Activation of lordosis in ovariectomized guinea pigs by free and esterified forms of estrone, estradiol-17β and estriol

Physiology and Behavior, Vol. 13, pp. 251-255. Brain Research Publications Inc., 1974. Printed in the U.S.A.

Activation of Lordosis in Ovariectomized Guinea Pigs by Free and Esterified Forms of Estrone, Estradiol- 170 and Estriol' H. H. F E D E R A N D R A E S I L V E R

Institute o f Animal Behavior, Rutgers University, 101 Warren Street, Newark, New Jersey 07102

(Received 20 March 1974) FEDER, H. H. AND R. SILVER. Activation of lordosis in ovariectomized guinea pigs by free and esterified forms of estrone, estradiol-17# and estriol. PHYSIOL. BEHAV. 13(2) 251-255, 1974. - The threshold doses of estradiol-17#, estxone, estriol, estradiol-17#-3-benzoate and estrone-3-benzoate required to activate lordosis in ovariectomized adult guinea pigs were determined by injecting these steroids in combination with progesterone. The doses of the 3 free steroids which activated lordosis in about 50% of the animals ranged from 20 to 50 ug/animal. In contrast, threshold doses of estradiol benzoate and estrone benzoate were only 0.4 and 1.7 tzg/animal, respectively. The data indicate that (1) conversion to estradiol-17/3 is not an absolute requirement for activation of lordosis and (2) esterified forms of estradiol and estrone are far more potent in inducing lordosis than the corresponding free forms of these steroids. The increased potency of the esterified estrogens may be attributable to their prolonged action. Testosterone propionate also induced lordosis in 6/18 ovariectomized guinea pigs when given in a dose of 500 t~g/animal for 7 days. Guinea pigs

Estrous behavior

Estradiol

Estrone

Estriol

Testosterone propionate

an e x t e n d e d period in the uterus once this triggering action has been initiated [ 18]. Investigations of the mechanism of action of estrogen on neural tissues mediating female behavior have been made f r o m this perspective. For example, studies with radioactive estrogenic substances have indicated that radioactivity is absent or minimal in brain tissues regulating behavior by the time lordosis behavior is displayed in cats and rats [9, 10, 15] in response to administration o f such substances. Other studies with antiestrogenic c o m p o u n d s and protein synthesis inhibitors given to ovariectomized rats d e m o n s t r a t e strong suppression of female behavior when the antiestrogen or inhibitor is given within 6 to 8 hr of estrogen injection, but diminished or no suppression of the behavior if the antiestrogen is given 12 to 24 hr after estrogen [1, 16, 19, 22]. These data suggest that the presence o f estrogen in neural tissues regulating sexual behavior is required for only a brief period during which it triggers a chain of events which eventually leads to expression of sexual behavior. The present experiments test some o f the assumptions m a d e a b o u t h o r m o n a l regulation of female behavior on the basis o f the m o d e l of h o r m o n e action provided by data on rat uterus. In these experiments, the generality of these

D E S C R I P T I O N S of the m e c h a n i s m of action of estrogen on rat uterine tissues have influenced thoughts a b o u t the way in which estrogens act on neural tissues mediating female sexual behavior. One principle which emerged f r o m the work on rat uterus was that estradiol-17•, rather than its metabolites, caused stimulation of the uterus [ 12]. While a m e t a b o l i t e such as estrone might produce uterine stimulation, it was t h o u g h t that this activity was due to conversion of estrone to estradiol and not to a direct action of estrone (although a recent report suggests direct actions of estrone and estriol on rat uterine tissue in vitro [ 17 ]. The e x t e n t to which this view has been incorporated into current thinking about estrogens and female behavior is reflected by the relative lack of i n f o r m a t i o n on the actions of estrogens o t h e r than estradiol-17~ on the activation of female behavioral responses. However, Beyer and colleagues [2] have r e p o r t e d that estradiol-17t~, estrone and estriol activate lordosis in ovariectomized rats. A n o t h e r principle which has emerged f r o m biochemical studies of estrogen action on rat uterus is that soon after estrogen reaches the uterus, it triggers a series of events mediated by estrophilic receptor proteins in c y t o p l a s m and nucleus [ 11 ]. The estrogen itself need not be retained for

