Effects of the antiestrogens,MER-25 and CI 628, on rat and hamster lordosis

Antiestrogens were used to test the hypothesis that estrogen exerts a “maintenance,” as well as a “priming,” effect on rat and hamster sexual receptivity as it apparently does for guinea pigs. MER-25 (75 or 150 mg/kg) significantly reduced rat LQ when given ?2 hr or 8 hr after EB injection. MER-25 given at 34 hr (2 hr prior to P) failed to diminish rat LQ. With hamsters, MER-25 in large doses (750 mg/kg) given either at ?2 hr or 34 hr reduced lordosis duration to 40% of controls, but this effect was confounded by severe illness among the MER-25 injected animals. Lower doses failed to block behavior, but still produced some toxicity. CI 628 (50 mg/kg) greatly reduced hamster lordosis duration and increased lordosis latency when given 0 hr, but not 34 hr, after EB. The results are consistent with similar previous work on rats and do not support the concept of estrogen “maintenance” in either rats or hamsters.     

Onset of the receptive and proceptive components of feminine sexual behavior in rats following the intravenous administration of progesterone

The present study was carried out in order to assess the time course of action of progesterone (P) in the facilitation of complete feminine sexual behavior. Female rats (estrogen primed via 5% E₂ Silastic capsules) were given 200 μg of P either intravenously (iv) or subcutaneously (sc), and tested for estrous behavior at $\text{1}\text{4}$, $\text{1}\text{2}$, 1, 2, and 4 hr after treatment. Among iv-treated animals, significant amounts of lordosis behavior were seen as early as $\text{1}\text{2}$ hr, and a dramatic rise in solicitation behavior was observed at 2 hr. Although sc-treated animals displayed significant amounts of lordosis and solicitation behavior at 2 hr, the behavior was not maximal until 4 hr. Intravenous administration of 400 μg P was equipotent to 200 μg P, whereas 50 μg of iv P was relatively ineffective. A dual mechanism hypothesis pertaining to progesterone's actions in the facilitation of both the receptive and preceptive components of feminine sexual behavior in rats is discussed.     

Long-term (39–51 hr) exposure to estradiol in silastic capsules and lordosis behavior of guinea pigs

Three dosage levels of estradiol (E₂) in Silastic capsules were administered to ovariectomized guinea pigs for periods of 39 or 51 hr. At 39 hr., a systemic injection of progesterone was given, and hourly testing began. Dose-dependency of various aspects of lordosis behavior was established at both time intervals, and the serum E₂ levels produced by the medium and high dosage levels bracketed those found at the proestrus peak in intact guinea pigs. Although there were no significant behavioral differences between the 39- and 51-hr groups at the individual dosage levels, when the data from all three dosage groups were collapsed, exposure for 51 rather than 39 hr was seen to produce increases in maximum lordosis and heat-duration measures. The results suggest that subtle facilitative effects of estrogen occur very late in the estrogen-conditioning process (even after progesterone is administered).     

Regulation of estrogen-stimulated lordosis behavior and hypothalamic progestin receptor induction by antiestrogens in female rats

Two estrogen antagonists, CI-628 (CI) and tamoxifen (TX), were used to examine the relationship between estrogen priming of lordosis behavior and progestin receptor induction in the hypothalamus-preoptic area (HPOA) of ovariectomized female rats. Lordosis behavior was assessed by measuring lordosis quotients (LQ) in response to injection of 2 micrograms of estradiol benzoate (EB) followed 48 hr later by 500 micrograms of progesterone (P). Behavior testing began 4 hr after P injection. The effects of antiestrogens were assessed by injecting CI and TX (1-2 mg) from 0 to 48 hr prior to EB. Levels of cytosol progestin receptor in the HPOA were determined by quantifying the specific binding of 0.5 nM [3H]R5020 to cytosols from animals receiving the same EB and antiestrogen treatments used in behavioral testing. TX given concurrently with or CI given 2 hr before EB abolished both lordosis behavior and induction of HPOA progestin receptors. In contrast, CI given 12 hr prior to EB abolished lordosis but permitted a 95% elevation in the concentration of progestin binding sites in the HPOA. TX or CI given 48 hr before EB resulted in moderate levels of lordosis (mean LQs from 56 to 69) and induction of HPOA progestin receptors from 85 to 130% above noninjected controls. However, CI given 24 hr prior to EB produced less than a 40% increase in brain R5020 binding even though lordosis behavior was equivalent to that seen in the 48-hr animals (mean LQ = 53). These data indicate that the effects of antiestrogens on female sexual behavior and on the synthesis of brain progestin receptors depend on which antiestrogen is used and the time interval between administration of estrogen and antiestrogen. They also demonstrate that under some conditions estrogen induction of cytosol progestin receptors in the HPOA can be dissociated from estrogen priming of lordosis behavior in rats.     

