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Effects of ovariectomy and constant light on responsiveness to estrogen in the rat
HORMONES
AND BEHAVIOR
4, 269-279
(1973)
Effects of Ovariectomy and Constant Light on Responsiveness to Estrogen in the Rat1 D. DAMASSA and J. M. DAVIDSON Department
OfPhysiology, Stanford University Stanford, California 94305
Medical School,
The hypothesis that the responsiveness of sexual hehavior and LH secretion to exogenous gonadal steroid treatment is dependent on the endogenous steroid environment existing prior to treatment was tested in female rats. The major finding was that estrogen was more effective in stimulating lordosis behavior when treatment was commenced immediately after ovariectomy than when it was delayed for 6 weeks. This indicates that the sensitivity of behavior regulating mechanisms in the female rat declines after removal of the “activating” hormones, as previously reported for testosterone in the male. Similar results were obtained in groups of animals whose pattern of steroid secretion prior to ovariectomy had been changed by 2 months’ exposure to constant light. The constant illumination itself showed no significant effect on behavioral responsiveness in spayed estrogen-treated rats. Results are also reported for plasma LH determinations and uterine weights in each of the experiments. Plasma LH levels were found to be lower under conditions of constant as compared to cycling light, both in spayed untreated and spayed estrogen-treated animals.
A commonly observed phenomenon in endocrinology is the decline in “target tissue” sensitivity after removal of the trophic hormone for that tissue (Liddle, Island, and Meador, 1962; Clar, Massons, and Robust&, 1967). Among the tissues which have been studied in this respect are those affected by gonadal steroids, including the putative hypothalamic structures which respond to variations in circulating steroid levels by regulating the expression of sexual behavior and the secretion of gonadotropic hormones (Davidson and Bloch, 1969; Davidson, 1969). In previous investigations, it was shown that during the 2 months after castration there is a marked decrease in the sensitivity of male rats to the sex behavior-activating effects of testosterone (Young, 1961; Davidson, 1972; J. M. Davidson, E. R. Smith, C. H. Rodgers, and G. J. Bloch, unpublished data). The primary purpose of this study was to determine if, after ovariectomy, changes in ‘Supported
by NIH Grant MH 21178.
269 Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form rcscrved.
270
DAMASSA
AND DAVIDSON
sensitivity to estrogen could be demonstrated in terms of sexual receptivity. Parallel observations were also made on “feedback” suppression of luteinizing hormone (LH) by estrogen and on uterine weight. In addition, the possibility that behavioral, feedback, or uterine sensitivity to estrogen might be affected by neuroendocrine perturbations involving altered estrogen levels was investigated by studying rats which had been exposed to 2 months of constant light. In these anovulatory animals, persistent vaginal cornification and sexual receptivity are presumably related to estrogen titers which are persistently above diestrous levels (Hardy, 1970; Naftolin, BrownGrant, and Corker, 1972). However, the steroid levels have not yet been studied fully, and it was desirable to determine whether the condition of persistent behavioral estrus is related to an increased sensitivity of the animal to circulating estrogen. Accordingly, the effects of daily estrogen administration were compared starting 1 day or 6 weeks after ovariectomy in rats exposed to cycling or to constant light. METHODS The subjects were 95 female Long-Evans* rats which were housed three or four per cage in temperature-controlled rooms. Illumination was supplied by incandescent lamps either on a 12-hr light cycle with lights off at 1100 hr (LD), or for 24 hr each day (LL). All animals received feed and water ad lib. The effects of daily estrogen administration were compared in two situations: treatment commencing on the day after ovariectomy (“maintenance” paradigm) or 6 weeks thereafter (“restoration” paradigm). Behavior tests were conducted between 1400 hr and 1700 hr, using dimly illuminated semicircular test cages (41 cm high, 76 cm wide, and 46 cm deep), on days 2, 4, 6, 7,8, 11, and 14 of hormone treatment (first injection 0900 hr on Day 1). A sexually experienced male was placed in each test cage and presented with a test female which was allowed to receive 11 mounts. Sexual receptivity was assessedby the lordosis quotient (LQ = [no of lordoses/no. of mounts] X 100) which was calculated from the female’s responses to the last 10 mounts. In order to facilitate the statistical evaluation of receptivity over the 14day hormone treatment period, a Total Lordosis Quotient (TLQ) was calculated for each animal (TLQ = C LQ’s/no. of tests). On the morning after the last test the animals were sacrificed. Blood was rapidly collected from the abdominal aorta under ether anesthesia, using heparinized syringes. Uteri were weighed after the removal of any luminal fluid. The plasma samples were assayed in triplicate for LH by radioimmunoassay (Niswender, Midgley, Monroe, and Reichert, 1968). Plasma LH values are reported in terms of the standard used: NIH LH RP-1 (potency = *Simonscn’s
Laboratories,
Gilroy,
CA.
ESTROGEN
SENSITIVITY
AFTER
SPAYING
271
0.03 NIH LH S-l). Samplesin which LH was nondetectable were assigned values of 10 rig/ml (sensitivity of the assayat 200 ~1 plasma). Unlessotherwise indicated, Student’s t test was used for statistical comparisons and all probabilities indicated are two-tailed. The null hypothesis was accepted fox P> 0.05. Experiment I-LD.
