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Facilitatory and inhibitory effects of supplementary estradiol benzoate given to ovariectomized, estrogen-primed guinea pigs
Hormones and Behavior 2, 307-314 (1971)
Facilitatory and Inhibitory Effects of Supplementary Estradiol Benzoate Given to Ovariectomized, Estrogen-Primed Guinea Pigs W. D. JOSLYN and H. H. FEDER’ Oregon Regional Primate Research Center, Department of Reproductive Physiology and Behavior, Beaverton, Oregon 9 7005
Ovariectomizcd guinea pigs were given a priming dose of estradiol bcnzoate followed 18, 36, or 96 hr later by concurrent injections of supplementary estradiol benzoate plus progesterone. The 36-hr group showed a prolongation of duration of estrus while the 18- and 96-hr groups showed an increased latency to response. These results show that the effect of supplementary estradiol is dependent upon the time interval permitted for priming. An additional study (using the 36-hr group) in which the supplementary estradiol was given at various times in relation to progcsteronc indicated that the estrogenic mechanisms regulating latency and duration of heat are experimentally separable. Latency seems primarily dependent on the amount of estrogen given as a priming dose. Duration, on the other hand, seems primarily dependent on the amount of estrogen available at the time of progesterone injection providing that the priming interval is optimal (i.e. 36 hr).
The priming dosage of estrogen given to ovariectomized guinea pigs to induce lordosis behavior is positively correlated with the duration and inversely related to the latency of this response when progesterone is injected 36 hr after the estrogen (Collins, Boling, Dempsey, and Young, 1938; Goy and Young, 1957). The sum of the latent period and the duration of estrus remains fairly constant (the latency and duration being inversely related to each other) indicating that latency and duration are measures of the same underlying physiological state and that both are controlled by estrogen dosage through the same mechanism. But the dosage of estrogen given to prime the ovariectomized animal is not the only hormonal variable affecting the quality of the lordosis response. The optimal interval between estrogen and progesterone injection of spayed guinea pigs is 24-48 hr (Dempsey, Hertz, and Young, 1936). If this interval is reduced to 18 hr or increased to 96 hr the latency to response after progesterone increases and the duration of heat decreases. If the interval is yet further reduced (e.g., 6 hr) or increased (e.g., 1Please address all correspondence to: II. H. Feder, Institute of Animal Behavior, Rutgers University, 101 Warren Street, Newark, NJ 07102.
307
308
JOSLYN AND FEDER
240 hr) very few animals display lordosis (Goy and Phoenix, 1965; Zucker, 1966; G. Eaton, J. A. Resko, and R. W. Goy, unpublished data). A further complication in the hormonal control of lordosis is introduced by the possibility that estrogen has a role in addition to that of priming or conditioning. Collins et al. (1938) found that when guinea pigs which had been estrogen-primed for 48 hr were given progesterone they had a latency of 3.4 hr and a duration of heat of 9.3 hr. However, when such animals were given a supplementary intravenous injection of an estrogenic extract (“theelin”) simultaneously with the progesterone the latency declined to 2.7 hr and the duration increased to 15.9 hr. The present experiments were designed to investigate this action of supplementary estrogen more fully by using standardized testing techniques and by systematically varying estrogen dosages and time intervals between estrogen and progesterone treatments.
