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The comparative potency of various steroids to complete the priming process for lordosis in guinea pigs
HORMONES
AND BEHAWOR
12, 299-308 (1979)
The Comparative Potency of Various Steroids Complete the Priming Process for Lordosis in Guinea Pigs WILLIAM Rutgers-The
to
A. WALKER' AND H. H. FEDER~
State University, Institute of Animal Behavior, Newark, New Jersey 07102
IO1 Warren Street,
Ovariectomized adult guinea pigs were treated with a regimen of estradiol benzoate (0.2 pg/animal estradiol benzoate at hr 0 and 19) that was shown to be minimally effective for the induction of lordosis. They were then treated with 10, 20, or 80 mg of enclomiphene, 5, 20, 40, or 100 pg of estradiol, or testosterone, cortisol, estrone, estriol, diethylstilbestrol, catechol estradiol, or catechol estrone (all at a dose equivalent to 5 pg of estradiol) at hr 28. At hr 39 all females were given 0.5 mg progesterone, and subsequently tested for Iordosis behavior. Of the various agents injected at hr 28 only estradiol (at all doses given), estrone, estriol, and diethylstilbestrol were effective in supporting display of lordosis behavior. The results indicate that the antiestrogen enclomiphene, the catechol estrogens, and at least some C,, and C,, steroids are weaker than E2 or ineffective in facilitating lordosis behavior when given late in the priming period. Because previous work had shown that enclomiphene has partial estrogenic effects on lordosis behavior when administered early in the priming period (i.e., at hr 0, 19), it is suggested the early and late phases of the priming process induced by EL entail qualitatively different neural processes.
In guinea pigs, the display of lordosis depends upon the sequential action of estrogen and progesterone. It is important that estrogen be allowed to act for a period of about 36-60 hr (period of estrogen priming) if the highest levels of lordosis are to be displayed after an injection of progesterone to ovariectomized subjects (Collins, Boling, Dempsey, and Young, 1938; Joslyn, Feder, and Goy, 1971; Eaton, Goy, and Resko, 1975; Walker and Feder, 1977a). Shorter intervals of estrogen priming prior to progesterone administration result in lower lordosis scores. In a previous study (Walker and Feder, 1977a), it was reported that the synthetic compound enclomiphene (ENC) could act as a partial estrogen mimic in the CNS by facilitating the induction of lordosis in guinea pigs. Normally, an 1I-hr interval between estradiol benzoate (E,B) and progesterone (P) is insufficient for the display of lordosis. However, when this II-hr EBI Present address: Roche Institute of Molecular Biology, Nutley, N. J. 2 To whom reprint requests should be sent. 299 0018-506X/79/03029910$01.00/0 Copyright @ 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.
300
WALKER
AND
FEDER
priming period was preceded by 28 hr of exposure to ENC, lordosis was induced in 62.5% of the females tested. On the other hand, if females were primed for the total interval of 39 hr (28 hr + 11 hr) with just ENC and then treated with P, no females displayed lordosis. The fact that E,B was necessary in the last 11 hr to complete the priming process initiated by ENC was considered as direct evidence for the importance of long-term maintenance of estrogen action in the central nervous system. Further, these behavioral results suggested that the neurochemical events that occur during the late portion of the priming period are stimulated in a relatively specific manner. That is, the late phase of the priming period is stimulated by E,B but not by ENC, while the early phase of the priming period is stimulated by E,B or ENC. The purposes of the present series of experiments were to: (1) determine whether the differences between the estrogenic actions of ENC and E,B (or the unesterified form, E2) are due to quantitative or qualitative factors; and (2) provide further support for the hypothesis that the neuronal events that occur late in the priming process are activated only in a specific manner by estrogen (Walker and Feder, 1977a,b). GENERAL
METHODS
Animals Adult Hartley strain, albino female guinea pigs (Camm Research Laboratories, Wayne, N. J.), that were ovariectomized between 53 and 70 days of age, were used throughout these studies. Ovariectomies were performed under a combination of Innovar-vet (Pitman-Moore, Washington Crossing, N.J., 0.05 ml per animal) and Equithesin (Jensen-Salsbery, St. Louis, MO., 0.6 ml per animal). After surgery, animals were group housed with no more than 10 members per group in 90 x 60 x 23-cm cages. Purina Guinea Pig Chow and water were available ad libitum, and cabbage was supplied once a week. The lights were on from 0500 to 1900 hr and the temperature was maintained at approximately 23°C. Behavioral Testing After surgery, the animals were maintained for a period of not less than 26 or more than 50 days. At the end of this period all subjects were screened for their ability to respond to E,B and P treatment. This screening test consisted of injecting all subjects with 1.6 pg E,B followed 39 hr later by 0.5 mg P (all injections were administered subcutaneously in sesame oil). Starting at the time of P administration, each animal was tested for lordosis hourly in the home cage using the manual manipulation technique described by Goy and Young (1957). Only the animals that displayed lordosis during the screening test were used for further behavioral testing. In some cases the screening test was not administered.
301
STEROIDS AND LORDOSIS BEHAVIOR
Rather, the animals were first used in a study designed to determine an E,B injection schedule that would be just subthreshold for induction of lordosis (see Table 2). Subsequently, these animals were randomly assigned to various experiments (Experiments 1 and 2) and treated the same as animals that had received the screening test. Fourteen days after screening (or threshold testing), the subjects were divided into experimental groups. Presumably, all E2B from the screening test is cleared from neural tissues and plasma by 120hr after injection (Eaton et al., 1975). No subject was used in more than one experimental treatment. In all experimental groups, each animal was tested for lordosis just prior to P administration and then hourly for a period of 15 hr. During this time the quantity and quality of lordosis responses were determined. Subjects displaying a 2-set or longer lordosis followed by other lordosis responses (not necessarily consecutive) were considered “in heat.” The quality factors of latency, duration, and maximum are defined as: Latency. The time in hr after P administration to the first display of a lordosis response that is 2 set or longer. Duration. The total number of hr that an animal displays 2-set or longer responses. Maximum. The number of seconds that the longest lordosis position is held. EXPERIMENT l-ENCLOMIPHENE: A “WEAK” “INCOMPLETE” ESTROGEN?