Seventeenof 35 60-day-old female rats were brlater ally ovariectomized (restoration group), and the remaining 18 rats were ovariectomized 6 weeks later (maintenance group). Estrogen replacement therapy was initiated for both groups on the day after ovariectomy of the maintenance group and consisted of 14 daily SCinjections of 17 0 estradiol benzoate (EB) in approximately 0.1 ml of sesame oil. Maintenance and restoration groups each received 0.1 or 0.4 pg EB/ 100 g body weight, and twc control groups each received 0.1 ml sesameoil. The doses of EB used are considered to produce circulating estrogen levels within the “physiological” range for Long-Evansrats (Davidson, Smith, Rodgers,and Bloch, 1968). Experiment 2-LL-LD. Fifty-two 60-day-old rats were placed in condi. tions of constant illumination, and eight others of the same age were left in cycling light. Sixty days later approximately 50% of the animals in both LL and LD groups were bilaterally ovariectomized (restoration), and 6 weekslater the remaining animals were ovariectomized (maintenance). On the next morning EB injections and testing were initiated as described above. The LD animals were included to control for the circumstance that the rats used in this experiment were 2 months older than those in Experiment 1, due to the period of exposure to LL. RESULTS
Female Sexual Behavior Experiment 1-LD. None of the oil-injected control animals showed lordosis behavior on any of the tests. Figure 1 shows the mean LQ values for the four groups receiving estrogen treatment and Table 1 shows TLQ values. With the low dose of EB (0.1 c(g/lOOg) no lordoses occurred until Day 6 of treatment, and the mean LQ on any one test never exceeded40 for either the maintenance or the restoration groups (Fig. 1A). The 0.4qg dose, on the other hand, produced a more rapid onset of sexual responsivenessand higher levels of receptivity peaking at LQ levels similar to those found in normally estrous females or ovariectomized animals treated with higher dosesof steroid (Kuehn and Beach, 1962; Davidson et al., 1968). At both dose levels, the mean LQ was higher for the maintenancethan for the restoration groups on every test where lordoses were present (Fig. 1). Mean TLQ (Table 1) was significantly greater for the maintenance group al
272
DAMASSA AND DAVIDSON TABLE I Effects of Estradiol Benzoate (EB) on Sexual Receptivity and Uterine Weight: Experiment 1 Uterine wt. (mg)
Dose EB
N
LD3 Maintenance
Oil 0.1 0.4
4 I 7
0 19.8 f 5.3 61.2 f 5.6
104 f 8.02 291.5 f 10.3 323.9 f 21.0
LD Restoration
Oil 0.1 0.4
3 7 7
0 11.4 + 6.3 35.5 i 3.2
66.5 f 7.0 236.0 i 30.0 280.2 i 12.2
~.-
TLQl
~[No. lordoses on all tests/no. mounts] X 100. 2Mean f SE. 3LD: cycling light.
the 0.4pg dose (P< 0.01). The difference was not significant for the O.l#g groups presumably due to the consistently low level of receptivity exhibited by these animals. By the end of the period of estrogen treatment, maintenance and restoration groups had approached similar mean LQ values, although the dose difference persisted. Experiment 2-LL-LD. In the control (oil-treated) groups of animals kept in constant Light, lordosis behavior (LQ = 80) was observed in only one animal (maintenance group) on Day 1 of testing. As in Experiment 1, mean LQ levels did not rise as rapidly and/or did not reach as high levels in the O.las in the 0.4pg groups; and in all cases (LL 0.1 pg and 0.4 pg; LD 0.4 pg) the responsiveness under maintenance and restoration conditions approached equality by the last day of testing. As in Experiment 1, the three maintenance groups studied had higher mean LQ levels on every test (including the first of the series) except the last one (Fig. 2). As shown in Table 2, for all 3 pairs of groups mean TLQ was significantly higher in the maintenance than in the restoration condition (P< 0.05 for 0.1 pg, LL; and P< 0.01 for 0.4 pg, LL and 0.4 pg, LD). There were, however, no significant differences at the 0.4~c(g level between the groups of constant light-exposed rats and the corresponding cycling light animals (Two factor analysis of variance, P> 0.05 for lighting conditions and <0.01 for maintenance vs restoration). Thus, while lordosis behavior was invariably higher in Experiment 2 than Experiment 1 (Tables 1 and 2) this cannot be attributed to lighting conditions, but presumably to the difference in ages of the animals.
273
ESTROGEN SENSITIVITY AFTER SPAYING SO (A)
0.1 pp
60
EBllOOpm
40
40
-
- --
M-
Maintenance Rtrtorotion
l I’ 0.
0
1 2
4
6
s
lo
12
14
Days
Fig. 1. Effects of postcastration interval on estrogenactivated sexual behavior in female rats (Experiment 1). EB injections were given each day throughout the period of testing. Maintenance: estradiol benzoate (EB) treatment from the day after ovariectomy. Restoration: treatment commencing 6 weeks after ovariectomy. L/M: lordoses/mounts. Vertical lines indicate single standard errors.
Luteinizing Hormone Measurements Experiment I-LD. Mean plasma LH values for this experiment are shown in Fig. 3. The oil-injected control groups showed markedly higher LH levels for the restoration than for the maintenance paradigm, as expected in view of the longer postcastration interval. This difference was maintained with both low and high doses of EB (restoration vs maintenance, P< 0.05 in both cases). In none of the groups was plasma LH suppressed to the basal level found in cycling rats, which in this laboratory is 20-30 &ml at times in the cycle other than the afternoon of proestrus (Smith, Bowers, and Davidson, 1973).