METHODS AND RESULTS General Procedures Adult female guinea pigs (N= 77) of a heterogenous stock (Topeka) were provided with food and water ad libitum in a room illuminated from 5 AM to 5 PM daily. They were maintained in groups of 8-10 animals per cage. Animals were ovariectomized (OVX) under ether or methoxyflurane (Metofane, Pitman-Moore) anesthesia at least 2 weeks prior to hormone treatment. All injections were given subcutaneously in 0.1 ml of sesame oil vehicle. When two injections were given “concurrently” they were given within 60 set of each other at different injection sites. Lordosis was detected and quantified by a manual stimulation technique (Young, Dempsey, Hagquist, and Boling, 1937). Tests for lordosis began at the time of progesterone injection and continued at hourly intervals until the subject no longer displayed lordosis. To be classified as “estrous” or “in heat” a subject had to exhibit a repeatable lordosis response of at least 1-set duration for two or more successive tests. Values for latency (l-n), length of maximum lordosis (set) and duration of heat (hr) were computed only for animals which met the criteria for being estrous. As far aspossible the samesubjects were used during four successive experiments, but not eiery individual participated in all of the four experiments. In a few cases animals had to be dropped from the study because of illness and new animals used as replacements. At least 3 weeks intervened between experiments, and when very high doses of estrogen were used (Experiment II) 10 weeks intervened. The chronological order in which the experiments were conducted was: I, IV, II, and III. Experiment I. Two groups of 10 OVX females each were given 3 pg estradiol benzoate (EB, Progynon, Schering) at hr 0 followed by 0.4 mg proges-
309
ESTROGEN AND LORDOSIS BEHAVIOR TABLE 1 Effect on Lordosis of Supplementary Estradiol Benzoate (3 pg at Hour 36) Given to Ovariectomized Guinea Pigs Treated with Estradiol Benzoate (EB) and Progesterone (P) Treatment (hr) 0
3MgEB
36 0.4mgP
0.4mgP f 3/aEB P value by t test 3figEB
N
% Animals in heat
Latency (hi-) 2+-SEM
+0.1
10
100
2.1
10
100
2.6 +O.l >0.30
Maximum lordosis (set) XrSEM
Duration of &at (hr) X+-SEM
18.2 +2.9
8.8 +0.3
24.1 I!I4.4 >0.20
12.2 f0.5 < 0.001
terone (P, Proluton, Schering) at hr 36. One group received no further treatment while the other group received 3 pg supplementary EB subcutaneously at hr 36 (concurrently with progesterone). The supplementary EB at hr 36 increased the duration of heat by 3.4 hr (P < 0.001) but did not affect latency or the length of the maximum lordosis (Table 1). Experiment I% The results of Experiment I indicated that duration of heat is dependent on the level of estrogen present in blood and/or brain at the time of progesterone administration. This possibility was tested in the following way. Forty-nine OVX guinea pigs were given 3 pg EB at Hour 0 and 0.4 mg P at Hour 36. The subjects were divided into five groups and given various dosages of supplementary EB. Group I (N= 9) was given no supplementary EB (controls); Group II (N= 10) received 9 1.18supplementary EB at Hour 0; Group III (N = 10) was given 48 yg of additional E3 at Hour 0; Group IV (N= 10) received 48 pg EB divided into three injections of 16 pg each at Hours 0, 12, and 24; Group V (N = 10) received 48 pg EB at Hour 36, concurrently with progesterone. The duration of heat was 8.3 hr for the control group (I) and increased steadily in Groups II through V to a mean of 16.4 hr (Table 2). The differences were significant by analysis of variance (F (4,44) = 6.25; P < 0.001). The results of t tests between individual means are shown in Table 2. Latency to onset of heat was almost identical for the control group (I) and the group given 48 pg EB at Hour 36 (V). For the groups given additional before Hour 36 (II, III, and IV), the latencies for heat decreased from control level (J? {4,44) = 3.39; P < 0.025; Table 2). Maximum lordosis was unaffected by any of the supplemental EB treatments. Experiment III. The interval of 36 hr between the priming dose of E and injection of P used in Experiment I and II is such that the animals are
16 ng EB
3fi~gEB + 48 fig EB
3eEB + .16pg EB
3pgEB
II
III
IV
V
P < 0.05 by t test
Means differing at
-
3/.%EB + gr*gEB
-
-
3pgEB
I
12
0
Group
10
0.4 mg P + 48 fig EB