OR
The estrogenic properties of ENC were demonstrated by the data presented in Table 1 (a replication of earlier work by Walker and Feder, 1977a). It was clear that an ll-hr interval between E,B and P was inadequate for the induction of lordosis, whereas treatment with ENC prior to an 1I-hr EB-priming period facilitated the display of lordosis in 60% of females. A control group demonstrated that if E,B was allowed to act for the full 39-hr period, the induction of lordosis was maximal (last group, TABLE 1 Enclomiphene, a Partial Estrogen Mimic, Facilitates the Induction of Lordosis by Estmdiol Benzoate” Time of injection 0 hr
19 hr
ENC E,B
ENC
28 hr
39 hr
N
Percentage in heat
J&B
P P P
10 10 10
10.0 60.0 100.0
W
0 E,B = Estradiol benzoate (1.6 pg). ENC = Enclomiphene (5.0 mg). P = Progesterone (0.5 mg).
302
WALKER AND FEDER
Table 1). Based on these data and the fact that ENC priming without any supplemental estrogen did not stimulate lordosis, it was concluded that ENC mimicked some of the early actions of estrogen but that the action of estrogen was necessary to complete the activation of later processes not stimulated by ENC (Walker and Feder, 1977a). This conclusion implied that ENC acted as an “incomplete” estrogen that mimicked only some of the events induced by estrogen. However, the results in Table 1 can also be accounted for by the hypothesis that ENC was simply a “weak” estrogen that mimicked E, completely in a qualitative sense but was far less potent in a quantitative sense than E,. The present study was designed to test whether ENC action was quantitatively or qualitatively different from Ez action. If ENC is merely a “weak” estrogen, then it should be possible to mimic ENC action by using low dosages of estrogen, and conversely, it should be possible to completely mimic estrogen action by using high dosages of ENC. Therefore, in this experiment, ovariectomized guinea pigs were treated with subthreshold doses of E2B (to mimic weak estrogenic stimulation) for the first 28 hr of a 39-hr priming period. Then, high dosages of ENC (to mimic full E, action) were administered for the last 11 hr of the priming process. (Note that this treatment is exactly the reverse of the original design used in Table 1, middle group.) If ENC is a weak rather than an incomplete estrogen, then this injection paradigm should induce lordosis. Results In order to mimic the proposed “weak” estrogenic effects of ENC during the first 28 hr of a 39-hr priming period, various doses of E,B were injected at 0 and 19 hr. At 39 hr, P (0.5 mg) was injected and the animals were tested for lordosis. Table 2 shows the results of this dose-threshold determination study. From these data, an injection schedule of 0.2 pg E2B at 0 and 19 hr was selected as being just subthreshold for the induction of TABLE 2 Determination of an l!$B Injection Schedule That Is Subthreshold for the Induction of Lordosis in Guinea Pigs” Time of injection 0 hr E,B E,B E,B E,B
(0.1) (0.2) (0.3) (0.5)
19 hr E,B E,B E,B E,B
(0.1) (0.2) (0.3) (0.5)
28 hr
39 hr
N
Percentage in heat
Oil Oil Oil Oil
P P P P
17 22 18 16
0.0 27.3 50.0 81.3
(2The dosage of estradiol benzoate (E,B) injected is indicated in parentheses (pg). P = Progesterone (0.5 mg). Oil = Sesame oil (0.1 ml).
303
STEROIDS AND LORDOSIS BEHAVIOR
lordosis (27.3% in heat). Therefore, this treatment should be equal to or better than the estrogenic stimulation provided by the 5-mg injections of ENC given at 0 and 19 hr as shown in Table 1. The results in Table 3 indicate that weak estrogenic stimulation provided by 0.2 pg E,B at 0 and 19 hr followed by large dosages of ENC (10, 20, or 80 mg) at 28 hr did not induce lordosis. However, the same subthreshold E,B stimulation followed by 1.6 pugE,B at 28 hr did induce lordosis in 77% of the females tested. EXPERIMENT
2-VARIOUS STEROIDS AND COMPLETION OF THE PRIMING PERIOD
If the differences between the estrogenic properties of ENC and E, are qualitative, then ENC facilitation of the priming action of an 1I-hr E,B-P interval must be due to the fact that ENC mimics some of the early actions of estrogen but that the action of estrogen is necessary to complete the activation of later processes not stimulated by ENC (Walker and Feder, 1977a). This idea is in agreement with other studies that imply that action of estrogen in the nervous system late in the priming period is necessary for the completion of the priming process and the subsequent display of lordosis (Joslyn et al., 1971; Feder and Morin, 1974; Walker and Feder, 1977a,b, 1979; Blaustein and Wade, 1977; Moreines and Powers, 1977). The purpose of Experiment 2 was to examine the hormonal specificity of the late portion of the priming process by injection of different classes of steroids during this period. The experimental strategy used in this study was to inject subthreshold doses of E,B at 0 and 19 hr. Then at 28 hr, various steroids were administered and assessed for their ability to mainTABLE 3 Failure of Enclomiphene (ENC) to Maintain the E,B-Priming Process When Injected into Guinea Pigs during the Late Portion of the Estrogen-Priming Period” Time of injection 0 hr
19 hr
J&B EzB &B
E.3 J%B J%B 63
E8
28 hr Oil ENC (10) ENC (20) ENC (80) E,B(1.6)
39 hr
N
Percentage in heat
P P P P P
9 9 8 8 9
0.0 0.0 25.0 0.0 77.8
a The dosages of enclomiphene (ENC) injected are indicated within the parentheses (mg). E,B = Estradiol benzoate (0.2 pg) unless otherwise indicated by parentheses (i.e., in the last group &B cl.6 pg] was injected at 28 hr). P = Progesterone (0.5 mg). Oil = Sesame oil (0.1 ml).