DAMASSA AND DAVIDSON
274 KJ-
O.Ipg
El31lOOgm
------
Mointenonce
--
Rerlorotion
(6) 0.4~9 0
4
6
6
IO
EB/lOOgm
12
I4
Days
Fig. 2. Behavioral observations, Experiment 2. (A) Effects of 0.1 pg EB/lOO g/day on sexual receptivity: maintenance and restoration conditions, constant light. (B) Effects of 0.4 pg/lOO g/day on sexual receptivity in constant (LL) or cycling (LD) light.
Experiment 2-LL-LD. Results are shown in Fig. 4. As in Experiment 1, the maintenance groups show significantly lower plasma LH than the restoration groups for all treatments (P< 0.01). Two-factor analysis of variance on the LL and LD, 0.4~c(g groups revealed significance for conditions of lighting (P< 0.05) and maintenance vs restoration (P < 0.01). Comparisons on individual groups showed significantly lower LH levels in the LL than the LD condition. P was < 0.01 for the maintenance group and < 0.05 for restoration (t tests). In order to justify comparisons between the two experiments, a two-factor analysis of variance was performed on the LD 0.4~pg groups of Experiment 2 and the similarly dosed groups of Experiment 1. No significant differences in plasma LH were found between the experiments. Paired comparisons were then made between the LD groups of Experiment 1 and the
FSTROGEN
SENSITIVITY TABLE
AFTER
275
SPAYING
2
Effects of Estradiol Benzoatc(EB) on Sexual Receptivity and Uterine Weight: Experiment 2 --
.-
LLl Maintenance
DoseEB -_ Oil 0.1
TLC’ --.-
1
2.0 f 2.0 41.6 f 6.9 79.7 f 3.0
182.5 f 22.1 418.7 f 23.8 413.0 f 25.1
0 22.2 f 5.5 61.4 f 5.3
102.1 f 14.4 290.7 f 10.9 373.1 f 20.1
10 10
0.4 LL
Oil
6
Restoration
0.1
10
0.4
9
85.0 LD
4
Restoration
Uterine wt. (mg) --.--
N
-f 1.9
56.5 + 8.3
362.0 f 5 1.9 301.7
f. 18.9
1LL: constantlight.
r
-
EB dose
0 PLO’4
700
Fig. 3. Effects of estrogentreatment on plasmaLH in spayedrats, Experiment 1. LL groups of Experiment 2. For all six comparisons mean plasma LH was higher in LD than in LL. This difference was significant in five of the six cases (P< O.Ol), the one exception being the O.l+g restoration comparison. Even though the animals of Experiment 2 were 60 days older than those of Experiment 1, these comparisons are presumed to be valid due to the
DAMASSA AND DAVIDSON
276 500 -
100 -
EE dose :
0 P9’d
.I pL9/d
.4/g/d
.4 /LQld
Fig. 4. Plasma LH values from Experiment 2. Effects of different dose regimens of estrogen on plasma LH in rats exposed to cycling or constant light.
similarity of values in the LD 0.4~pg groups of Experiment identically treated but younger rats of Experiment 1.
2 and the
Uterine Weight Experiment I-LD. Table 1 shows the mean uterine weights for the different groups of this experiment. In all cases the maintenance groups showed heavier uteri than the restoration. This difference was, however, only significant (P< 0.01) in the oil-injected controls. Experiment 2-LL-LD. As in Experiment 1, mean uterine weights were greater in the maintenance groups, in aII cases (Table 2). In the LL condition these differences were significant after treatment with oil (P< 0.05) 0.1 pg EB (P< 0.001) and 0.4pg EB (P< 0.01) injections. The LD (0.4c(g EB) animals did not show significant differences. The uterine weights of the LD groups (0.4 pg) were lower than those of the comparable LL groups. Two-factor analysis of variance on these data revealed significant differences for maintenance vs restoration (P < 0.01) as well as for lighting conditions (P < 0.05).