-
-
10
10
0.4mgP
0.4mgP
10
9
N
16 /.qgEB
-
0.4mgP
0.4 mg P
-
36
24
Treatment (hr)
-
100
100
100
100
100
% Animals in heat
I vs II,III,IV III vs v IVVSV
3.4 f0.3
2.7 2 0.2
2.8 +-0.1
2.9 + 0.2
3.4 +0.2
Latency (W F+sEM
ns by analysis of variance
13.1 i: 1.6
14.7 f 1.7
11.9 rt 1.6
14.4 -+2.2
15.0 + 1.6
Maximum lonjosis (set) XkSEM
Effect on Lordosis of Supplementary E&radio1 Benzoate Given in Various Doses and at Various Times to Ovariectomized Guinea Pigs Treated with Estradiol Benzoate (EB) and Progesterone (P)
TABLE 2
I vs III,IV,V II vs IV II vs v
16.4 + 1.9
14.8 + 1.3
11.9 f 1.1
10.3 rt 0.9
8.3 f 0.8
Duration of &eat (hr) X-+SEM
Pl
E
3 F 2
311
ESTROGEN AND LORDOSIS BEHAVIOR
“optimally primed” to respond with a display of lordosis behavior. Experiment III was performed to determine how animals which are not optimally primed respond to injection of supplementary EB. An interval of 18 hr between priming dose of EB and injection of P was chosen. With this time interval most animals display lordosis, but the latencies are longer and the durations of heat shorter than with the standard 36-hr interval (Collins et al, 1938; G. Eaton, J. A. Resko, and R. W. Goy, unpublished data). Forty-nine OVX females were given 3 pg EB at Mour 0 and 0.4 mg P at Hour 18. The subjects were randomly assigned to three groups. Control animals (M = 17) were given no supplementary EB; one experimental group was given 3 1-18supplementary EB at Hour 18, concurrently with P (N = 16); another experimental group was given 12 pg supplementary EB at Hour 18 concurrently with P (N = 16). Almost all animals in the three groups responded(88-94%) by displaying behavioral estrus (Table 3). The only statistically reliable effect was an increased latency to heat for the 12-gg group (F (2,42) = 6.81; P < 0.005) which differed from both the control and the 3-pg supplementary EB groups (t test subsequent to analysis of variance: P < 0.01 and P < 0.02, respectively). Experiment IV. Wewished to determine whether the effects of supplementary EB given at the time of priming were incomplete by dint of the interval bctween the priming doseof EB and administration of P being too long rather than too short (e.g., 18 hr as in Experiment III). Fifty-one OVX guinea pigs were given 3 yg EB at Hour 0 and 0.4 mg P at Hour 96. The females were then TABLE 3 Effect on Lordosis of Varying Doses of Supplementary Estradiol Benzoate at Hour 18 Given to Ovariectomized Guinea Pigs Treated with Estradiol Benzoate (EB) and Progesterone (P) Treatment (hr) Group I
0 3figEB
II
3/aEB
III
3 l.lg
Maximum Iordosis (set) X+SEM
Duration of *at (hr) XTSEM
18
N
0.4 mgP
17
94
5.2 f0.1
8.8 + 0.9
6.2 c 0.5
16
94
5.5 20.2
8.6 f 1.6
6.1 +O.7
16
88
6.2 2~0.3
8.9 + 1.0
7.0 kO.5
-
-
I vs III II vs III
ns by analysis of variance
ns by analysis of variance
0.4mgP + 3/aEB 0.4 mg P + 12 pg EB
Means differing at
P
Latency % Animals _ (hr) in heat X+SEM
312
JOSLYN AND FEDER TABLE 4 Effect on Lordosis of Varying Doses of Supplementary Estradiol Benzoate Given at Hour 96 to Ovariectomized Guinea Pigs Treated wtih Estradiol Benzoate (EB) and Progesterone (P) Treatment (hr)
Group I
0 3pgEB
II
3pgEB
III
3ngEB
Means differing at P
Latency % Animals (hr) in heat ~-+SEM
Maximum lordosis (set) X+SEM
Duration of heat (hr) X-+SEM
96
N
0.4mgP
15
87
6.4 + 0.1
8.6 +1.4
3.8 + 0.3
20
70
7.3 +0.1
6.0 f 0.9
5.5 f0.6
16
69
8.9 f 0.3
6.1 f 0.8
4.9 +0.7
-
-
I vs 11,111 ns by analysis of variance II vs III
0.4 mg P + 3/.acEB 0.4 mg P f 12 pg EB
ns by analysis of variance
divided into three groups. Group I was given no supplementary EB (controls, N= 15); Group II was given 3 gg supplementary EB at Hour 96 (N= 20) and Group III was given 12 yg EB at Hour 96 (N = 16). The percentage of subjects showing heat in the three groups was 87, 70, and 69, respectively (Table 4). These percentages did not differ significantly using a chi-square test. However, both the 3- and the 12-ng supplementary EB treatments significantly increased the latency to heat (F (2,35) = 33.60; P < 0.001). Subsequent t tests revealed that the control group differed from both the 3+g group (P < 0.01) and the 12-pg group (P < 0.001) and the 3- and 12,ug groups also differed from one another (P < 0.001). Thus, the effect of additional EB on latency under the conditions of this experiment was dose dependent.