Supported by Research Scientist Development Award NIMH-29006 (H.H.F.), Research Grant NIH-HD 04467 (H.H.F.) and by Grants from Rutgers University Research Council (R.S.) and the Alfred P. Sloan Foundation. We thank Dr. P. Perlman of Schering Inc. (Bloomfield, N. J.) for generous supplies of testosterone propionate and progesterone. Contribution No. 190 of the Institute of Animal Behavior. Purity of estriol was confirmed by melting point and by thin-layer chromatography. 251

252

F E D E R AND S I L V E R

assumptions was tested by using a species o t h e r than the rat (guinea pigs) and gauging the effectiveness of estrogenic c o m p o u n d s o t h e r than estradiol-17~ in activating female behavior. Additionally, the idea that only a short pulse of estrogen is necessary for activating female behavior was examined by using both esterified (long lasting) and nonesterified forms of estrogens. The assumption that esterified estrogen is longer lasting than free estrogen rests on our finding that 24 hr after injection of free tritiated estradiol the guinea pig h y p o t h a l a m u s contains less than 1/10 the radioactivity found at 0.5 hr [8]. In contrast, Eaton, Resko and Goy (personal c o m m u n i c a t i o n ) found a higher percentage of radioactivity was retained by guinea pig hypothalamus 3 6 h r after injection of tritiated estradiol benzoate. Because some workers have demonstrated a facilitatory effect of testosterone on lordosis behavior in rats and rabbits (e.g., [ 3 , 2 1 ] ) we also administered testosterone to ovariectomized guinea pigs in order to test its p o t e n c y in facilitating lordosis in this species. METHOD

Female Hartley strain guinea pigs were obtained from a commercial supplier (Carom Research, Wayne, N. J.) and were ovariectomized under barbiturate (Equithesin) anesthesia. The animals were housed in groups of 6 to 8 per cage and given free access to Purina guinea pig chow and water. A 14:10 hr light-dark cycle was maintained throughout the experiment. T w o to 3 weeks after ovariectomy, the animals (weighing an average of about 500 g) were assigned at r a n d o m to experimental groups. Each group received a test for sexual receptivity after injection with varying doses of estradiol-17¢, estrone, estriol, estradiol-17~-3-benzoate, estrone-3-benzoate (estrogens obtained from Steraloids, Pawling, N. Y.) testosterone propionate (Oreton, Schering) or sesame oil vehicle followed by an injection of 0.5 mg progesterone (Proluton, Schering) to all animals (dosages and timing of injections are shown in Tables 1 and 2). Injections were subcutaneous. The animals were given 10 hourly tests for estrous behavior starting i m m e d i a t e l y after the progesterone injection by using the manual stimulation m e t h o d of Young et al. [23]. The duration of lordosis maintained during manual stimulation was measured in seconds, and the latency to the first positive response (in hr) after the progesterone injection was also recorded. An animal was considered in heat if she displayed lordosis of at least 2 sec duration in at least 2 consecutive hours of testing. Percentages of animals in heat were recorded for each treatment group. Most experimental groups were comprised of animals receiving only one set of injections. However, the 20, 25, 50 and 100 ug estradiol and estrone groups contained animals previously tested with these steroids at lower dose levels. In these cases of repeated testing, the tests were separated by an interval of two weeks. RESULTS