DISCUSSION The primary question to which this study addressed itself-whether there is a change in sensitivity to the lordosis behavior-activating effect of estrogen with increasing time after ovariectomy-,was clearly answered. In each of five conditions under which the lordosis response to EB injections was studied, the
ESTROGEN
SENSITIVITY
AFTER
SPAYING
277
treatment was more effective when initiated shortly after surgery than when a delay of 6 weeks intervened. In this respect, estrogen effects on female sexual behavior resemble testosterone effects in the male (see Davidson, 1972). It should be noted, however, that while in the present study 2 weeks of EB administration sufficed to obliterate differences in lordosis behavior between the maintenance and restoration conditions, significant differences were still present in the male after 8 weeks of daily treatment with testosterone propionate (Davidson, 1972). Estradiol benzoate replacement therapy produced no significant differences in lordosis behavior between constant light- and cycling light-exposed animals when all conditions except light exposure were similar (Experiment 2). The greater responsiveness of animals in Experiment 2 as compared to Experiment 1 was thus presumably due to their being 2 months older. In both experiments, feedback suppression of LH secretion by EB was also investigated. It was not surprising that the oil-treated controls for the restoration groups showed higher plasma LH levels than for the maintenance groups, since the restoration controls had been deprived of ovarian steroids for 8 weeks compared to only 2 weeks in maintenance groups, and plasma LH shows a steadily increasing rise after ovariectomy (Gay and Midgley, 1969 and unpublished experiments in this laboratory). If we attempt to compare estrogen feedback sensitivity under the two conditions, the difficulty arises that comparisons are made from different starting levels of plasma LH. Nevertheless, LH was measured only after 14 days of EB injection. Since a single dose of 0.5 c(g EB/rat has been found to significantly reduce plasma LH 24 hr after injection (Ajika, Krulich, Fawcett, and McCann, 1972), one might expect a 14-day treatment with larger doses to have obliterated initial differences in LH between the maintenance and restoration groups, as was the case for differences in behavior. That the differences remained after 2 weeks of treatment suggests that LH is in fact less suppressible in long-term castrates than in the maintenance situation. However, further work is needed to substantiate this conclusion; the experimental design used was primarily directed at the behavioral question. Similar considerations apply to the observation that plasma LH was apparently more suppressible by EB in constant light-exposed than in cycling light-exposed animals. The control group data from that experiment also indicate that after ovariectomy plasma LH rises more slowly and/or plateaus earlier in constant light than in cycling light. This conclusion is consistent with data presented recently by. Tapper, Naftolin, and Brown-Grant (1972). We conclude that either constant light has a direct inhibitory effect on LH secretion, or that the altered steroid environment in constant light-exposed animals has an effect on the capacity for LH release which persists after ovariectomy. This study provides no information on the mechanism underlying the
278
DAMASSA AND DAVIDSON
postcastration changes observed. Although a number of models are possible, our preferred interpretation is that receptor mechanisms in the relevant target tissues decline in sensitivity as time elapses after removal of the trophic hormone, possibly due to atrophy of certain structures and/or breakdown of biosynthetic mechanisms. We recognize that the definition of sensitivity is problematic, particularly when responses are measured from different starting points as seen in the above discussion of the LH data. Although the LQ level of the controls was the same in maintenance and restoration conditions (i.e., zero), this would probably not be the case were it possible to measure the “substrate” for the behavioral action of estrogen. Our concern in this report is, in fact, merely to show that differences exist in relation to time after ovariectomy, as measured by behavioral, LH, and uterine weight responses. Two weeks of EB treatment appears to reverse the change for lordosis behavior, but not for LH and uterine weight.
ACKNOWLEDGMENTS Radioimmunoassay reagents were kindlyssupplied by Drs. G. D. Niswcndcr, L. E. Reichert, and A. F. Parlow (NIAMD Rat Pituitary Hormone Program). REFERENCES Ajika, K., Krulich, L., Fawcett, C. P., and McCann, S. M. (1972). Effects of estrogen on plasma and pituitary gonadotropins and prolactin, and on hypothalamic releasing and inhibiting factors. Neuroendocrinology 9, 304-315. Clar, J., Massons, J. M., and Robust&T. (1967). Action de 10s andr6genos sobre prdstata y vesiculas scminales en la rata macho castrada. Rev. &pan. Fisiol. 23, 115-t 16. Davidson, J. M. (1969). Feedback regulation of gonadotropin secretion. In W. F. Ganong and L. Martini (Eds.), Frontiers in Neuroendocrinology, pp. 343-388, Oxford University Press, New York. Davidson, J. M. (1972). Hormones and reproductive behavior. In H. Balin and S. Glasser @As.), Reproductive Biology, pp. 877-918, Excerpta Medica, Amsterdam, The Netherlands. Davidson, J. RI., and Bloch, C. J. (1969). Neuroendoerine aspects of male reproduction. Biol. Reprod. I, Suppl. 1, 67-92. Davidson, J. M., Smith, E. R., Rodgers, C. H., and Bloch, G. J. (1968). Relative thresholds of behavioral and somatic responses to estrogen. Physiol. Behov. 3, 227-229. Gay, V. L., and Midgley, A. R. (1969). Response of the adult rat to orchidectomy and ovariectomy as determined by LH radioimmunoassay. Endocrinology 84, 1359-1364. Hardy, D. F. (1970). The effect of constant light on the estrous cycle and behavior of the female rat. Physiol. Behav. 5, 421425. Kuehn, R. E., and Beach, F. A. (1962). Quantitative measurement of sexual receptivity in female rats. Behaviour 21, 282-299. Liddle, G. W., Island, D., and Meador, C. K. (1962). Normal and abnormal regulation of corticotropin secretion in man. Rec. Progr. Horm. Res 18, 125-153.
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Naftolin, F., Brown-Grant, K., and Corker, C. S. (1972). Plasma and pituitary luteinizing hormone and peripheral plasma oestradiol concentrations in the normal oestrous cycle of the rat and after experimental manipulation of the cycle. /. EndocrinoZ. 53, 17-30, 1972. Niswender, G. D., Midgley, A. R., Jr., Monroe, S. E., and Reichert, L. E., Jr. (1968). Radioimmunoassay for rat luteinizing hormone with antiovine LH serum and ovine LH-’ 3’ 1. Proc. Sot. Exp. Biol. Med. 128, 807-81 I. Smith, E. R., Bowers, C. Y., and Davidson, J. M. (1973). Circulating levels of plasma gonadotropins in 4- and S-day cycling rats. Endocrinology, in press. Tapper, C. M., Naftolin, F., and BrownGrant, K. (1972). Influence of the reproductive state at the time of operation on the early response to ovariectomy in the rat. J. Endocrinol.
5 3, 4 7-5 7.