DISCUSSION Ovariectomized guinea pigs given estrogen priming followed at selected intervals by progesterone also recieved supplementary injections of estrogen in various doses and ‘at various times relative to progesterone injection. When the interval between the priming estrogen and the progesterone was 36 hr (“optimal” priming) the only detectable effect of supplementary estrogen given concurrently with progesterone was facilitatory in that the duration of heat was prolonged (Experiment I). This partially confirms the earlier work of Collins et al. (1938). In contrast to these authors, we were unable to detect a shortening of the latency to heat. Collins et al. used an intravenous injection
ESTROGEN
AND LORDOSIS
BEHAVIOR
313
route and administered unesterified estrogen. Both of these factors would favor speedier incorporation of estrogen into brain tissues mediating behavior than our subcutaneous injection of esterified estradiol. However, even when we attempted to compensate for this by administering estradiol benzoate (SC) 2 hr before progesterone latency was not shortened while duration of heat was significantly prolonged (W. D. Joslyn, unpublished data). When the interval between the priming dose of estrogen and the injection of progesterone was either shortened to 18 hr or lengthened to 96 hr (“non-optimal” priming) there was no facilitator-y action of supplementary estradioi on duration of heat (Experiments III and IV). Instead, the oniy apparent effect was inhibitory in that latency to heat was lengthened. Furthermore, this inhibitory effect of supplementary estradiol was strengthened with increasing estradiol dosage. The mechanisms mediating these paradoxical facilitatory and inhibitory actions of supplementary estradiol are unknown. The supplementary estradiol could be affecting (a) the conditioning process set in motion by the earlier priming dose of estrogen, (b) the uptake or metabolism of the concurrently administered progesterone, or (c) neural sites not previously conditioned by the priming dose of estradiol and not concerned with progesterone action. It is even uncertain that the supplementary estradiol given at nonoptimal priming intervals (18 or 96 hr) exerts its inhibitory action in similar ways during both intervals. Some clarification of the manner in which supplementary estradiol influences lordosis may be obtained through studies of brain uptake of radioactive steroids. The data from Experiment 11 suggest overlapping, but separable mechanisms through which estrogen determines the latency to heat and the duration of heat in optimally primed animals. Latency appears to be regulated by the amount of estrogen present during the early stages of the priming process, since supplementary estradiol given during the first 24 hr after initial priming decreased latency, but estradiol given in the later stages of the priming process at 34 hr (W. D. Joslyn, unpublished data) or at 36 hr had no effect on latency. On the other hand, duration of heat seems to be modulated by the amount of estrogen present near the end of the priming process at 36 hr. Thus, if the groups in Experiment 11 are arranged in the order of amount of estradiol logically expected to be present at Hour 36: it can be seen that duration of heat increased with increases in the expected amount of estrogen.
ACKNOWLEDGMENTS This work was supported by Grant HD-04467 to H. H. F. from the National Institute of Child Health and Human Development and by a Career Development Award (K2-MH-29006) to H. H. F. from the National Institute of Mental Health. We thank Dr. R. W. Coy and Dr. C. H. Phoenix for their encouragement. Publication number 552 of the Oregon Regional Primate Research, Center. Additional funds supporting this work were provided hy Grant MH-21312 to R. W. Coy and Grant PR-11063 to the ORPRC.
314
JOSLYN AND FEDER REFERENCES
Collins, V. J., Boling, J. L., Dempsey, E. W., and Young, W. C. (1938). Some quantitative studies of experimentally induced sexual receptivity in spayed guinea pigs. Endocrinology 23, 188-196. Dempsey, E. W., Hertz, R., and Young, W. C. (1936). The experimental induction of oestrus (sexual receptivity) in the normal and ovariectomized guinea pig. Amer. J. Physiol.
116, 201-209.
Goy, R. W., and Phoenix, C. H. (1965). Inhibitory actions of progesterone. Amer. Zool. 5, 725 (abstr.). Goy, R. W., and Young, W. C. (1957). Strain differences in the behavioral responses of female guinea pigs to alpha-estradiol benzoate and progesterone. Behavior 10, 340-354.
Young, W. C., Dempsey, E. determination of heat in Zucker, 1. (1966). Facilitatory of spayed guinea pigs. J.
W., Hagquist, C. W., and Baling, J. L. (1937). The the guinea pig. J. Lab. Clin. Med. 23, 300-302. and inhibitory effects of progesterone on sexual responses Comp. Physiol. Psychol. 62, 376-381.