Comparison of the effects of estradiol-17beta and the synthetic estrogen, RU-2858, on lordosis behavior in adult female rats and guinea pigs

Female sexual behavior (as assessed by lordosis responses) in adult ovariectomized guinea pigs and rats was measured after a single subcutaneous injection of either the synthetic estrogen RU-2858 or estradiol-17β (E) and a subsequent injection of progesterone (P). Thresholds for responsiveness to each estrogen were determined over a range of doses from 1 to 640 μg/kg of body weight. RU-2858 was able to stimulate lordosis at doses several times lower than E in both guinea pigs and rats. In general, guinea pigs were less sensitive to both estrogenic compounds than rats and were particularly insensitive to E. Approximately 40% of guinea pigs given RU-2858 displayed lordosis prior to P injection. Subsequent P injection did not greatly enhance the intensity of lordosis responses in these animals. These guinea pigs were also more sensitive than the remaining 60% of RU-2858-injected guinea pigs on several indexes of lordosis intensity (e.g., lordosis threshold, duration of heat, duration of individual lordosis responses). Rats did not respond behaviorally to RU-2858 without a subsequent P injection nearly as frequently as did guinea pigs. At the doses used in this study, E did not facilitate lordosis behavior in rats or guinea pigs in the absence of a subsequent P injection.     

Blockade of lordosis by androst-1,4,6-triene-3,17-dione (ATD) and tamoxifen in female hamsters primed with testosterone propionate

Normal female hamsters display lordosis after testosterone propionate (TP) plus progesterone (P) treatments. Such effect is probably mediated through aromatization of testosterone (T) into estradiol. If so, then an aromatase inhibitor (ATD) or an estrogen antagonist (tamoxifen, TAM) should be able to block the activational effect of T on lordosis. To test this hypothesis, 48 ovariectomized female hamsters were assigned into six groups which, according to treatments received, were ATD + TP, TAM + TP, OIL + TP, ATD + EB (estradiol benzoate), TAM + EB, and OIL + EB groups. The groups received assigned treatments for 2 days and were injected with P on the third day. Five minutes of behavior test was conducted 4 hr after P injection. The OIL + TP, OIL + EB, and ATD + EB groups all had averaged total lordosis duration (TLD) longer than 200 sec. The TLD of the TAM + EB group was only 117 sec. The ATD + TP and TAM + TP groups showed almost no lordosis. The results showed that the estrogen antagonist (TAM) impaired lordosis no matter whether the animals were primed with TP or EB, but the aromatase inhibitor (ATD) blocked lordosis only in TP primed females. It is concluded that the aromatization of T to estrogen is required for testosterone activation of lordosis in female hamsters.     

Inhibitory actions of progesterone on hormonal induction of estrus in female guinea pigs.

Biphasic actions of progesterone (P) were studied in three groups of guinea pigs injected with 3 μg estradiol benzoate (EB) followed 36 hr later with either .05, 0.4, or 5.0 mg P. All animals in each group displayed estrous behavior within 5–7 hr after P. Groups differed, however, in their response to a second sequence of 3 μg EB and 0.4 mg P initiated 9 hr after the first or prior P. Percentages of females displaying estrous responses were 100, 50, and 0 in the .05, 0.4, and 5.0 mg groups, respectively.Radioimmunoassay of P at varying times after the first or prior injection showed that concentrations in the plasma declined to baseline within 9–18 hr for animals given prior injections of .05 mg P, within 36 hr for those given 0.4 mg P, and remained elevated throughout for 45 or more hr for those given 5.0 mg P.A second experiment showed that the inhibitory effect of 1.0 mg P given to spayed females without prior estrogenization (direct inhibition) was inversely related to the time elapsing between that injection and the initiation of a sequential regimen consisting of 6 μg EB and 0.4 mg P, and also demonstrated that 0.05, 0.1, 0.25, 0.5, or 1.0 mg P given 2 hr before the sequential regimen results in a dose-related inhibition. Thus, prior facilitation of lordosis is in no way essential for inhibition with exogenous P, and variables that regulate degree of biphasic inhibition also regulate direct inhibition.     