Young, W. C. (1961). The hormones and mating behavior. In W. C. Young (Ed.), Sex and Internal Secretions, Vol. II, p. 1184, Williams & Wilkins, Baltimore.
AND BEHAVIOR
4, 269-279
(1973)
Effects of Ovariectomy and Constant Light on Responsiveness to Estrogen in the Rat1 D. DAMASSA and J. M. DAVIDSON Department
OfPhysiology, Stanford University Stanford, California 94305
Medical School,
The hypothesis that the responsiveness of sexual hehavior and LH secretion to exogenous gonadal steroid treatment is dependent on the endogenous steroid environment existing prior to treatment was tested in female rats. The major finding was that estrogen was more effective in stimulating lordosis behavior when treatment was commenced immediately after ovariectomy than when it was delayed for 6 weeks. This indicates that the sensitivity of behavior regulating mechanisms in the female rat declines after removal of the “activating” hormones, as previously reported for testosterone in the male. Similar results were obtained in groups of animals whose pattern of steroid secretion prior to ovariectomy had been changed by 2 months’ exposure to constant light. The constant illumination itself showed no significant effect on behavioral responsiveness in spayed estrogen-treated rats. Results are also reported for plasma LH determinations and uterine weights in each of the experiments. Plasma LH levels were found to be lower under conditions of constant as compared to cycling light, both in spayed untreated and spayed estrogen-treated animals.
A commonly observed phenomenon in endocrinology is the decline in “target tissue” sensitivity after removal of the trophic hormone for that tissue (Liddle, Island, and Meador, 1962; Clar, Massons, and Robust&, 1967). Among the tissues which have been studied in this respect are those affected by gonadal steroids, including the putative hypothalamic structures which respond to variations in circulating steroid levels by regulating the expression of sexual behavior and the secretion of gonadotropic hormones (Davidson and Bloch, 1969; Davidson, 1969). In previous investigations, it was shown that during the 2 months after castration there is a marked decrease in the sensitivity of male rats to the sex behavior-activating effects of testosterone (Young, 1961; Davidson, 1972; J. M. Davidson, E. R. Smith, C. H. Rodgers, and G. J. Bloch, unpublished data). The primary purpose of this study was to determine if, after ovariectomy, changes in ‘Supported
by NIH Grant MH 21178.
269 Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form rcscrved.
270
DAMASSA
AND DAVIDSON
sensitivity to estrogen could be demonstrated in terms of sexual receptivity. Parallel observations were also made on “feedback” suppression of luteinizing hormone (LH) by estrogen and on uterine weight. In addition, the possibility that behavioral, feedback, or uterine sensitivity to estrogen might be affected by neuroendocrine perturbations involving altered estrogen levels was investigated by studying rats which had been exposed to 2 months of constant light. In these anovulatory animals, persistent vaginal cornification and sexual receptivity are presumably related to estrogen titers which are persistently above diestrous levels (Hardy, 1970; Naftolin, BrownGrant, and Corker, 1972). However, the steroid levels have not yet been studied fully, and it was desirable to determine whether the condition of persistent behavioral estrus is related to an increased sensitivity of the animal to circulating estrogen. Accordingly, the effects of daily estrogen administration were compared starting 1 day or 6 weeks after ovariectomy in rats exposed to cycling or to constant light. METHODS The subjects were 95 female Long-Evans* rats which were housed three or four per cage in temperature-controlled rooms. Illumination was supplied by incandescent lamps either on a 12-hr light cycle with lights off at 1100 hr (LD), or for 24 hr each day (LL). All animals received feed and water ad lib. The effects of daily estrogen administration were compared in two situations: treatment commencing on the day after ovariectomy (“maintenance” paradigm) or 6 weeks thereafter (“restoration” paradigm). Behavior tests were conducted between 1400 hr and 1700 hr, using dimly illuminated semicircular test cages (41 cm high, 76 cm wide, and 46 cm deep), on days 2, 4, 6, 7,8, 11, and 14 of hormone treatment (first injection 0900 hr on Day 1). A sexually experienced male was placed in each test cage and presented with a test female which was allowed to receive 11 mounts. Sexual receptivity was assessedby the lordosis quotient (LQ = [no of lordoses/no. of mounts] X 100) which was calculated from the female’s responses to the last 10 mounts. In order to facilitate the statistical evaluation of receptivity over the 14day hormone treatment period, a Total Lordosis Quotient (TLQ) was calculated for each animal (TLQ = C LQ’s/no. of tests). On the morning after the last test the animals were sacrificed. Blood was rapidly collected from the abdominal aorta under ether anesthesia, using heparinized syringes. Uteri were weighed after the removal of any luminal fluid. The plasma samples were assayed in triplicate for LH by radioimmunoassay (Niswender, Midgley, Monroe, and Reichert, 1968). Plasma LH values are reported in terms of the standard used: NIH LH RP-1 (potency = *Simonscn’s
Laboratories,
Gilroy,
CA.
ESTROGEN
SENSITIVITY
AFTER
SPAYING
271
0.03 NIH LH S-l). Samplesin which LH was nondetectable were assigned values of 10 rig/ml (sensitivity of the assayat 200 ~1 plasma). Unlessotherwise indicated, Student’s t test was used for statistical comparisons and all probabilities indicated are two-tailed. The null hypothesis was accepted fox P> 0.05. Experiment I-LD.