Facilitatory and Inhibitory Effects of Supplementary Estradiol Benzoate Given to Ovariectomized, Estrogen-Primed Guinea Pigs W. D. JOSLYN and H. H. FEDER’ Oregon Regional Primate Research Center, Department of Reproductive Physiology and Behavior, Beaverton, Oregon 9 7005
Ovariectomizcd guinea pigs were given a priming dose of estradiol bcnzoate followed 18, 36, or 96 hr later by concurrent injections of supplementary estradiol benzoate plus progesterone. The 36-hr group showed a prolongation of duration of estrus while the 18- and 96-hr groups showed an increased latency to response. These results show that the effect of supplementary estradiol is dependent upon the time interval permitted for priming. An additional study (using the 36-hr group) in which the supplementary estradiol was given at various times in relation to progcsteronc indicated that the estrogenic mechanisms regulating latency and duration of heat are experimentally separable. Latency seems primarily dependent on the amount of estrogen given as a priming dose. Duration, on the other hand, seems primarily dependent on the amount of estrogen available at the time of progesterone injection providing that the priming interval is optimal (i.e. 36 hr).
The priming dosage of estrogen given to ovariectomized guinea pigs to induce lordosis behavior is positively correlated with the duration and inversely related to the latency of this response when progesterone is injected 36 hr after the estrogen (Collins, Boling, Dempsey, and Young, 1938; Goy and Young, 1957). The sum of the latent period and the duration of estrus remains fairly constant (the latency and duration being inversely related to each other) indicating that latency and duration are measures of the same underlying physiological state and that both are controlled by estrogen dosage through the same mechanism. But the dosage of estrogen given to prime the ovariectomized animal is not the only hormonal variable affecting the quality of the lordosis response. The optimal interval between estrogen and progesterone injection of spayed guinea pigs is 24-48 hr (Dempsey, Hertz, and Young, 1936). If this interval is reduced to 18 hr or increased to 96 hr the latency to response after progesterone increases and the duration of heat decreases. If the interval is yet further reduced (e.g., 6 hr) or increased (e.g., 1Please address all correspondence to: II. H. Feder, Institute of Animal Behavior, Rutgers University, 101 Warren Street, Newark, NJ 07102.
307
308
JOSLYN AND FEDER
240 hr) very few animals display lordosis (Goy and Phoenix, 1965; Zucker, 1966; G. Eaton, J. A. Resko, and R. W. Goy, unpublished data). A further complication in the hormonal control of lordosis is introduced by the possibility that estrogen has a role in addition to that of priming or conditioning. Collins et al. (1938) found that when guinea pigs which had been estrogen-primed for 48 hr were given progesterone they had a latency of 3.4 hr and a duration of heat of 9.3 hr. However, when such animals were given a supplementary intravenous injection of an estrogenic extract (“theelin”) simultaneously with the progesterone the latency declined to 2.7 hr and the duration increased to 15.9 hr. The present experiments were designed to investigate this action of supplementary estrogen more fully by using standardized testing techniques and by systematically varying estrogen dosages and time intervals between estrogen and progesterone treatments.