Erythroid colony formation and erythropoietin activity in mice treated with estradiol benzoate

In mice administered with estradiol benzoate (EB) at 0.2 or 5 μg dose levels, the number of erythroid colony-forming units (CFU-E) in marrow declines progressively starting from 12 through 48 hr. On the other hand hypoxic erythropoietin (Ep) production, although potentiated in animals primed with relatively large dosages of EB (5–25 μg), is significantly diminished after treatment with lower amounts of estrogen (0.2 μg). In mice primed with large amounts of EB, the enhancement of Ep activity apparently compensates the depleting influence on marrow CFU-E, thus leading to a nearnormal erythroid response to hypoxia. On the other hand, a sharp drop of this parameter is observed in animals primed with the lower dosage (0.2 μg), $\text{i.e.}$ the depleting influence on the CFU-E pool is not compensated here by potentiation of the Ep response.     

Estrogen and progesterone interactions influencing sexual and social behavior in the brown lemming, Lemmus trimucronatus.

The effects of estrogen and progesterone on the social and sexual behavior of brown lemmings, Lemmus trimucronatus, were investigated. The behavior of hormone-treated and untreated ovariectomized females and sexually vigorous males was observed in six consecutive daily 5-min dyadic encounters. Sexual receptivity, as measured by lordosis, and other social behaviors including nasonasal contact, boxing postures, allogrooming, perineal investigation, and male mounting increased following 48 hr of exposure to daily injections of 0.5 μg estradiol benzoate (EB). Lordosis in EB-primed females was not facilitated or inhibited by short-term (4 hr) exposure to 0.5 mg progesterone (P). Long-term (greater than 24 hr) exposure to P apparently inhibited lordosis and other social behaviors in EB-treated females, although males continued to attempt to mount these females. In EB-treated females a dramatic increase in threat-leaps, directed by the female toward the male, was observed within 4 hr of P injection. Threat-leaps declined when P was withdrawn. Threat-leaps were also observed in ovariectomized females after prolonged exposure to P only (0.5 mg/day). Vaginal perforation and cornification were first apparent 48 hr after EB injection. P-alone treated ovariectomized females also showed vaginal perforation but cornified cells were infrequent and these animals did not show lordosis.     

Hysterectomy-induced facilitation of lordosis behavior in the rat

The present study investigated the effect of hysterectomy on hormone-induced lordosis behavior. Lordosis quotients (LQ) were measured in hysterectomized-ovariectomized (HO) and ovariectomized-sham hysterectomized (OSH) rats after several treatments including either estradiol benzoate (EB) alone or EB plus progesterone (P) 44 hr later. Testing consisted of placing the females with sexually active males 48 hr after EB. In Experiment 1, HO animals treated with 5 μg/kg EB and 0.5 mg P had significantly higher LQs than OSH animals; groups treated with 10 μg/kg plus P were not different. Experiment 2 showed that a single injection of 50 μg/kg EB resulted in equally high levels of receptivity in both groups. The LQs of HO animals injected with 3 μg/kg for 4 days did not differ from those of OSH animals; however, the administration of 0.5 mg P 24 hr after the fourth EB injection resulted in significantly higher LQs in the HO group (Experiment 3). In Experiment 4, HO rats injected with 5 μg/kg EB and 0.1 mg P 44 hr later displayed higher levels of lordosis behavior than OSH animals. It was concluded that hysterectomy facilitated the lordosis behavior of ovariectomized rats injected with both EB and P and that the mechanism for this potentiation remains to be determined.     