WALKER AND FEDER
304
TABLE 4 Effect of Various Dosages of Free Estradiol (E2) on Maintenance of the %B-Priming Process When Injected Late in the Priming Period” Time of injection 19 hr
0 hr
28 hr
EzB
E,B
EZ(5)
E,B E,B W
E,B E,B E,B
Ez (20) E, (40) Es (lW
39 hr
N
Percentage in heat
P P P P
8 8 8 6
87.5 75.0 50.0 66.7
a The dosages of free estradiol (E,) injected are indicated within the parentheses (pg). E,B = Estradiol benzoate (0.2 pg). P = Progesterone (0.5 mg).
tain and complete the priming process. Lordosis behavior was tested after injecting P at 39 hr. Results In order to assess the behavioral potency of different classes of steroids it was first necessary to find a dosage of free E, that would be able to maintain the priming process when injected at 28 hr. Table 4 presents the results of this study. In all groups E,B (0.2 pg) was injected at 0 and 19 hr (this is the subthreshold injection schedule developed in Experiment 1, Table 2). At 28 hr, various dosages of unesterified E, (5,20,40, or 100 pg) were injected. P (0.5 mg) was administered at 39 hr. The lowest dosage of E, tested, 5 (ug, appeared to be adequate to maintain and complete the priming process. Therefore, in Experiment 3, the maintenance of the priming process by cortisol and testosterone was assessedby injection, at 28 hr, of dosages of these steroids that were equimolar to 5 kg E, (1.825 x lo-’ M). The data in Table 5 indicate that neither cortisol nor testosterone stimulated the priming process. Five micrograms of E, significantly increased the percentage of females displaying lordosis. EXPERIMENT
3-VARIOUS ESTROGENS AND COMPLETION OF THE PRIMING PROCESS
The purpose of this experiment was to extend our understanding of hormonal specificity during the late phase of the estrogen priming process. In this regard, various estrogens given late in the priming process were assessed in relation to the induction of lordosis. Results
In all groups, 0.2 /*g E,B was injected at 0 and 19 hr. At 28 hr, various estrogens were injected at a dose equimolar to 5 pg E, (1.825 x lop2 M).
305
STEROIDS AND LORDOSIS BEHAVIOR TABLE 5 Effect of Different Classes of Steroids on Maintenance of the E,B-Priming Process When Injected in the Late Portion of the Priming Period” Time of injection 0 hr
19 hr
28 hr
E,B EtB E,B W
W J&B E,B EzB
Oil tort Test E, (5)
39 hr
N
Percentage in heat
P P P P
10 8 8 14
20.0 12.5 25.0 78.5*
n The amounts of cortisol (Cort) and testosterone (Test) injected were equimolar to the 5 pg of E, (1.825 x lo-’ M) injected at 28 hr in the last group. E,B = Estradiol benzoate (0.2 pg). P = Progesterone (0.5 mg). Oil = Sesame oil (0.1 ml). * P < 0.01 versus first group (Fisher exact probability test).
At 39 hr, P (0.5 mg) was injected and the animals were tested for lordosis. The different estrogens administered were: estrone (E,), EP, estriol (E3), and the synthetic estrogen, diethylstilbestrol (DES). In addition, the estrogenic potencies of catechol estrone (20H-E,) and catechol estradiol (20H-E,) were assessed. Samples of both catechol estrogens were obtained from Steraloids, Inc. In order to check the purity of the commercially synthesized catechol estrogens, additional samples (kindly supplied by Dr. T. Gentry at the Institute for Steroid Research, Montefiore Hospital) of catechol estrogens were used. From Table 6 it is clear that El, EP, E,, and DES all have equal potency for completing the priming initiated by the subthreshold E,B injections. The catechol estrogens did not maintain the priming process which indicates that they possess little, if any, estrogenic potency in this situation. The facilitatory effect of the 20H-E, supplied by Steraloids, Inc., is most likely referable to its contamination with free E, because the 20H-E, synthesized and supplied by Dr. Gentry had no estrogenic activity. In subsequent studies (J. Wilcox and H. H. Feder, unpublished data) it has been shown that 2.5 ,ug E, given at 28 hr resulted in lordosis in 67% of the females and 1.Opg E, given at 28 hr resulted in lordosis in 33% of the females primed with 0.2 pg EB at 0 and 19 hr (N = 9/group). Because 20H-E,, 20H-EB, cortisol, and testosterone (at a dose equimolar to 5 pg E,) induced lordosis in fewer than 33% of the females, it appears that these substances are far less potent (if they are active at all) than E, in completing the priming process. This study does not establish that these substances are absolutely devoid of activity, but only that they are significantly less potent than E,.
W
W EzB
&B
P P
P P
20H-E,b 20H-Ezb
20H-E,c 20H-EzC
P P P P P
39 hr
Oil E, JS Es DES
28 hr
9 18
N
11.1 0.0
10.0 -c -
6.6 rt 0.3
0.4 0.2 0.4 0.5
0.0 77.8
!I c k e
5.4 5.9 6.3 5.7
Latency (z -c SE, hr)
0.0 80.0 80.0 88.9 77.8
Percentage in heat rt r 2 -t
0.5 0.6 0.4 0.9
4.0 2 -
6.0 r 0.7
5.9 6.1 4.4 5.4
Duration (2 2 SE, hr)
4.0 2 -
8.3 5 1.1
_f 0.6 t 0.5 t 0.5 k 2.1
6.3 6.1 5.3 8.9
Maximum (2 -t SE, set)
a Each of the various estrogens injected at hr 28 was administered in a dose equivalent to 5 pg Ez (1.835 x IO-’ M). E, (estrone), E, (estriol), DES (diethylstilbestrol), 20H-E, (catechol estrone), and 20H-E2 (catechol estradiol). E,B = Estradiol benzoate (0.2 pg). P = progesterone (0.5 mg). b Compounds were supplied by Steraloids, Inc. c Compounds were supplied by Dr. T. Gentry, Montefiore Hospital, New York.
