housed individually under a reversed light-dark cycle. As in Experiment 1, the females were subjected to two consecutive weekly “sensitization” treatments. Each treatment consisted of administration of 1Oygm EB/rat and then 500 pgrn P/rat two days later. All the hormones were administered by subcutaneous injection in 0.1 ml sesame oil in the pre-experimental as well as the experimental treatments. Each sensitization treatment was followed by the behavioral test described below. All of the subjects showed a high degree of receptivity following the second sensitization treatment. Two weeks after this treatment the subjects were no longer receptive when tested, indicating that residual depots of steroids, if any, were not sufficient to induce lordosis. Experimental Treatment Starting more than 2 wk after the last sensitization injection, all the females were subjected to seven weekly hormone treatments. On the Day 1 of each of the first 6 wk the females were injected subcutaneously with 0 (oil alone), 1, 3, 5, 10 or 20 pgrn EB/rat, in that order. Each Day 1 injection was followed by an injection of 5001.tgrn P/rat on Day 3. In the seventh week, each female was given seven daily injections of 10 pgm EB/rat (10 X 7), and on the last day an injection of 500/.4gm P was also given. Test for Lordosis Approximately 6 hr after each P injection, several hours after the beginning of dark phase of light cycle, the females were tested for lordosis. The procedure of the test is the same as that described in Experiment 1, except that the quality of lordosis in response to mounts by male was not scored. Lordosis quotient and percentage response were defined and calculated in the same ways as in Experiment 1. Results and Discussion The results of the behavioral tests are summarized in Table 5. When not preceded with estrogen priming (0 ggrn EB/rat), progesterone treatment at a dosage of 500 pgm/rat did not induce lordosis in any of the 11 females. Lordosis was observed only when the female was primed with estrogen. As the dose of the priming estrogen was increased while the dose of progesterone was kept constant, both LQ and PR increased monotonically.
272
KOW AND PFAFF TABLE 5 Lordosis Quotient (LQ) and Percentage Response (PR) of Ovariectomized Female Rats to Mounts by Male 6 hr after Progesterone, Following Various Doses of Estrogen Priming (?it SE;n = 11) Estradiol benzoate priming dose (clpm/rat)
LQ PR (%)
-__-
0
1
3
5
10
20
10x7
0 +O
10.8 25.4
36.0 +8.6
68.6 k11.4
80.6 +8.5
86.0 *8.1
95.7 t1.6
45
82
91
100
100
0
91
The results of this experiment show that estrogen priming is essential for progesterone to facilitate lordosis in ovariectomized female rats. In this respect progesterone is similar to the second estrogen treatment.
GENERAL DISCUSSION Experiment 1 demonstrated that estrogen can facilitate sexual receptivity in female rats in the type of paradigm used to show the facilitatory effect of progesterone. This facilitatory action of estrogen may be exerted directly on the central nervous system, rather than indirectly through stimulating the release of adrenal progesterone or other adrenal hormones (Experiment 2). Thus, in the induction of sexual receptivity in female rats estrogen can act in two modes: priming and facilitation. From the results of Experiment 1, one can see that there are differences between these two modes of estrogen action. Priming action requires small dosage (< 3 @gm EB/rat) and long latency (Z 24 hr), but not previous estrogen treatment; whereas facilitating action tends to require larger dosage (see Table 2 for significance levels), short latency (4 to 8 hr), and estrogen priming. Such a short latency for facilitation has never been found for the priming action of estrogen. Even under conditions intended to minimize latency of the priming action, lordosis was not observed until more than 16 hr after the injection of estradiol (Green et al., 1970). These differences suggest that the two modes of actions may be achieved by estrogen acting on neural substrates with two different mechanisms. This suggestion, together with the findings that there are two types of estrogen accumulation in the brains of rats, as well as guinea pigs (see Introduction), makes the following proposal theoretically feasible. To a female rat, given a single small dose (3 pgm EB or less) of estrogen, the hormone may be taken
MODES OF ESTROGEN
ACTION
273