The results clearly indicate that all three free estrogens tested (estradiol-17¢~, estrone, e s t r i o l ) i n d u c e d display of lordosis behavior in ovariectomized guinea pigs when given in c o m b i n a t i o n with progesterone (Table 1). No striking differences in threshold among these 3 free estrogens were

detected. Each activated lordosis in about 40% of animals at a dose level of 20 ug/animal, and each had a p p r o x i m a t e l y the same effec~t on latency to lordosis and m a x i m u m lordosis duration (Tables 1 and 2). The esterified estrogens were far more effective than the free estrogens in inducing lordosis. This result is consistent with very early work on guinea pigs demonstrating that the rank order of effectiveness was dihydroxyestrin b e n z o a t e > t h e e l i n in oil>theelin in water for activation of lordosis in ovariectomized animals [5,6]. Estrone benzoate in a dose of 1.7 ug/animal induced lordosis in 77.8% of animals in the present experiment, while estradiol benzoate induced lordosis in 55.6% of animals at a dose of only 0.4 ug/animal (Table 1). When it is borne in mind that only 72% of the molecular weight of estrone benzoate or estradiol benzoate is attributable to the steroid moiety, the difference between threshold doses of free estrogens and esterified estrogens becomes even more magnified. Thus, 1.7 ug of estrone benzoate contains only 1.2 ug of estrone and 0.4 ug of estradiol benzoate contains only 0.3 ug estradiol. The esterified estrogens also seemed to produce longer m a x i m u m lordosis durations than the corresponding free forms of the steroids, an effect especially striking in the case of estradiol (Tables 1 and 2). Even though the threshold doses for free estradiol and free estrone do not appear to differ markedly (in fact, free estrone seems to be somewhat more effective than free estradiol at the 25 ug levels, Tables 1 and 2), it is clear that the benzoate form of estradiol is about four times more p o t e n t in inducing lordosis than the benzoate form of estrone (Tables 1 and 2). Testosterone propionate (500 ug/day/7days) was also shown to have some ability to activate lordosis in ovariectomized guinea pigs. Six of 18 females given this t r e a t m e n t of testosterone propionate in c o m b i n a t i o n with a single injection (0.5 mg) of progesterone d i s p l a y e d lordosis. Latency to lordosis was 6.2 + 0.5 hr (X +- SEM) and maxi m u m lordosis duration was 5.8 -+ 0.9 sec (X +- SEM). None of six control females given sesame oil vehicle for 7 days and an injection of 0.5 mg progesterone showed lordosis. Although the difference between these two groups with regard to % in heat did not reach statistical significance (Table 2), it seems as though a slight facilitatory effect of testosterone on lordosis is suggested.

DISCUSSION

The first feature to emerge from this study is the finding that estrone and estriol, as well as estradiol-17t~, activate female sexual behavior in ovariectomized guinea pigs. Perhaps the ability of estrone to activate lordosis is attributable to conversion of estrone to estradiol. This conversion has been d o c u m e n t e d [20]. Additionally, after injection of radioactive estrone to ovariectomized guinea pigs about half of the radioactivity recovered from h y p o t h a l a m u s is in the form of estradiol-17~ (H. H. Feder, unpublished data). However, estriol is said not to be convertible to estradiol-17~, at least in rat uterine tissue [17]. If subsequent studies with radioactive estriol should show no radioactivity recoverable from guinea pig h y p o t h a l a m u s in the form of estradiol-17t3, then one would be forced to conclude that conversion to estradiol-17¢~ is not a prerequisite for estriol-induced lordosis. This speculation can be carried one step further. lf, as suggested, lordosis is activated through the mediation

E S T R O G E N S A N D G U I N E A PIG L O R D O S I S

253 TABLE 1

INDUCTION OF LORDOSIS IN OVARIECTOMIZED GUINEA PIGS BY VARYING DOSES OF FREE AND ESTERFIED ESTROGENS. AN INJECTION OF 0.5 MG PROGESTERONE WAS GIVEN TO EACH ANIMAL 40 HR AFTER ESTROGEN INJECTION.