W
E,B W
JLB &B EzB E,B
W
E,B
W W W EzB
%B
19 hr
0 hr
Time of injection
TABLE 6 Effect of Various Estrogens on Maintenance of the E,B-Priming Process”
s
STEROIDS
AND
LORDOSIS
BEHAVIOR
DISCUSSION
The present series of experiments was based on the findings of a previous study that indicated that the action of estrogen in the nervous system was necessary in the late portion of the estrogen-priming process for the completion of this process. This was demonstrated by the fact that the synthetic compound, enclomiphene (ENC), initiated the estrogenpriming process but that the action of E, was required during the last 11 hr of the priming period for the successful induction of lordosis (Walker and Feder, 1977a,b). It was concluded that “ENC can mimic some of the early actions of estrogen but that ultimately the presence of estrogen is necessary to complete the activation of later processes not stimulated by ENC” (Walker and Feder, 1977a). This conclusion implied a qualitative difference between the estrogenic properties of ENC and E, (i.e., ENC is an “incomplete” estrogen). The alternative interpretation that ENC was different from estrogen in a merely quantitative sense (i.e., ENC is a “weak” estrogen) was also discussed but data were not available to eliminate one or the other interpretation. The present study was designed, in part, to clarify whether ENC is an “incomplete” or a “weak” estrogen. If ENC is a “weak” estrogen, then a large enough dose of ENC should effectively induce lordosis. To test this, female guinea pigs were primed with subthreshold dosages of E,B for the first 28 hr of a 39-hr period. Then, either E,B (1.6 pg) or a high dosage of ENC (IO, 20, or 80 mg) was injected at 28 hr and P (0.5 mg) at 39 hr. The results clearly demonstrated that ENC did not maintain or complete the priming events at this stage. Injection of E,B at this time induced the display of lordosis, as expected. These results in combination with the previous report (Walker and Feder, 1977a)demonstrate that ENC mimics the action of estrogen during the early part of the priming process but does not replace the action of estrogen during the late phase of the priming process. Our tentative interpretation of these results is that estrogen action during the priming process could be characterized as activating qualitatively different processes during various portions of the priming period. The data also indicate that, in terms of a behavioral endpoint, ENC acts in a manner qualitatively different from E, (i.e., ENC is an “incomplete” rather than a “weak” estrogen). However, at this time we cannot say whether the biochemical events that underlie sexual behavior are also differentially affected by ENC and E, in a qualitative or in a purely quantitative fashion. Experiments 2 and 3 investigated the specificity requirements of the late phase of the priming process by testing either different classes of steroids (corticoid or androgen) or various types of estrogens. These studies revealed that cortisol and testosterone (at the dosage used) did not activate the processes associated with the late portion of the priming period.
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WALKER AND FEDER
However, E,, E2, E3, and DES had equal potency in completing the priming process at the dosage used. Catechol estrone and catechol estradiol did not complete the priming process and it appears that these compounds are without estrogenic activity as measured in this situation. In summary, our basic finding is that the synthetic antiestrogen ENC enhances early, but not late events involved in the priming of neural tissues that mediate guinea pig lordosis. E2, on the other hand, enhances early and late phases of the priming process. This distinction between lordosis-promoting properties of ENC and E, is potentially an important tool. By comparing the effects of ENC and E, on various biochemical markers of estrogen action (e.g., RNA polymerase II activity, synthesis of estrogen-induced protein, and induction of progestin receptor synthesis), one may reveal a difference in the biochemical action of ENC that is responsible for its characterization as an “incomplete” estrogen. ACKNOWLEDGMENTS This research was supported by Research Grant NIH-HD-04467 and NIMH Career Development Award NIMH-29006 (both to H.H.F.). Contribution 321 of the Institute of Animal Behavior.
REFERENCES
Blaustein,J. D., and Wade, G. N. (1977). Sequential inhibition of sexual behavior by progesterone in female rats: comparison with a synthetic anti-estrogen. .I. Comp. Physiol.
Psychol.
91, 752-760.
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-l%. Eaton, G. G., Goy, R. W., and Resko, J. A. (1975). Brain uptake and metabolism of estradiol benzoate and estrus behavior in ovariectomized guinea pigs. Horm. Behav. 6, 81-97. Feder, H. H., and Morin, L. P. (1974). Suppression of lordosis in guinea pigs by ethamoxytriphetol (MER-25) given at long intervals (34-35 hr) after estradiol benzoate treatment. Horm. Behav.
5, 63-72.
Goy, R. W., and Young, W. C. (1957). Strain differences in the behavioral responses of female guinea pigs to alpha-estradiol benzoate and progesterone. Behaviour 10, 340353.
Joslyn, W. D., Feder, H. H., and Goy, R. W. (1971). Estrogen conditioning and progesterone facilitation of lordosis in guinea pigs. Physiol. Behav. 7, 477-482. Moreines, J., and Powers, J. B. (1977). Effects of acute ovariectomy on lordosis response of female rats. Physiol. Behav. 19, 277-283. Walker, W. A., and Feder, H. H. (1977a). Inhibitory and facilitatory effects of various anti-estrogens on the induction of female sexual behavior by estradiol benzoate in guinea pigs. Brain Res. 134, 455-465. Walker, W. A., and Feder, H. H. (1977b). Anti-estrogen effects on estrogen accumulation in brain cell nuclei: Neurochemical correlates of estrogen action on female sexual behavior in guinea pigs. Brain Res. 134, 467-478. Walker, W. A., and Feder, H. H. (1979). Long-term effects of estrogen action are crucial for the display of lordosis in female guinea pigs: Antagonism by anti-estrogens and correlations with in vitro cytoplasmic binding affinity. Endocrinology 104, 89-96.