up mainly by the saturable hypothalamic and limbic estrogen-concentrating neurons (see references in Introduction) and act to prime the animal. If large or repeated doses of estrogen were given, estrogen accumulated by unsaturable, extrahypothalamic sites might act to facilitate receptivity. This proposal may serve as an explanation for how estrogen alone, with large, or repeated doses, but not a single, small dose, induces sexual receptivity. Together with the prolonged (24 hr) increase in receptivity observed in Experiment 1, following the second estrogen injection, the above proposal can explain the ambiguity of progesterone’s biphasic effect observed in female rats. In this species, the biphasic effect of progesterone can be observed when the dosage of the priming estrogen is low (Nadler, 1970) but not when it is high (Zucker, 1967; Edwards, Whalen, and Nadler, 1968). It is possible that in this species progesterone does have a biphasic effect, and when a small dosage of priming estrogen was used, the facilitating effect of progesterone alone, and hence the biphasic effect, was observed. But, when large dosages of priming estrogen were used, receptivity may be facilitated by both progesterone and estrogen, and since estrogen can induce a secondary, prolonged increase of receptivity the second (inhibitory) phase of the progesterone effect might be masked. In this respect it is interesting to note that in estrogen-primed, ovariectomized guinea pigs the heat duration induced by progesterone can be prolonged if a supplemental dose of estrogen is given simultaneously with progesterone (Collins, Boling, Dempsey, and Young, 1938; Joslyn and Feder, 1971). With the demonstration that estrogen can facilitate receptivity using a paradigm similar to that for progesterone, it is important to examine whether the same mechanisms are employed by the two ovarian hormones. As shown in Experiment 1, the receptivity-facilitating effect of the second estrogen treatment is similar to that of progesterone in having a comparable short latency and in requiring estrogen priming. With intracerebral implantation technique it has been demonstrated that both hormones can achieve this facilitatory effect by acting at the same neural loci in the midbrain reticular formation (Ross, Claybaugh, Clemens, and Gorski, 1971). Moreover, the lordosis-facilitating effect is not the only action through which estrogen can mimic progesterone. In the estrogen-primed, ovariectomized rats a sharp increase of plasma luteinizing hormone (LH) not only can be induced by injection of progesterone (Caligaris, Astrada, and Taleisnik, 1968; Kalra, Krulich, Quijada, Kalra, Fawcett, and McCann, 1970; Kalra, Kalra, Krulich, Fawcett, and McCann, 1972), but can also be induced by an injection of estrogen (Cahgaris, Astrada, and Taleisnik, 1971; Kalra and McCann, 1973). The LH release effects of estrogen and progesterone seem to be similar in mechanism (Kalra and McCann, 1973) and latency. The LH release in female rats induced by cerebral cortex stimulation has been shown to be blocked not only by an injection of progesterone but by estrogen as well (Taleisnik, Caligaris, and DeOlmos, 1962).
274
KOW AND PFAFF
There are differences between the effects of repeated estrogen treatment and progesterone. As discussed in Experiment 1, the second estrogen treatment, but not progesterone, can induce a secondary increase in receptivity observable 24 hr after the treatment. In this connection, it is interesting to note that in estrogen-primed, ovariectomized rats estrogen injection induced LH release not only at 6 hr but also at around 29 hr (Caligaris, Astrada, and Taleisnik, 1971; Kalra and McCann, 1973). Another difference also discussed in Experiment 1 is the dose-response relationship. Whereas at high dosages the second estrogen treatment is less effective than progesterone in facilitating receptivity, at low dosages the situation seems to be reversed. Finally, there are indirect pieces of evidence suggesting that the second estrogen treatment and progesterone employ different mechanisms in facilitating sexual receptivity. In male rats castrated as adults, feminine mating behavior can be induced with high dose of estrogen alone in repeated treatments, but can not be facilitated with progesterone, not even with a very high dosage (see review by Kow, Malsbury, and Pfaff, 1974). Female hamsters and guinea pigs are behaviorally more sensitive to progesterone than are rats (see Introduction in Wade, Harding, and Feder, 1973) but it is very difficult to induce receptivity with estrogen alone (hamsters: Ciaccio and Lisk, 1967; guinea pigs: Boling, Young, and Dempsey, 1938). Thus, while some evidence suggests that progesterone and repeated estrogen treatments employ the same mechanisms in facilitating receptivity, other evidence suggests otherwise. The best explanation at present seems to be that estrogen employs more than one mechanism in facilitating sexual receptivity, and one of these mechanisms is also employed by progesterone. In summary, it was found that, in the induction of lordosis, estrogen can have two kinds of action: priming and facilitatory. For estrogen to show a facilitatory action the presence of the adrenal gland is not essential. To the extent that this facilitatory action of estrogen requires large dosages, its physiological significance has not been evaluated. However, in the study of estrogen action on the central nervous system one must be aware of the fact that estrogen could act in more than one way. ACKNOWLEDGMENTS The authors express their appreciation to Mr. M. Montgomery for his technical assistance and preparation of illustrations. This study was supported by NIH Fellowship No. NS.53743 (L.-M. Kow). REFERENCES Barfield, M. A., and Lisk, R D. (1974). Relative contributions of ovarian and adrenal progesterone to the timing of heat in the 4day cyclic rat. Endocrinology 94, 571-575.