Steroid

Estrone

Estradiol-17~

Estriol

Estrone-3-benzoate

Estradiol-I 7#3-benzoate

Dose (~g/animal)

N

% In Heat

Latency to Lordosis* (Hr, X ± sere)

Maximum Lordosis Duration* (sec, X ± sem)

7.0 ± 0.0

5

16

12.5

4.0 ± 1.0

10

9

0

-

-

20

8

37.5

7.0 ± 0.6

16.0 ± 2.5

25

9

66.7

6.7 + 0.9

8.7 ± 2.6

50

22

59.1

4.5 ± 0.4

13.3 ± 2.2

100

15

66.7

4.0 -+ 0.7

15.8 ± 2.5

5

15

0

10

24

4.2

20

15

25

9

50

-

-

5.0

5.0

46.6

5.0 ± 0.6

7.0 ± 1.2

11.0

7.0

8.0

24

62.5

5.0 +- 0.2

6.5 ± 1.7

100

14

42.8

5.5 ± 0.8

10.0 ± 3.0

10

7

0

-

20

8

37.5

6.0 ± 2.0

6.7 ± 2.4

50

6

100.0

5.0 ± 0.6

12.8 ± 1.6

0.4

4

25.0

6.0

14.0

0.8

6

0

-

-

1.7

9

77.8

6.6±0.6

12.4+2.6

5.0

7

100.0

5.4±0.5

13.0±2.4

0.2

5

0

-

-

0.4

9

55.6

5.8±0.5

10.0±3.5

0.8

11

63.6

5.8±0.4

21.0±5.0

1.7

10

70.0

4.7±0.3

50.7±7.7

3.3

10

80.0

5.6±0.9

30.1±5.1

*These figures based only on animals which displayed lordosis.

o f nuclear or c y t o p l a s m i c r e c e p t o r m o l e c u l e s in t h e h y p o t h a l a m u s [ 1 9 ] , t h e n for estriol t o be effective in activating lordosis the h y p o t h a l a m u s w o u l d have t o c o n t a i n either (a) nuclear or c y t o p l a s m i c r e c e p t o r s specific for estriol or (b) nuclear or c y t o p l a s m i c r e c e p t o r s w h i c h n o r m a l l y r e s p o n d t o estradiol-17# b u t can be f o o l e d i n t o a c c e p t i n g closely related steroids such as estriol or e s t r o n e . The ability o f t e s t o s t e r o n e p r o p i o n a t e to activate lordosis in s o m e female guinea pigs m a y be a t t r i b u t a b l e t o c o n v e r s i o n o f t h e a n d r o g e n t o estradiol, as has b e e n d e m o n s t r a t e d f o r rats [21] and r a b b i t s [ 3 ] . However, we c a n n o t rule o u t t h e possibility t h a t t h e same n o n - s p e c i f i c i t y t h a t p e r m i t t e d estriol t o i n d u c e lordosis also p e r m i t s testost e r o n e p e r s e to activate this r e s p o n s e in guinea pigs. A s e c o n d f e a t u r e emerging f r o m t h e data in this experim e n t is t h a t esterified f o r m s o f estradiol and e s t r o n e

( u n f o r t u n a t e l y we were unable t o o b t a i n estriol b e n z o a t e ) were far m o r e p o t e n t in activating lordosis t h a n the corres p o n d i n g free f o r m s o f t h e s e steroids. Thus, a dose o f 20 ~g o f free estradiol was required to activate lordosis in 7/15 guinea pigs, b u t a dose o f only 0.4 tzg o f estradiol b e n z o a t e resulted in lordosis in 5/9 animals. Similarly, 25 ~g o f free e s t r o n e was n e e d e d for lordosis in 6/9 animals, while o n l y 1.7 tzg o f e s t r o n e b e n z o a t e was n e e d e d for lordosis in 7/9 animals (for similar data in rats see [14] ). These data seem i n c o n s i s t e n t w i t h the idea t h a t e s t r o g e n simply triggers a chain o f b i o c h e m i c a l events in the nervous s y s t e m and t h a t e s t r o g e n may disappear very rapidly f r o m neural tissues and still activate lordosis b e h a v i o r m a n y h o u r s later. The triggering h y p o t h e s i s is suggested by Bullock's s t u d y [4] o n guinea pigs in w h i c h s u b c u t a n e o u s salistic capsules containing estradiol-17# were w i t h d r a w n after only 0.5 h r and