METHODS AND RESULTS General Procedures Adult female guinea pigs (N= 77) of a heterogenous stock (Topeka) were provided with food and water ad libitum in a room illuminated from 5 AM to 5 PM daily. They were maintained in groups of 8-10 animals per cage. Animals were ovariectomized (OVX) under ether or methoxyflurane (Metofane, Pitman-Moore) anesthesia at least 2 weeks prior to hormone treatment. All injections were given subcutaneously in 0.1 ml of sesame oil vehicle. When two injections were given “concurrently” they were given within 60 set of each other at different injection sites. Lordosis was detected and quantified by a manual stimulation technique (Young, Dempsey, Hagquist, and Boling, 1937). Tests for lordosis began at the time of progesterone injection and continued at hourly intervals until the subject no longer displayed lordosis. To be classified as “estrous” or “in heat” a subject had to exhibit a repeatable lordosis response of at least 1-set duration for two or more successive tests. Values for latency (l-n), length of maximum lordosis (set) and duration of heat (hr) were computed only for animals which met the criteria for being estrous. As far aspossible the samesubjects were used during four successive experiments, but not eiery individual participated in all of the four experiments. In a few cases animals had to be dropped from the study because of illness and new animals used as replacements. At least 3 weeks intervened between experiments, and when very high doses of estrogen were used (Experiment II) 10 weeks intervened. The chronological order in which the experiments were conducted was: I, IV, II, and III. Experiment I. Two groups of 10 OVX females each were given 3 pg estradiol benzoate (EB, Progynon, Schering) at hr 0 followed by 0.4 mg proges-
309
ESTROGEN AND LORDOSIS BEHAVIOR TABLE 1 Effect on Lordosis of Supplementary Estradiol Benzoate (3 pg at Hour 36) Given to Ovariectomized Guinea Pigs Treated with Estradiol Benzoate (EB) and Progesterone (P) Treatment (hr) 0
3MgEB
36 0.4mgP
0.4mgP f 3/aEB P value by t test 3figEB
N
% Animals in heat
Latency (hi-) 2+-SEM
+0.1
10
100
2.1
10
100
2.6 +O.l >0.30
Maximum lordosis (set) XrSEM
Duration of &at (hr) X+-SEM
18.2 +2.9
8.8 +0.3
24.1 I!I4.4 >0.20
12.2 f0.5 < 0.001
terone (P, Proluton, Schering) at hr 36. One group received no further treatment while the other group received 3 pg supplementary EB subcutaneously at hr 36 (concurrently with progesterone). The supplementary EB at hr 36 increased the duration of heat by 3.4 hr (P < 0.001) but did not affect latency or the length of the maximum lordosis (Table 1). Experiment I% The results of Experiment I indicated that duration of heat is dependent on the level of estrogen present in blood and/or brain at the time of progesterone administration. This possibility was tested in the following way. Forty-nine OVX guinea pigs were given 3 pg EB at Hour 0 and 0.4 mg P at Hour 36. The subjects were divided into five groups and given various dosages of supplementary EB. Group I (N= 9) was given no supplementary EB (controls); Group II (N= 10) received 9 1.18supplementary EB at Hour 0; Group III (N = 10) was given 48 yg of additional E3 at Hour 0; Group IV (N= 10) received 48 pg EB divided into three injections of 16 pg each at Hours 0, 12, and 24; Group V (N = 10) received 48 pg EB at Hour 36, concurrently with progesterone. The duration of heat was 8.3 hr for the control group (I) and increased steadily in Groups II through V to a mean of 16.4 hr (Table 2). The differences were significant by analysis of variance (F (4,44) = 6.25; P < 0.001). The results of t tests between individual means are shown in Table 2. Latency to onset of heat was almost identical for the control group (I) and the group given 48 pg EB at Hour 36 (V). For the groups given additional before Hour 36 (II, III, and IV), the latencies for heat decreased from control level (J? {4,44) = 3.39; P < 0.025; Table 2). Maximum lordosis was unaffected by any of the supplemental EB treatments. Experiment III. The interval of 36 hr between the priming dose of E and injection of P used in Experiment I and II is such that the animals are
16 ng EB
3fi~gEB + 48 fig EB
3eEB + .16pg EB
3pgEB
II
III
IV
V
P < 0.05 by t test
Means differing at
-
3/.%EB + gr*gEB
-
-
3pgEB
I
12
0
Group
10
0.4 mg P + 48 fig EB