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Beach, F. A. (1942). Importance of progesterone to induction of sexual receptivity in spayed female rats. Proc. Sot. Expt. Biol. Med. 51, 361-371. Boling, J. L., and Blandau, R. J. (1939). The estrogen-progesterone induction of mating responses in the spayed female rat. Endocrinology 25, 359-364. Boling, J. L., Young, W. C., and Dempsey, E. W. (1938). Miscellaneous experiments on the estrogen-progesterone induction of heat in the spayed guinea pig. Endocrinology 23, 182-187. Caligaris, L., Astrada, J. J., and Taleisnik, S. (1968). Stimulating and inhibiting effects of progesterone on the release of luteinizing hormone. Acta Endocrinol. 59, 177-185. Caligaris, L., Astrada, J. J., and Taleisnik, S. (1971). Release of luteinizing hormone induced by estrogen injection into ovariectomized rats. Endocrinology 88, 810-815. Ciaccio, L, A., and Lisk, R. D. (1967). Facilitation and inhibition of estrous behavior in the spayed female golden hamster (Mesocricetus auratus). Amer. Zool. 7, 712 (abstr). Collins, V. J., Baling, J. L., Dempsey, E. W., and Young, W. C. (1938). Some quantitative studies of experimentally induced sexual receptivity in spayed guinea pigs. Endocrinology 23, 188-196. Davidson, J. M., Rodgers, C. R, Smith, E. R., and Bloch, G. J. (1968). Stimulation of female sex behavior in adrenalectomized rats with estrogen alone. Endocrinology 82, 193-195. Edwards, D. A., Whalen, R. E., and Nadler, R. D. (1968). induction of estrus: estrogen-progesterone interactions. Physiol. Behav. 3, 29-33. Eisenfeld, A. J., and Axelrod, J. (1965). Selectivity of estrogen distribution in tissues. J. Pharm Exp. Ther. 150, 469-475. Eisenfeld, A. J., and Axehod, J. (1966). Effect of steroid hormones, ovariectomy, estrogen pretreatment, sex, and immaturity on the distribution of 3H-estradiol. Endocrinology
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Pfaff, D. W., and Keiner, M. (1973). Atlas of estradiol-concentrating cells in the central nervous system of the female rat. J. Camp. Ncurol. 151, 121-158. Powers, J. B. (1970). Hormonal control of sexual receptivity during the estrous cycle of the rat. Physiol. Behav. 5, 831-835. Powers, J. B., and Valenstein, E. S. (1972). Individual differences in sexual responsiveness to estrogen and progesterone in ovariectomized rats. Physiol. Behav. 8, 673-676. Ross, J., Claybaugh, C., Clemens, L G., and Gorski, R. A. (1971). Short latency induction of estrous behavior with intracerebral gonadal hormones in ovariectomized rats. Endocrinology 89, 32-38. Taleisnik, S., Caligaris, L., and De Olmos, J. (1962). Luteinizing hormone release by cerebral cortex stimulation. Amer. J. Physiol. 203, 1109-l 112. Wade, G. N., and Feder, H. H. (1972a). [ 1,2-3H] Progesterone uptake by guinea pig brain and uterus: differential localization, time-course of uptake and metabolism, and effects of age, sex, estrogen-priming and competing steroids. Brain Res. 45, 525-543. Wade, G. N., and Feder, H. H. (1972b). Uptake of (1,2-‘H) 20a-hydroxypregn4cn-3one, (1,2 -3H) corticosterone, and (6,7-3H) estradiol-17p by guinea pig brain and uterus: comparison of uptake of (1,2-3H) progesterone. Brain Res. 45, 545-554. Wade, G. N., Harding C. I;., and Fcder, H. H. (1973). Neural uptake of (1,2-“H) progesterone in ovariectomized rats, guinea pigs, and hamsters: correlation with species differences in behavioral responsiveness. Brain Res. 61, 357-367. Whalen, R. E. and Gorzalka, B. B. (1972). The effects of progesterone and its metabolites on the induction of sexual receptivity in rats. Horm. Behav. 3, 221-226. Whalen, R. E. (1974). Estrogen-progesterone induction of mating in female rats. Harm. Behav. 5, 157-162. Zucker, I. (1967). Actions of progesterone in the control of sexual receptivity of the spayed female rat. J. Comp. Physiol. Psychol. 63, 313-316.