254

F E D E R AND S I L V E R TABLE2 STATISTICAL COMPARISONS BETWEEN SELECTED GROUPS OF OVARIECTOMIZED GUINEA PIGS GIVEN STEROID TREATMENTS TO INDUCE LORDOSIS BEHAVIOR. DATA ON WHICH THESE STATISTICS ARE BASED ARE IN TABLE 1.

% in Heat (by x 2 or Fischer exact probability)

Latency to Lordosis (by 2-tailed t-test)

Maximum Lordosis Duration (by 2-tailed t-test)

3.3 tzg estradiol benzoate vs 5.0 ~g estradiol

p<0.01 *

NO TEST

NO TEST

5.0 #g estrone benzoate vs 5.0 ~tg estrone

p<0.01*

p>0.10

p>0.10

0.8 t~g estradiol benzoate vs 0.8 ~tg estrone benzoate

p = 0.02*

NO TEST

NO TEST

0.4/~g estradiol benzoate vs 0.4 tzg estrone benzoate

p = 0.34

p>0.10

p>0.10

100/~g estradiol vs 100/2g estrone

p = 0.18

p>0.10

p>0.10

50/~g estradiol vs 50/~g estrone vs 50 gg estriol

p>0.05 for all comparisons

p>0.10 for all comparisons

p>0.05 for all comparisons

25 tzg estradiol vs 25 ~tg estrone

p = 0.02*

20 ttg estradiol vs 20 tzg estrone vs 20 tzg estriol

p = 0.51 for all comparisons

Comparison

testosterone propionate vs oil

NO TEST

p>0.05 for all comparisons

p = 0.13

NO TEST

NO TEST

p<0.01 for estradiol vs estrone* p>0.10 for estradiol vs estriol NO TEST

In squares labelled "NO TEST" there was no statistical test because of the absence of responsive animals in one of the groups under consideration. *Indicates statistically significant difference.

yet lordosis was displayed m a n y h o u r s later. If the triggering h y p o t h e s i s were valid, o n e w o u l d n o t e x p e c t that esterified (long lasting) steroids w o u l d be so m u c h m o r e effective t h a n the free forms. This finding suggests t h a t in a d d i t i o n t o early triggering e f f e c t s o f estrogen, p r o l o n g e d p r e s e n c e o f e s t r o g e n also c o n t r i b u t e s to sexual behavior in female guinea pigs. F u r t h e r evidence for such a m a i n t e n a n c e effect o f e s t r o g e n in guinea pigs c o m e s f r o m studies showing that s u p p l e m e n t a r y injections o f estradiol b e n z o a t e given 36 hr after the original dose o f estradiol

b e n z o a t e p r o l o n g d u r a t i o n o f heat [ 5,13] and f r o m studies s h o w i n g that the a n t i e s t r o g e n MER-25 suppresses lordosis w h e n given 3 4 - 4 6 hr after estradiol b e n z o a t e priming [ 7 ] . There are also s o m e data on antiestrogens in rats w h i c h suggest a m a i n t e n a n c e effect o f estrogen [ 2 2 ] . At this time, we c a n n o t resolve t h e a p p a r e n t discrepancies b e t w e e n the data suggesting a m a i n t e n a n c e effect o f estrogen on lordosis behavior in guinea pigs with the silastic i m p l a n t data o f Bullock [ 4 ] .

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3. Beyer, C. and N. Vidal. Inhibitory action of MER-25 on androgen induced oestrous behavior in the ovariectomized rabbit. J. Endocr. 51: 401-402, 1971. 4. Bullock, D. W. Induction of heat in ovariectomized guinea pigs by brief exposure to estrogen and progesterone. Hormones Behav. 1: 137-143, 1970.

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