AND BEHAWOR
12, 299-308 (1979)
The Comparative Potency of Various Steroids Complete the Priming Process for Lordosis in Guinea Pigs WILLIAM Rutgers-The
to
A. WALKER' AND H. H. FEDER~
State University, Institute of Animal Behavior, Newark, New Jersey 07102
IO1 Warren Street,
Ovariectomized adult guinea pigs were treated with a regimen of estradiol benzoate (0.2 pg/animal estradiol benzoate at hr 0 and 19) that was shown to be minimally effective for the induction of lordosis. They were then treated with 10, 20, or 80 mg of enclomiphene, 5, 20, 40, or 100 pg of estradiol, or testosterone, cortisol, estrone, estriol, diethylstilbestrol, catechol estradiol, or catechol estrone (all at a dose equivalent to 5 pg of estradiol) at hr 28. At hr 39 all females were given 0.5 mg progesterone, and subsequently tested for Iordosis behavior. Of the various agents injected at hr 28 only estradiol (at all doses given), estrone, estriol, and diethylstilbestrol were effective in supporting display of lordosis behavior. The results indicate that the antiestrogen enclomiphene, the catechol estrogens, and at least some C,, and C,, steroids are weaker than E2 or ineffective in facilitating lordosis behavior when given late in the priming period. Because previous work had shown that enclomiphene has partial estrogenic effects on lordosis behavior when administered early in the priming period (i.e., at hr 0, 19), it is suggested the early and late phases of the priming process induced by EL entail qualitatively different neural processes.
In guinea pigs, the display of lordosis depends upon the sequential action of estrogen and progesterone. It is important that estrogen be allowed to act for a period of about 36-60 hr (period of estrogen priming) if the highest levels of lordosis are to be displayed after an injection of progesterone to ovariectomized subjects (Collins, Boling, Dempsey, and Young, 1938; Joslyn, Feder, and Goy, 1971; Eaton, Goy, and Resko, 1975; Walker and Feder, 1977a). Shorter intervals of estrogen priming prior to progesterone administration result in lower lordosis scores. In a previous study (Walker and Feder, 1977a), it was reported that the synthetic compound enclomiphene (ENC) could act as a partial estrogen mimic in the CNS by facilitating the induction of lordosis in guinea pigs. Normally, an 1I-hr interval between estradiol benzoate (E,B) and progesterone (P) is insufficient for the display of lordosis. However, when this II-hr EBI Present address: Roche Institute of Molecular Biology, Nutley, N. J. 2 To whom reprint requests should be sent. 299 0018-506X/79/03029910$01.00/0 Copyright @ 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.
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AND
FEDER
priming period was preceded by 28 hr of exposure to ENC, lordosis was induced in 62.5% of the females tested. On the other hand, if females were primed for the total interval of 39 hr (28 hr + 11 hr) with just ENC and then treated with P, no females displayed lordosis. The fact that E,B was necessary in the last 11 hr to complete the priming process initiated by ENC was considered as direct evidence for the importance of long-term maintenance of estrogen action in the central nervous system. Further, these behavioral results suggested that the neurochemical events that occur during the late portion of the priming period are stimulated in a relatively specific manner. That is, the late phase of the priming period is stimulated by E,B but not by ENC, while the early phase of the priming period is stimulated by E,B or ENC. The purposes of the present series of experiments were to: (1) determine whether the differences between the estrogenic actions of ENC and E,B (or the unesterified form, E2) are due to quantitative or qualitative factors; and (2) provide further support for the hypothesis that the neuronal events that occur late in the priming process are activated only in a specific manner by estrogen (Walker and Feder, 1977a,b). GENERAL
METHODS
Animals Adult Hartley strain, albino female guinea pigs (Camm Research Laboratories, Wayne, N. J.), that were ovariectomized between 53 and 70 days of age, were used throughout these studies. Ovariectomies were performed under a combination of Innovar-vet (Pitman-Moore, Washington Crossing, N.J., 0.05 ml per animal) and Equithesin (Jensen-Salsbery, St. Louis, MO., 0.6 ml per animal). After surgery, animals were group housed with no more than 10 members per group in 90 x 60 x 23-cm cages. Purina Guinea Pig Chow and water were available ad libitum, and cabbage was supplied once a week. The lights were on from 0500 to 1900 hr and the temperature was maintained at approximately 23°C. Behavioral Testing After surgery, the animals were maintained for a period of not less than 26 or more than 50 days. At the end of this period all subjects were screened for their ability to respond to E,B and P treatment. This screening test consisted of injecting all subjects with 1.6 pg E,B followed 39 hr later by 0.5 mg P (all injections were administered subcutaneously in sesame oil). Starting at the time of P administration, each animal was tested for lordosis hourly in the home cage using the manual manipulation technique described by Goy and Young (1957). Only the animals that displayed lordosis during the screening test were used for further behavioral testing. In some cases the screening test was not administered.
301
STEROIDS AND LORDOSIS BEHAVIOR
Rather, the animals were first used in a study designed to determine an E,B injection schedule that would be just subthreshold for induction of lordosis (see Table 2). Subsequently, these animals were randomly assigned to various experiments (Experiments 1 and 2) and treated the same as animals that had received the screening test. Fourteen days after screening (or threshold testing), the subjects were divided into experimental groups. Presumably, all E2B from the screening test is cleared from neural tissues and plasma by 120hr after injection (Eaton et al., 1975). No subject was used in more than one experimental treatment. In all experimental groups, each animal was tested for lordosis just prior to P administration and then hourly for a period of 15 hr. During this time the quantity and quality of lordosis responses were determined. Subjects displaying a 2-set or longer lordosis followed by other lordosis responses (not necessarily consecutive) were considered “in heat.” The quality factors of latency, duration, and maximum are defined as: Latency. The time in hr after P administration to the first display of a lordosis response that is 2 set or longer. Duration. The total number of hr that an animal displays 2-set or longer responses. Maximum. The number of seconds that the longest lordosis position is held. EXPERIMENT l-ENCLOMIPHENE: A “WEAK” “INCOMPLETE” ESTROGEN?