Seventeenof 35 60-day-old female rats were brlater ally ovariectomized (restoration group), and the remaining 18 rats were ovariectomized 6 weeks later (maintenance group). Estrogen replacement therapy was initiated for both groups on the day after ovariectomy of the maintenance group and consisted of 14 daily SCinjections of 17 0 estradiol benzoate (EB) in approximately 0.1 ml of sesame oil. Maintenance and restoration groups each received 0.1 or 0.4 pg EB/ 100 g body weight, and twc control groups each received 0.1 ml sesameoil. The doses of EB used are considered to produce circulating estrogen levels within the “physiological” range for Long-Evansrats (Davidson, Smith, Rodgers,and Bloch, 1968). Experiment 2-LL-LD. Fifty-two 60-day-old rats were placed in condi. tions of constant illumination, and eight others of the same age were left in cycling light. Sixty days later approximately 50% of the animals in both LL and LD groups were bilaterally ovariectomized (restoration), and 6 weekslater the remaining animals were ovariectomized (maintenance). On the next morning EB injections and testing were initiated as described above. The LD animals were included to control for the circumstance that the rats used in this experiment were 2 months older than those in Experiment 1, due to the period of exposure to LL. RESULTS
Female Sexual Behavior Experiment 1-LD. None of the oil-injected control animals showed lordosis behavior on any of the tests. Figure 1 shows the mean LQ values for the four groups receiving estrogen treatment and Table 1 shows TLQ values. With the low dose of EB (0.1 c(g/lOOg) no lordoses occurred until Day 6 of treatment, and the mean LQ on any one test never exceeded40 for either the maintenance or the restoration groups (Fig. 1A). The 0.4qg dose, on the other hand, produced a more rapid onset of sexual responsivenessand higher levels of receptivity peaking at LQ levels similar to those found in normally estrous females or ovariectomized animals treated with higher dosesof steroid (Kuehn and Beach, 1962; Davidson et al., 1968). At both dose levels, the mean LQ was higher for the maintenancethan for the restoration groups on every test where lordoses were present (Fig. 1). Mean TLQ (Table 1) was significantly greater for the maintenance group al
272
DAMASSA AND DAVIDSON TABLE I Effects of Estradiol Benzoate (EB) on Sexual Receptivity and Uterine Weight: Experiment 1 Uterine wt. (mg)
Dose EB
N
LD3 Maintenance
Oil 0.1 0.4
4 I 7
0 19.8 f 5.3 61.2 f 5.6
104 f 8.02 291.5 f 10.3 323.9 f 21.0
LD Restoration
Oil 0.1 0.4
3 7 7
0 11.4 + 6.3 35.5 i 3.2
66.5 f 7.0 236.0 i 30.0 280.2 i 12.2
~.-
TLQl
~[No. lordoses on all tests/no. mounts] X 100. 2Mean f SE. 3LD: cycling light.
the 0.4pg dose (P< 0.01). The difference was not significant for the O.l#g groups presumably due to the consistently low level of receptivity exhibited by these animals. By the end of the period of estrogen treatment, maintenance and restoration groups had approached similar mean LQ values, although the dose difference persisted. Experiment 2-LL-LD. In the control (oil-treated) groups of animals kept in constant Light, lordosis behavior (LQ = 80) was observed in only one animal (maintenance group) on Day 1 of testing. As in Experiment 1, mean LQ levels did not rise as rapidly and/or did not reach as high levels in the O.las in the 0.4pg groups; and in all cases (LL 0.1 pg and 0.4 pg; LD 0.4 pg) the responsiveness under maintenance and restoration conditions approached equality by the last day of testing. As in Experiment 1, the three maintenance groups studied had higher mean LQ levels on every test (including the first of the series) except the last one (Fig. 2). As shown in Table 2, for all 3 pairs of groups mean TLQ was significantly higher in the maintenance than in the restoration condition (P< 0.05 for 0.1 pg, LL; and P< 0.01 for 0.4 pg, LL and 0.4 pg, LD). There were, however, no significant differences at the 0.4~c(g level between the groups of constant light-exposed rats and the corresponding cycling light animals (Two factor analysis of variance, P> 0.05 for lighting conditions and <0.01 for maintenance vs restoration). Thus, while lordosis behavior was invariably higher in Experiment 2 than Experiment 1 (Tables 1 and 2) this cannot be attributed to lighting conditions, but presumably to the difference in ages of the animals.
273
ESTROGEN SENSITIVITY AFTER SPAYING SO (A)
0.1 pp
60
EBllOOpm
40
40
-
- --
M-
Maintenance Rtrtorotion
l I’ 0.
0
1 2
4
6
s
lo
12
14
Days
Fig. 1. Effects of postcastration interval on estrogenactivated sexual behavior in female rats (Experiment 1). EB injections were given each day throughout the period of testing. Maintenance: estradiol benzoate (EB) treatment from the day after ovariectomy. Restoration: treatment commencing 6 weeks after ovariectomy. L/M: lordoses/mounts. Vertical lines indicate single standard errors.
Luteinizing Hormone Measurements Experiment I-LD. Mean plasma LH values for this experiment are shown in Fig. 3. The oil-injected control groups showed markedly higher LH levels for the restoration than for the maintenance paradigm, as expected in view of the longer postcastration interval. This difference was maintained with both low and high doses of EB (restoration vs maintenance, P< 0.05 in both cases). In none of the groups was plasma LH suppressed to the basal level found in cycling rats, which in this laboratory is 20-30 &ml at times in the cycle other than the afternoon of proestrus (Smith, Bowers, and Davidson, 1973).