-
-
10
10
0.4mgP
0.4mgP
10
9
N
16 /.qgEB
-
0.4mgP
0.4 mg P
-
36
24
Treatment (hr)
-
100
100
100
100
100
% Animals in heat
I vs II,III,IV III vs v IVVSV
3.4 f0.3
2.7 2 0.2
2.8 +-0.1
2.9 + 0.2
3.4 +0.2
Latency (W F+sEM
ns by analysis of variance
13.1 i: 1.6
14.7 f 1.7
11.9 rt 1.6
14.4 -+2.2
15.0 + 1.6
Maximum lonjosis (set) XkSEM
Effect on Lordosis of Supplementary E&radio1 Benzoate Given in Various Doses and at Various Times to Ovariectomized Guinea Pigs Treated with Estradiol Benzoate (EB) and Progesterone (P)
TABLE 2
I vs III,IV,V II vs IV II vs v
16.4 + 1.9
14.8 + 1.3
11.9 f 1.1
10.3 rt 0.9
8.3 f 0.8
Duration of &eat (hr) X-+SEM
Pl
E
3 F 2
311
ESTROGEN AND LORDOSIS BEHAVIOR
“optimally primed” to respond with a display of lordosis behavior. Experiment III was performed to determine how animals which are not optimally primed respond to injection of supplementary EB. An interval of 18 hr between priming dose of EB and injection of P was chosen. With this time interval most animals display lordosis, but the latencies are longer and the durations of heat shorter than with the standard 36-hr interval (Collins et al, 1938; G. Eaton, J. A. Resko, and R. W. Goy, unpublished data). Forty-nine OVX females were given 3 pg EB at Mour 0 and 0.4 mg P at Hour 18. The subjects were randomly assigned to three groups. Control animals (M = 17) were given no supplementary EB; one experimental group was given 3 1-18supplementary EB at Hour 18, concurrently with P (N = 16); another experimental group was given 12 pg supplementary EB at Hour 18 concurrently with P (N = 16). Almost all animals in the three groups responded(88-94%) by displaying behavioral estrus (Table 3). The only statistically reliable effect was an increased latency to heat for the 12-gg group (F (2,42) = 6.81; P < 0.005) which differed from both the control and the 3-pg supplementary EB groups (t test subsequent to analysis of variance: P < 0.01 and P < 0.02, respectively). Experiment IV. Wewished to determine whether the effects of supplementary EB given at the time of priming were incomplete by dint of the interval bctween the priming doseof EB and administration of P being too long rather than too short (e.g., 18 hr as in Experiment III). Fifty-one OVX guinea pigs were given 3 yg EB at Hour 0 and 0.4 mg P at Hour 96. The females were then TABLE 3 Effect on Lordosis of Varying Doses of Supplementary Estradiol Benzoate at Hour 18 Given to Ovariectomized Guinea Pigs Treated with Estradiol Benzoate (EB) and Progesterone (P) Treatment (hr) Group I
0 3figEB
II
3/aEB
III
3 l.lg
Maximum Iordosis (set) X+SEM
Duration of *at (hr) XTSEM
18
N
0.4 mgP
17
94
5.2 f0.1
8.8 + 0.9
6.2 c 0.5
16
94
5.5 20.2
8.6 f 1.6
6.1 +O.7
16
88
6.2 2~0.3
8.9 + 1.0
7.0 kO.5
-
-
I vs III II vs III
ns by analysis of variance
ns by analysis of variance
0.4mgP + 3/aEB 0.4 mg P + 12 pg EB
Means differing at
P
Latency % Animals _ (hr) in heat X+SEM
312
JOSLYN AND FEDER TABLE 4 Effect on Lordosis of Varying Doses of Supplementary Estradiol Benzoate Given at Hour 96 to Ovariectomized Guinea Pigs Treated wtih Estradiol Benzoate (EB) and Progesterone (P) Treatment (hr)
Group I
0 3pgEB
II
3pgEB
III
3ngEB
Means differing at P
Latency % Animals (hr) in heat ~-+SEM
Maximum lordosis (set) X+SEM
Duration of heat (hr) X-+SEM
96
N
0.4mgP
15
87
6.4 + 0.1
8.6 +1.4
3.8 + 0.3
20
70
7.3 +0.1
6.0 f 0.9
5.5 f0.6
16
69
8.9 f 0.3
6.1 f 0.8
4.9 +0.7
-
-
I vs 11,111 ns by analysis of variance II vs III
0.4 mg P + 3/.acEB 0.4 mg P f 12 pg EB
ns by analysis of variance
divided into three groups. Group I was given no supplementary EB (controls, N= 15); Group II was given 3 gg supplementary EB at Hour 96 (N= 20) and Group III was given 12 yg EB at Hour 96 (N = 16). The percentage of subjects showing heat in the three groups was 87, 70, and 69, respectively (Table 4). These percentages did not differ significantly using a chi-square test. However, both the 3- and the 12-ng supplementary EB treatments significantly increased the latency to heat (F (2,35) = 33.60; P < 0.001). Subsequent t tests revealed that the control group differed from both the 3+g group (P < 0.01) and the 12-pg group (P < 0.001) and the 3- and 12,ug groups also differed from one another (P < 0.001). Thus, the effect of additional EB on latency under the conditions of this experiment was dose dependent.