OR
The estrogenic properties of ENC were demonstrated by the data presented in Table 1 (a replication of earlier work by Walker and Feder, 1977a). It was clear that an ll-hr interval between E,B and P was inadequate for the induction of lordosis, whereas treatment with ENC prior to an 1I-hr EB-priming period facilitated the display of lordosis in 60% of females. A control group demonstrated that if E,B was allowed to act for the full 39-hr period, the induction of lordosis was maximal (last group, TABLE 1 Enclomiphene, a Partial Estrogen Mimic, Facilitates the Induction of Lordosis by Estmdiol Benzoate” Time of injection 0 hr
19 hr
ENC E,B
ENC
28 hr
39 hr
N
Percentage in heat
J&B
P P P
10 10 10
10.0 60.0 100.0
W
0 E,B = Estradiol benzoate (1.6 pg). ENC = Enclomiphene (5.0 mg). P = Progesterone (0.5 mg).
302
WALKER AND FEDER
Table 1). Based on these data and the fact that ENC priming without any supplemental estrogen did not stimulate lordosis, it was concluded that ENC mimicked some of the early actions of estrogen but that the action of estrogen was necessary to complete the activation of later processes not stimulated by ENC (Walker and Feder, 1977a). This conclusion implied that ENC acted as an “incomplete” estrogen that mimicked only some of the events induced by estrogen. However, the results in Table 1 can also be accounted for by the hypothesis that ENC was simply a “weak” estrogen that mimicked E, completely in a qualitative sense but was far less potent in a quantitative sense than E,. The present study was designed to test whether ENC action was quantitatively or qualitatively different from Ez action. If ENC is merely a “weak” estrogen, then it should be possible to mimic ENC action by using low dosages of estrogen, and conversely, it should be possible to completely mimic estrogen action by using high dosages of ENC. Therefore, in this experiment, ovariectomized guinea pigs were treated with subthreshold doses of E2B (to mimic weak estrogenic stimulation) for the first 28 hr of a 39-hr priming period. Then, high dosages of ENC (to mimic full E, action) were administered for the last 11 hr of the priming process. (Note that this treatment is exactly the reverse of the original design used in Table 1, middle group.) If ENC is a weak rather than an incomplete estrogen, then this injection paradigm should induce lordosis. Results In order to mimic the proposed “weak” estrogenic effects of ENC during the first 28 hr of a 39-hr priming period, various doses of E,B were injected at 0 and 19 hr. At 39 hr, P (0.5 mg) was injected and the animals were tested for lordosis. Table 2 shows the results of this dose-threshold determination study. From these data, an injection schedule of 0.2 pg E2B at 0 and 19 hr was selected as being just subthreshold for the induction of TABLE 2 Determination of an l!$B Injection Schedule That Is Subthreshold for the Induction of Lordosis in Guinea Pigs” Time of injection 0 hr E,B E,B E,B E,B
(0.1) (0.2) (0.3) (0.5)
19 hr E,B E,B E,B E,B
(0.1) (0.2) (0.3) (0.5)
28 hr
39 hr
N
Percentage in heat
Oil Oil Oil Oil
P P P P
17 22 18 16
0.0 27.3 50.0 81.3
(2The dosage of estradiol benzoate (E,B) injected is indicated in parentheses (pg). P = Progesterone (0.5 mg). Oil = Sesame oil (0.1 ml).
303
STEROIDS AND LORDOSIS BEHAVIOR
lordosis (27.3% in heat). Therefore, this treatment should be equal to or better than the estrogenic stimulation provided by the 5-mg injections of ENC given at 0 and 19 hr as shown in Table 1. The results in Table 3 indicate that weak estrogenic stimulation provided by 0.2 pg E,B at 0 and 19 hr followed by large dosages of ENC (10, 20, or 80 mg) at 28 hr did not induce lordosis. However, the same subthreshold E,B stimulation followed by 1.6 pugE,B at 28 hr did induce lordosis in 77% of the females tested. EXPERIMENT
2-VARIOUS STEROIDS AND COMPLETION OF THE PRIMING PERIOD
If the differences between the estrogenic properties of ENC and E, are qualitative, then ENC facilitation of the priming action of an 1I-hr E,B-P interval must be due to the fact that ENC mimics some of the early actions of estrogen but that the action of estrogen is necessary to complete the activation of later processes not stimulated by ENC (Walker and Feder, 1977a). This idea is in agreement with other studies that imply that action of estrogen in the nervous system late in the priming period is necessary for the completion of the priming process and the subsequent display of lordosis (Joslyn et al., 1971; Feder and Morin, 1974; Walker and Feder, 1977a,b, 1979; Blaustein and Wade, 1977; Moreines and Powers, 1977). The purpose of Experiment 2 was to examine the hormonal specificity of the late portion of the priming process by injection of different classes of steroids during this period. The experimental strategy used in this study was to inject subthreshold doses of E,B at 0 and 19 hr. Then at 28 hr, various steroids were administered and assessed for their ability to mainTABLE 3 Failure of Enclomiphene (ENC) to Maintain the E,B-Priming Process When Injected into Guinea Pigs during the Late Portion of the Estrogen-Priming Period” Time of injection 0 hr
19 hr
J&B EzB &B
E.3 J%B J%B 63
E8
28 hr Oil ENC (10) ENC (20) ENC (80) E,B(1.6)
39 hr
N
Percentage in heat
P P P P P
9 9 8 8 9
0.0 0.0 25.0 0.0 77.8
a The dosages of enclomiphene (ENC) injected are indicated within the parentheses (mg). E,B = Estradiol benzoate (0.2 pg) unless otherwise indicated by parentheses (i.e., in the last group &B cl.6 pg] was injected at 28 hr). P = Progesterone (0.5 mg). Oil = Sesame oil (0.1 ml).