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274 KJ-
O.Ipg
El31lOOgm
------
Mointenonce
--
Rerlorotion
(6) 0.4~9 0
4
6
6
IO
EB/lOOgm
12
I4
Days
Fig. 2. Behavioral observations, Experiment 2. (A) Effects of 0.1 pg EB/lOO g/day on sexual receptivity: maintenance and restoration conditions, constant light. (B) Effects of 0.4 pg/lOO g/day on sexual receptivity in constant (LL) or cycling (LD) light.
Experiment 2-LL-LD. Results are shown in Fig. 4. As in Experiment 1, the maintenance groups show significantly lower plasma LH than the restoration groups for all treatments (P< 0.01). Two-factor analysis of variance on the LL and LD, 0.4~c(g groups revealed significance for conditions of lighting (P< 0.05) and maintenance vs restoration (P < 0.01). Comparisons on individual groups showed significantly lower LH levels in the LL than the LD condition. P was < 0.01 for the maintenance group and < 0.05 for restoration (t tests). In order to justify comparisons between the two experiments, a two-factor analysis of variance was performed on the LD 0.4~pg groups of Experiment 2 and the similarly dosed groups of Experiment 1. No significant differences in plasma LH were found between the experiments. Paired comparisons were then made between the LD groups of Experiment 1 and the
FSTROGEN
SENSITIVITY TABLE
AFTER
275
SPAYING
2
Effects of Estradiol Benzoatc(EB) on Sexual Receptivity and Uterine Weight: Experiment 2 --
.-
LLl Maintenance
DoseEB -_ Oil 0.1
TLC’ --.-
1
2.0 f 2.0 41.6 f 6.9 79.7 f 3.0
182.5 f 22.1 418.7 f 23.8 413.0 f 25.1
0 22.2 f 5.5 61.4 f 5.3
102.1 f 14.4 290.7 f 10.9 373.1 f 20.1
10 10
0.4 LL
Oil
6
Restoration
0.1
10
0.4
9
85.0 LD
4
Restoration
Uterine wt. (mg) --.--
N
-f 1.9
56.5 + 8.3
362.0 f 5 1.9 301.7
f. 18.9
1LL: constantlight.
r
-
EB dose
0 PLO’4
700
Fig. 3. Effects of estrogentreatment on plasmaLH in spayedrats, Experiment 1. LL groups of Experiment 2. For all six comparisons mean plasma LH was higher in LD than in LL. This difference was significant in five of the six cases (P< O.Ol), the one exception being the O.l+g restoration comparison. Even though the animals of Experiment 2 were 60 days older than those of Experiment 1, these comparisons are presumed to be valid due to the
DAMASSA AND DAVIDSON
276 500 -
100 -
EE dose :
0 P9’d
.I pL9/d
.4/g/d
.4 /LQld
Fig. 4. Plasma LH values from Experiment 2. Effects of different dose regimens of estrogen on plasma LH in rats exposed to cycling or constant light.
similarity of values in the LD 0.4~pg groups of Experiment identically treated but younger rats of Experiment 1.
2 and the
Uterine Weight Experiment I-LD. Table 1 shows the mean uterine weights for the different groups of this experiment. In all cases the maintenance groups showed heavier uteri than the restoration. This difference was, however, only significant (P< 0.01) in the oil-injected controls. Experiment 2-LL-LD. As in Experiment 1, mean uterine weights were greater in the maintenance groups, in aII cases (Table 2). In the LL condition these differences were significant after treatment with oil (P< 0.05) 0.1 pg EB (P< 0.001) and 0.4pg EB (P< 0.01) injections. The LD (0.4c(g EB) animals did not show significant differences. The uterine weights of the LD groups (0.4 pg) were lower than those of the comparable LL groups. Two-factor analysis of variance on these data revealed significant differences for maintenance vs restoration (P < 0.01) as well as for lighting conditions (P < 0.05).