Hormonal regulation of macrophage collagenase activity.

Whereas peritoneal macrophages from nonpregnant guinea pigs were stimulated $\text{in vitro}$ by endotoxin to produce collagenase on the second day of culture, those from pregnant guinea pigs were incapable of this response. However, if the cells from pregnant animals were preincubated for one day prior to endotoxin stimulation, collagenase activity could be detected. Injection of either estrogen or progesterone into guinea pigs at doses comparable to those found during pregnancy prior to removal of the peritoneal cells also inhibited the $\text{in vitro}$ stimulation of collagenase production. The addition of these hormones $\text{in vitro}$ revealed that at 5 × 10^?6 M estrogen and progesterone inhibited 53% and 100% respectively of the collagenase activity. Addition of both hormones at a final concentration of 5 × 10^?7 M of each inhibited 87% of the activity indicating a synergistic effect since this concentration of either hormone alone was ineffective.     

The inhibition of steroid-induced sexual behavior by intrahypothalamic actinomycin-D

Lordosis behavior can be elicited in the ovariectomized rat after treatment with estradiol benzoate (EB) and progesterone (P) injections, but the EB must act for an extended period before P can facilitate this behavior. The possibility that this action of EB involves the stimulation of RNA or protein synthesis was tested by implanting actinomycin D (Act-D) directly into the preoptic area, one probable site of estrogen action. A total dose of 0.18 μg Act-D in bilateral cocoa butter pellets significantly inhibited lordosis behavior when implanted 12 hr after the injection of 3 μg. but not 15 μg EB. Implantation of this dose of Act-D subcutaneously, or intrahypothalamically 32 hr after EB injection, was without effect. Act-D placed in the ventromedial hypothalamus also suppressed lordosis, but implants in the caudate nucleus were without effect. At the time of the behavioral tests the animals were in excellent condition as determined by calculation of a health score, and no physical lesions were evident at the site of the implants. However, it was impossible to test the reversibility of this suppression of lordosis behavior since the animals became ill and many died within 1–2 weeks of implantation. The present results are consistent with, but not proof of, the concept that RNA synthesis may be essential for steroidinduced sexual behavior.     

Differential effects of the anti-estrogen MER-25 and of three 5alpha-reduced androgens on mounting and lordosis behavior in the rat.

Four experiments were performed in order to evaluate further the hypothesis that androgen must be aromatized to estrogen for the activation of masculine sexual behavior in the male rat. In Experiment 1 it was found that the anti-estrogen MER-25 failed to disrupt mounting behavior in castrated males which simultaneously received testosterone propionate (TP). However, in Experiment 2 it was found that MER-25 as weil as 3β-androstanediol effectively activated masculine behavior in castrated males treated simultaneously with dihydrotestosterone propionate. Both MER-25 and 3β-androstanediol had previously been shown to display an affinity for cytoplasmic estradiol-17β receptors present in male rat anterior hypothalamus. In Experiments 3 and 4, performed with ovariectomized females, it was found that whereas MER-25 antagonized the stimulatory effect of estradiol benzoate (EB) on lordosis behavior, 3β-androstanediol did not. In addition, 5α-dihydrotestosterone and 3α-androstanediol, two compounds which had previously been shown to have almost no affinity for estradiol-17β receptors in the hypothalamus, both inhibited the stimulatory effect of EB on lordosis. It is concluded that the fact that anti-estrogens suppress lordosis induced in females with either EB or TP, but fail to disrupt TP-induced mounting behavior in male rats does not argue against the aromatization hypothesis for masculine sexual behavior.     