WALKER AND FEDER
304
TABLE 4 Effect of Various Dosages of Free Estradiol (E2) on Maintenance of the %B-Priming Process When Injected Late in the Priming Period” Time of injection 19 hr
0 hr
28 hr
EzB
E,B
EZ(5)
E,B E,B W
E,B E,B E,B
Ez (20) E, (40) Es (lW
39 hr
N
Percentage in heat
P P P P
8 8 8 6
87.5 75.0 50.0 66.7
a The dosages of free estradiol (E,) injected are indicated within the parentheses (pg). E,B = Estradiol benzoate (0.2 pg). P = Progesterone (0.5 mg).
tain and complete the priming process. Lordosis behavior was tested after injecting P at 39 hr. Results In order to assess the behavioral potency of different classes of steroids it was first necessary to find a dosage of free E, that would be able to maintain the priming process when injected at 28 hr. Table 4 presents the results of this study. In all groups E,B (0.2 pg) was injected at 0 and 19 hr (this is the subthreshold injection schedule developed in Experiment 1, Table 2). At 28 hr, various dosages of unesterified E, (5,20,40, or 100 pg) were injected. P (0.5 mg) was administered at 39 hr. The lowest dosage of E, tested, 5 (ug, appeared to be adequate to maintain and complete the priming process. Therefore, in Experiment 3, the maintenance of the priming process by cortisol and testosterone was assessedby injection, at 28 hr, of dosages of these steroids that were equimolar to 5 kg E, (1.825 x lo-’ M). The data in Table 5 indicate that neither cortisol nor testosterone stimulated the priming process. Five micrograms of E, significantly increased the percentage of females displaying lordosis. EXPERIMENT
3-VARIOUS ESTROGENS AND COMPLETION OF THE PRIMING PROCESS
The purpose of this experiment was to extend our understanding of hormonal specificity during the late phase of the estrogen priming process. In this regard, various estrogens given late in the priming process were assessed in relation to the induction of lordosis. Results
In all groups, 0.2 /*g E,B was injected at 0 and 19 hr. At 28 hr, various estrogens were injected at a dose equimolar to 5 pg E, (1.825 x lop2 M).
305
STEROIDS AND LORDOSIS BEHAVIOR TABLE 5 Effect of Different Classes of Steroids on Maintenance of the E,B-Priming Process When Injected in the Late Portion of the Priming Period” Time of injection 0 hr
19 hr
28 hr
E,B EtB E,B W
W J&B E,B EzB
Oil tort Test E, (5)
39 hr
N
Percentage in heat
P P P P
10 8 8 14
20.0 12.5 25.0 78.5*
n The amounts of cortisol (Cort) and testosterone (Test) injected were equimolar to the 5 pg of E, (1.825 x lo-’ M) injected at 28 hr in the last group. E,B = Estradiol benzoate (0.2 pg). P = Progesterone (0.5 mg). Oil = Sesame oil (0.1 ml). * P < 0.01 versus first group (Fisher exact probability test).
At 39 hr, P (0.5 mg) was injected and the animals were tested for lordosis. The different estrogens administered were: estrone (E,), EP, estriol (E3), and the synthetic estrogen, diethylstilbestrol (DES). In addition, the estrogenic potencies of catechol estrone (20H-E,) and catechol estradiol (20H-E,) were assessed. Samples of both catechol estrogens were obtained from Steraloids, Inc. In order to check the purity of the commercially synthesized catechol estrogens, additional samples (kindly supplied by Dr. T. Gentry at the Institute for Steroid Research, Montefiore Hospital) of catechol estrogens were used. From Table 6 it is clear that El, EP, E,, and DES all have equal potency for completing the priming initiated by the subthreshold E,B injections. The catechol estrogens did not maintain the priming process which indicates that they possess little, if any, estrogenic potency in this situation. The facilitatory effect of the 20H-E, supplied by Steraloids, Inc., is most likely referable to its contamination with free E, because the 20H-E, synthesized and supplied by Dr. Gentry had no estrogenic activity. In subsequent studies (J. Wilcox and H. H. Feder, unpublished data) it has been shown that 2.5 ,ug E, given at 28 hr resulted in lordosis in 67% of the females and 1.Opg E, given at 28 hr resulted in lordosis in 33% of the females primed with 0.2 pg EB at 0 and 19 hr (N = 9/group). Because 20H-E,, 20H-EB, cortisol, and testosterone (at a dose equimolar to 5 pg E,) induced lordosis in fewer than 33% of the females, it appears that these substances are far less potent (if they are active at all) than E, in completing the priming process. This study does not establish that these substances are absolutely devoid of activity, but only that they are significantly less potent than E,.
W
W EzB
&B
P P
P P
20H-E,b 20H-Ezb
20H-E,c 20H-EzC
P P P P P
39 hr
Oil E, JS Es DES
28 hr
9 18
N
11.1 0.0
10.0 -c -
6.6 rt 0.3
0.4 0.2 0.4 0.5
0.0 77.8
!I c k e
5.4 5.9 6.3 5.7
Latency (z -c SE, hr)
0.0 80.0 80.0 88.9 77.8
Percentage in heat rt r 2 -t
0.5 0.6 0.4 0.9
4.0 2 -
6.0 r 0.7
5.9 6.1 4.4 5.4
Duration (2 2 SE, hr)
4.0 2 -
8.3 5 1.1
_f 0.6 t 0.5 t 0.5 k 2.1
6.3 6.1 5.3 8.9
Maximum (2 -t SE, set)
a Each of the various estrogens injected at hr 28 was administered in a dose equivalent to 5 pg Ez (1.835 x IO-’ M). E, (estrone), E, (estriol), DES (diethylstilbestrol), 20H-E, (catechol estrone), and 20H-E2 (catechol estradiol). E,B = Estradiol benzoate (0.2 pg). P = progesterone (0.5 mg). b Compounds were supplied by Steraloids, Inc. c Compounds were supplied by Dr. T. Gentry, Montefiore Hospital, New York.