DISCUSSION The primary question to which this study addressed itself-whether there is a change in sensitivity to the lordosis behavior-activating effect of estrogen with increasing time after ovariectomy-,was clearly answered. In each of five conditions under which the lordosis response to EB injections was studied, the
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treatment was more effective when initiated shortly after surgery than when a delay of 6 weeks intervened. In this respect, estrogen effects on female sexual behavior resemble testosterone effects in the male (see Davidson, 1972). It should be noted, however, that while in the present study 2 weeks of EB administration sufficed to obliterate differences in lordosis behavior between the maintenance and restoration conditions, significant differences were still present in the male after 8 weeks of daily treatment with testosterone propionate (Davidson, 1972). Estradiol benzoate replacement therapy produced no significant differences in lordosis behavior between constant light- and cycling light-exposed animals when all conditions except light exposure were similar (Experiment 2). The greater responsiveness of animals in Experiment 2 as compared to Experiment 1 was thus presumably due to their being 2 months older. In both experiments, feedback suppression of LH secretion by EB was also investigated. It was not surprising that the oil-treated controls for the restoration groups showed higher plasma LH levels than for the maintenance groups, since the restoration controls had been deprived of ovarian steroids for 8 weeks compared to only 2 weeks in maintenance groups, and plasma LH shows a steadily increasing rise after ovariectomy (Gay and Midgley, 1969 and unpublished experiments in this laboratory). If we attempt to compare estrogen feedback sensitivity under the two conditions, the difficulty arises that comparisons are made from different starting levels of plasma LH. Nevertheless, LH was measured only after 14 days of EB injection. Since a single dose of 0.5 c(g EB/rat has been found to significantly reduce plasma LH 24 hr after injection (Ajika, Krulich, Fawcett, and McCann, 1972), one might expect a 14-day treatment with larger doses to have obliterated initial differences in LH between the maintenance and restoration groups, as was the case for differences in behavior. That the differences remained after 2 weeks of treatment suggests that LH is in fact less suppressible in long-term castrates than in the maintenance situation. However, further work is needed to substantiate this conclusion; the experimental design used was primarily directed at the behavioral question. Similar considerations apply to the observation that plasma LH was apparently more suppressible by EB in constant light-exposed than in cycling light-exposed animals. The control group data from that experiment also indicate that after ovariectomy plasma LH rises more slowly and/or plateaus earlier in constant light than in cycling light. This conclusion is consistent with data presented recently by. Tapper, Naftolin, and Brown-Grant (1972). We conclude that either constant light has a direct inhibitory effect on LH secretion, or that the altered steroid environment in constant light-exposed animals has an effect on the capacity for LH release which persists after ovariectomy. This study provides no information on the mechanism underlying the
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postcastration changes observed. Although a number of models are possible, our preferred interpretation is that receptor mechanisms in the relevant target tissues decline in sensitivity as time elapses after removal of the trophic hormone, possibly due to atrophy of certain structures and/or breakdown of biosynthetic mechanisms. We recognize that the definition of sensitivity is problematic, particularly when responses are measured from different starting points as seen in the above discussion of the LH data. Although the LQ level of the controls was the same in maintenance and restoration conditions (i.e., zero), this would probably not be the case were it possible to measure the “substrate” for the behavioral action of estrogen. Our concern in this report is, in fact, merely to show that differences exist in relation to time after ovariectomy, as measured by behavioral, LH, and uterine weight responses. Two weeks of EB treatment appears to reverse the change for lordosis behavior, but not for LH and uterine weight.
ACKNOWLEDGMENTS Radioimmunoassay reagents were kindlyssupplied by Drs. G. D. Niswcndcr, L. E. Reichert, and A. F. Parlow (NIAMD Rat Pituitary Hormone Program). REFERENCES Ajika, K., Krulich, L., Fawcett, C. P., and McCann, S. M. (1972). Effects of estrogen on plasma and pituitary gonadotropins and prolactin, and on hypothalamic releasing and inhibiting factors. Neuroendocrinology 9, 304-315. Clar, J., Massons, J. M., and Robust&T. (1967). Action de 10s andr6genos sobre prdstata y vesiculas scminales en la rata macho castrada. Rev. &pan. Fisiol. 23, 115-t 16. Davidson, J. M. (1969). Feedback regulation of gonadotropin secretion. In W. F. Ganong and L. Martini (Eds.), Frontiers in Neuroendocrinology, pp. 343-388, Oxford University Press, New York. Davidson, J. M. (1972). Hormones and reproductive behavior. In H. Balin and S. Glasser @As.), Reproductive Biology, pp. 877-918, Excerpta Medica, Amsterdam, The Netherlands. Davidson, J. RI., and Bloch, C. J. (1969). Neuroendoerine aspects of male reproduction. Biol. Reprod. I, Suppl. 1, 67-92. Davidson, J. M., Smith, E. R., Rodgers, C. H., and Bloch, G. J. (1968). Relative thresholds of behavioral and somatic responses to estrogen. Physiol. Behov. 3, 227-229. Gay, V. L., and Midgley, A. R. (1969). Response of the adult rat to orchidectomy and ovariectomy as determined by LH radioimmunoassay. Endocrinology 84, 1359-1364. Hardy, D. F. (1970). The effect of constant light on the estrous cycle and behavior of the female rat. Physiol. Behav. 5, 421425. Kuehn, R. E., and Beach, F. A. (1962). Quantitative measurement of sexual receptivity in female rats. Behaviour 21, 282-299. Liddle, G. W., Island, D., and Meador, C. K. (1962). Normal and abnormal regulation of corticotropin secretion in man. Rec. Progr. Horm. Res 18, 125-153.
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Naftolin, F., Brown-Grant, K., and Corker, C. S. (1972). Plasma and pituitary luteinizing hormone and peripheral plasma oestradiol concentrations in the normal oestrous cycle of the rat and after experimental manipulation of the cycle. /. EndocrinoZ. 53, 17-30, 1972. Niswender, G. D., Midgley, A. R., Jr., Monroe, S. E., and Reichert, L. E., Jr. (1968). Radioimmunoassay for rat luteinizing hormone with antiovine LH serum and ovine LH-’ 3’ 1. Proc. Sot. Exp. Biol. Med. 128, 807-81 I. Smith, E. R., Bowers, C. Y., and Davidson, J. M. (1973). Circulating levels of plasma gonadotropins in 4- and S-day cycling rats. Endocrinology, in press. Tapper, C. M., Naftolin, F., and BrownGrant, K. (1972). Influence of the reproductive state at the time of operation on the early response to ovariectomy in the rat. J. Endocrinol.
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Young, W. C. (1961). The hormones and mating behavior. In W. C. Young (Ed.), Sex and Internal Secretions, Vol. II, p. 1184, Williams & Wilkins, Baltimore.