Brain uptake and metabolism of estradiol benzoate and estrous behavior in ovariectomized guinea pigs

Groups of spayed guinea pigs were injected sc with tritiated estradiol benzoate in oil and killed at intervals varying from 12 to 120 hr later. The quantities of radioactivity with the mobility of estrone (E₁), estradiol-17β (E₂), and estriol (E₃) were estimated in plasma, hypothalamus, cortex, and cerebellum. Radiometabolites extracted from the hypothalamus and the cortex were identified by derivative formation and by isotope dilution techniques. The hypothalamus contained larger quantities of E₂ than any of the other tissues studied. The same pattern of uptake and decay of radioactivity was observed in all tissues. Concentration of total radioactivity was greatest 12 hr after injection and declined fairly regularly to minimal value at 120 hr. Unlike the hypothalamus and the cerebellum, in the cortex a large proportion of the radioactivity was present as E₁. ³H-estradiol benzoate was metabolized to ³H-estradiol by blood in vitro suggesting that the esterified form of the hormone is long lasting because of its slow release from the site of injection rather than its long half-life in the blood.Additional groups of spayed guinea pigs were tested for lordosis in response to fingering after injection of estradiol benzoate followed by progesterone at intervals varying from 12 to 120 hr. The expression of lordosis varied in a complex manner as a function of the interval between the injection of estradiol benzoate and progesterone. Maximum measures of lordosis were obtained when the interval between injections was 36 hr. The relation between behavior and the neural uptake of estrogens suggests that both the duration of estrogen action and the concentration of estrogens at the time the behavior is being displayed determine the character of the response.     

Influence of the blood concentration of prolactin on the length of the sensitive period for establishing maternal behavior in sheep at parturition

The fading of postpartum maternal interest for the neonate (sensitive period) in ewes separated from their young at lambing is delayed when parturition is induced with 20 mg of estradiol benzoate (EB). An experiment was carried out to investigate the role of prolactin in this phenomenon. The sensitive period was studied in three groups of parturient ewes. In all groups lambs were removed at birth and reintroduced to their mothers 24 hr later. Maternal acceptance was tested at this time. In group 1 (dexamethasone D), ewes were induced to lamb with dexamethasone (15 mg im). In group 2 (EB), ewes were treated with 20 mg of estradiol benzoate (im). In group 3 (EB + CB 154) ewes received 20 mg of EB as in group 2 and 1 mg of CB 154 (sc) every 12 hr to prevent the enhanced secretion of prolactin which normally occurs after EB injection. The concentration of prolactin was highest in group 2 (EB), lowest in group 3 (EB + CB), and intermediate in group 1 (D) (p ? 0.001 between groups). By contrast, the proportion of ewes showing maternal behavior was similar in groups 2 and 3 ($\text{15}\text{23}$ and $\text{17}\text{22}$), both of which differed from group 1 ($\text{3}\text{22}\text{; p ? 0.005}$). It is concluded that the lengthening of the sensitive period for establishing maternal behavior in sheep following EB induced parturition is not related with high levels of prolactin in the peripheral circulation.     

Endogenous progesterone and lordosis behavior in male rats given estrogen alone

Male rats castrated as adults were given successive doses of estradiol benzoate (EB) combined or not, with dexamethasone (DEXA) at the end of estrogen treatment. Two experiments were done to determine if progesterone (P) of adrenocortical origin was involved in the display of lordosis behavior under these experimental circumstances. There was a significant rise in blood P concentration in animals given 0.5 and 1.0 microgram EB when compared with oil-control injected animals, an effect which was completely suppressed by DEXA treatment. An increase in the proportion of estrogen treated animals displaying lordosis responses to male mounts was found with increasing doses of EB and paralleled the effects of EB on P adrenocortical secretion. However, the number of feminized animals given 1 microgram EB + DEXA was reduced to the level corresponding to the effects of 0.5 microgram EB on lordosis behavior. These data show that the secretion of P by the adrenals is involved in the expression of lordosis behavior in castrated male rats primed with repeated doses of estrogen.     

Stimulation of the estrogen synthesizing system of the immature rat ovary by exogenous and endogenous gonadotropins