W
E,B W
JLB &B EzB E,B
W
E,B
W W W EzB
%B
19 hr
0 hr
Time of injection
TABLE 6 Effect of Various Estrogens on Maintenance of the E,B-Priming Process”
s
STEROIDS
AND
LORDOSIS
BEHAVIOR
DISCUSSION
The present series of experiments was based on the findings of a previous study that indicated that the action of estrogen in the nervous system was necessary in the late portion of the estrogen-priming process for the completion of this process. This was demonstrated by the fact that the synthetic compound, enclomiphene (ENC), initiated the estrogenpriming process but that the action of E, was required during the last 11 hr of the priming period for the successful induction of lordosis (Walker and Feder, 1977a,b). It was concluded that “ENC can mimic some of the early actions of estrogen but that ultimately the presence of estrogen is necessary to complete the activation of later processes not stimulated by ENC” (Walker and Feder, 1977a). This conclusion implied a qualitative difference between the estrogenic properties of ENC and E, (i.e., ENC is an “incomplete” estrogen). The alternative interpretation that ENC was different from estrogen in a merely quantitative sense (i.e., ENC is a “weak” estrogen) was also discussed but data were not available to eliminate one or the other interpretation. The present study was designed, in part, to clarify whether ENC is an “incomplete” or a “weak” estrogen. If ENC is a “weak” estrogen, then a large enough dose of ENC should effectively induce lordosis. To test this, female guinea pigs were primed with subthreshold dosages of E,B for the first 28 hr of a 39-hr period. Then, either E,B (1.6 pg) or a high dosage of ENC (IO, 20, or 80 mg) was injected at 28 hr and P (0.5 mg) at 39 hr. The results clearly demonstrated that ENC did not maintain or complete the priming events at this stage. Injection of E,B at this time induced the display of lordosis, as expected. These results in combination with the previous report (Walker and Feder, 1977a)demonstrate that ENC mimics the action of estrogen during the early part of the priming process but does not replace the action of estrogen during the late phase of the priming process. Our tentative interpretation of these results is that estrogen action during the priming process could be characterized as activating qualitatively different processes during various portions of the priming period. The data also indicate that, in terms of a behavioral endpoint, ENC acts in a manner qualitatively different from E, (i.e., ENC is an “incomplete” rather than a “weak” estrogen). However, at this time we cannot say whether the biochemical events that underlie sexual behavior are also differentially affected by ENC and E, in a qualitative or in a purely quantitative fashion. Experiments 2 and 3 investigated the specificity requirements of the late phase of the priming process by testing either different classes of steroids (corticoid or androgen) or various types of estrogens. These studies revealed that cortisol and testosterone (at the dosage used) did not activate the processes associated with the late portion of the priming period.
308
WALKER AND FEDER
However, E,, E2, E3, and DES had equal potency in completing the priming process at the dosage used. Catechol estrone and catechol estradiol did not complete the priming process and it appears that these compounds are without estrogenic activity as measured in this situation. In summary, our basic finding is that the synthetic antiestrogen ENC enhances early, but not late events involved in the priming of neural tissues that mediate guinea pig lordosis. E2, on the other hand, enhances early and late phases of the priming process. This distinction between lordosis-promoting properties of ENC and E, is potentially an important tool. By comparing the effects of ENC and E, on various biochemical markers of estrogen action (e.g., RNA polymerase II activity, synthesis of estrogen-induced protein, and induction of progestin receptor synthesis), one may reveal a difference in the biochemical action of ENC that is responsible for its characterization as an “incomplete” estrogen. ACKNOWLEDGMENTS This research was supported by Research Grant NIH-HD-04467 and NIMH Career Development Award NIMH-29006 (both to H.H.F.). Contribution 321 of the Institute of Animal Behavior.
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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-l%. Eaton, G. G., Goy, R. W., and Resko, J. A. (1975). Brain uptake and metabolism of estradiol benzoate and estrus behavior in ovariectomized guinea pigs. Horm. Behav. 6, 81-97. Feder, H. H., and Morin, L. P. (1974). Suppression of lordosis in guinea pigs by ethamoxytriphetol (MER-25) given at long intervals (34-35 hr) after estradiol benzoate treatment. Horm. Behav.
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Goy, R. W., and Young, W. C. (1957). Strain differences in the behavioral responses of female guinea pigs to alpha-estradiol benzoate and progesterone. Behaviour 10, 340353.
Joslyn, W. D., Feder, H. H., and Goy, R. W. (1971). Estrogen conditioning and progesterone facilitation of lordosis in guinea pigs. Physiol. Behav. 7, 477-482. Moreines, J., and Powers, J. B. (1977). Effects of acute ovariectomy on lordosis response of female rats. Physiol. Behav. 19, 277-283. Walker, W. A., and Feder, H. H. (1977a). Inhibitory and facilitatory effects of various anti-estrogens on the induction of female sexual behavior by estradiol benzoate in guinea pigs. Brain Res. 134, 455-465. Walker, W. A., and Feder, H. H. (1977b). Anti-estrogen effects on estrogen accumulation in brain cell nuclei: Neurochemical correlates of estrogen action on female sexual behavior in guinea pigs. Brain Res. 134, 467-478. Walker, W. A., and Feder, H. H. (1979). Long-term effects of estrogen action are crucial for the display of lordosis in female guinea pigs: Antagonism by anti-estrogens and correlations with in vitro cytoplasmic binding affinity. Endocrinology 104, 89-96.