DISCUSSION Ovariectomized guinea pigs given estrogen priming followed at selected intervals by progesterone also recieved supplementary injections of estrogen in various doses and ‘at various times relative to progesterone injection. When the interval between the priming estrogen and the progesterone was 36 hr (“optimal” priming) the only detectable effect of supplementary estrogen given concurrently with progesterone was facilitatory in that the duration of heat was prolonged (Experiment I). This partially confirms the earlier work of Collins et al. (1938). In contrast to these authors, we were unable to detect a shortening of the latency to heat. Collins et al. used an intravenous injection
ESTROGEN
AND LORDOSIS
BEHAVIOR
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route and administered unesterified estrogen. Both of these factors would favor speedier incorporation of estrogen into brain tissues mediating behavior than our subcutaneous injection of esterified estradiol. However, even when we attempted to compensate for this by administering estradiol benzoate (SC) 2 hr before progesterone latency was not shortened while duration of heat was significantly prolonged (W. D. Joslyn, unpublished data). When the interval between the priming dose of estrogen and the injection of progesterone was either shortened to 18 hr or lengthened to 96 hr (“non-optimal” priming) there was no facilitator-y action of supplementary estradioi on duration of heat (Experiments III and IV). Instead, the oniy apparent effect was inhibitory in that latency to heat was lengthened. Furthermore, this inhibitory effect of supplementary estradiol was strengthened with increasing estradiol dosage. The mechanisms mediating these paradoxical facilitatory and inhibitory actions of supplementary estradiol are unknown. The supplementary estradiol could be affecting (a) the conditioning process set in motion by the earlier priming dose of estrogen, (b) the uptake or metabolism of the concurrently administered progesterone, or (c) neural sites not previously conditioned by the priming dose of estradiol and not concerned with progesterone action. It is even uncertain that the supplementary estradiol given at nonoptimal priming intervals (18 or 96 hr) exerts its inhibitory action in similar ways during both intervals. Some clarification of the manner in which supplementary estradiol influences lordosis may be obtained through studies of brain uptake of radioactive steroids. The data from Experiment 11 suggest overlapping, but separable mechanisms through which estrogen determines the latency to heat and the duration of heat in optimally primed animals. Latency appears to be regulated by the amount of estrogen present during the early stages of the priming process, since supplementary estradiol given during the first 24 hr after initial priming decreased latency, but estradiol given in the later stages of the priming process at 34 hr (W. D. Joslyn, unpublished data) or at 36 hr had no effect on latency. On the other hand, duration of heat seems to be modulated by the amount of estrogen present near the end of the priming process at 36 hr. Thus, if the groups in Experiment 11 are arranged in the order of amount of estradiol logically expected to be present at Hour 36: it can be seen that duration of heat increased with increases in the expected amount of estrogen.
ACKNOWLEDGMENTS This work was supported by Grant HD-04467 to H. H. F. from the National Institute of Child Health and Human Development and by a Career Development Award (K2-MH-29006) to H. H. F. from the National Institute of Mental Health. We thank Dr. R. W. Coy and Dr. C. H. Phoenix for their encouragement. Publication number 552 of the Oregon Regional Primate Research, Center. Additional funds supporting this work were provided hy Grant MH-21312 to R. W. Coy and Grant PR-11063 to the ORPRC.
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JOSLYN AND FEDER REFERENCES
Collins, V. J., Boling, J. L., Dempsey, E. W., and Young, W. C. (1938). Some quantitative studies of experimentally induced sexual receptivity in spayed guinea pigs. Endocrinology 23, 188-196. Dempsey, E. W., Hertz, R., and Young, W. C. (1936). The experimental induction of oestrus (sexual receptivity) in the normal and ovariectomized guinea pig. Amer. J. Physiol.
116, 201-209.
Goy, R. W., and Phoenix, C. H. (1965). Inhibitory actions of progesterone. Amer. Zool. 5, 725 (abstr.). Goy, R. W., and Young, W. C. (1957). Strain differences in the behavioral responses of female guinea pigs to alpha-estradiol benzoate and progesterone. Behavior 10, 340-354.
Young, W. C., Dempsey, E. determination of heat in Zucker, 1. (1966). Facilitatory of spayed guinea pigs. J.
W., Hagquist, C. W., and Baling, J. L. (1937). The the guinea pig. J. Lab. Clin. Med. 23, 300-302. and inhibitory effects of progesterone on sexual responses Comp. Physiol. Psychol. 62, 376-381.