Immature rat ovaries increase their secretion of estradiol (E₂) when stimulated by gonadotropins but only after a lag period of several hours. Once established, estrogen secretion can be maintained, or increased, by the continued presence of gonadotropin. A combination of ovine FSH+LH given at 2 hr intervals stimulated the estrogen synthesizing system ($\text{ESS}$) of the ovary and serum E₂ showed a pronounced rise between 16 and 20 hrs after the initial injection. When given every 2 hrs for 5 doses (0–8 hrs) serum E₂ was undetectable. However, it was increased if 20 IU PMS was injected at the time of the last dose of FSH+ LH. Endogenous FSH&LH, increased by hourly injections of LH-releasing hormone for a period of 8 hrs, stimulated the $\text{ESS}$; serum E₂ increased at the expected time when this treatment was followed by an injection of PMS.Anti-PMS antiserum given 12 hrs after PMS, prevented the expected rise in serum E₂ at 24 hrs. However, FSH, LH or a combination of the two given every 2 hrs beginning at the time of the anti-PMS produced an increase in E₂ secretion; the combination was more effective than either hormone alone.These results are consistent with the interpretation that a combined FSH-LH action is responsible for induction of the $\text{ESS}$ in the immature rat ovary. The combination of hormones is also very effective in maintaining estrogen secretion but some function appears possible with FSH or LH alone.     

Independence of progesterone-induced facilitation and inhibition of lordosis behavior in ovariectomized guinea pigs

In ovariectomized Hartley guinea pigs, 15 μg progesterone was shown to facilitate lordosis in 48% of animals when administered 36 hr after a 3.3 μg injection of estradiol benzoate. This dose of progesterone also inhibited lordosis behavior in 65% of animals administered an additional 0.6 mg progesterone at 60 hr. Significant inhibition of lordosis response to the 0.6 mg progesterone existed among animals in which lordosis was not facilitated by the initial 15 μg dose of progesterone. These results show that progesterone-induced inhibition can occur without prior facilitation of lordosis as tested by the manual stimulation technique.     

The effect of antiestrogens on the nuclear binding of the estrogen receptor

Experiments were designed to determine whether or not various antiestrogens in direct competition with estradiol-17β (E₂) would inhibit the translocation of the estrogen receptor complex from the cytoplasm to nuclei in rat uterine tissue. Incubation of the antiestrogens CI-628, $\text{cis}$-clomiphene, U-11,100A and MER-25 with rat uteri caused the nuclear uptake of the antiestrogen receptor complex which was greatest for most antiestrogens at concentrations of 1 × 10^?6 to 1 × 10^?5M. At higher concentrations of CI-628, $\text{cis}$-clomiphene, and U-11,100A the nuclear binding of the antiestrogen receptor complex was greatly decreased. Incubation of the antiestrogens with E₂ resulted in a dramatic inhibition of the nuclear uptake of the estrogen receptor. $\text{Trans}$-clomiphene, a weak estrogen, did not inhibit the movement of the uterine cytoplasmic receptor into the nuclear fraction.     

Progestin receptors in substance P-immunoreactive neurons in the hypothalamus of male guinea pigs after behaviorally effective estradiol pulse treatment

Pulsatile administration of estradiol effectively primes orchidectomized (ORCH) male guinea pigs to display progesterone-facilitated lordosis. In contrast, a single injection of estradiol benzoate (EB) is not behaviorally effective. In ovariectomized female guinea pigs, estradiol pulses induce progestin receptors selectively in substance P neurons in the ventrolateral hypothalamus (VLH), a site at which estradiol primes females to respond behaviorally to progesterone. To test the hypothesis that behaviorally effective estradiol pulses induce progestin receptors selectively in substance P neurons in the VLH in males, ORCH animals received a single injection of EB 40 h before, or two pulses of estradiol-17β, 39 and 11 h before perfusion. Colchicine was administered intracerebroventricularly prior to perfusion. The only difference found between the two estradiol treatment groups was a higher number of progestin receptorimmunoreactive (PR-IR) cells in the rostral VLH of estradiol pulse-treated males. There were no significant differences in the number of PR-IR cells in the mid- or caudal VLH, nor in the number of substance P-immunoreactive (SP-IR) neurons in the VLH/ventromedial hypothalamus (VMH) of animals receiving the two estradiol treatments. Furthermore, the percentage of PR-IR cells in the VLH also immunoreactive for SP did not differ between the estradiol pulse- (22%–25%) and the EB-injected animals (22%–32%). These data do not support the hypothesis that administration of behaviorally effective estradiol pulses, as compared to behaviorally ineffective EB injections, induce progestin receptors selectively in substance P neurons in the VLH of male guinea pigs.