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.     

The reversible inhibition of steroid-induced sexual behavior by intracranial cycloheximide

Cycloheximide(Cyclo), an inhibitor of protein synthesis by a direct action on protein synthesis at the ribosomal level, was used to reversibly inhibit estrogen-induced sexual receptivity. Cyclo (100 μg per rat) was infused into the preoptic area(POA) of ovariectomized rats at varying times before, simultaneously with, and after 3 μg of subcutaneous estradiol benzoate (EB). All animals received 0.5 mg progesterone (P) 36 hr after EB, and were tested for sexual receptivity 4–6 hr after P. The females were placed with stud males and a lordosis quotient was computed for each female (lordosis quotient = number of lordosis responses/20 mounts by the male × 100). Females receiving Cyclo 6 hr before, simultaneously with, or 12 hr after EB showed significantly lower levels of sexual receptivity when compared to females receiving Cyclo 36 hr before and 18 and 24 hr after EB. When those animals that showed low levels of sexual behavior after Cyclo infusion were reprimed with EB and P 7 days later and presented with a male they showed high levels of sexual receptivity. Thus, the effect of Cyclo was reversible. Only Cyclo infusions into the POA (bilateral) and third ventricle were effective in suppressing sexual behavior. Caudate nucleus, lateral ventricle, and unilateral POA infusions were without effect.The data presented are in agreement with earlier work that utilized actinomycin D to inhibit steroid-induced sexual behavior. Cyclo was found to be less toxic than actinomycin D. All of the available evidence is consistent with the hypothesis that estrogen stimulates RNA and/or protein synthesis in its facilitation of sexual behavior in the female rat.     

Specificity and neural sites of action of anisomycin in the reduction or facilitation of female sexual behavior in rats

These experiments were designed to investigate the role of neuronal protein synthesis in the hormonal activation of female sexual behavior using intracranial implants of the protein synthesis inhibitor, anisomycin. In the first experiment, female rats receiving bilateral cannulae implants in the medial preoptic area (POA), septal region (SEPT), ventromedial hypothalamus (VMH), or midbrain central gray (CG) were injected with 2.5 micrograms estradiol benzoate (EB), followed 48 hr later by 500 micrograms progesterone (P). Females receiving anisomycin in the VMH at the time of EB injection had lower levels of lordosis and darting compared to tests without anisomycin. Sexual behavior was unaffected in females receiving anisomycin implants in the POA, SEPT, or CG. In a second experiment, we replicated the finding that anisomycin could attenuate lordotic responsivity when placed in the VMH of female rats injected with 2.5 micrograms EB and 500 micrograms P. In addition, we found that POA implants of anisomycin could facilitate lordosis in females given a low dose of EB (1.25 microgram) plus 500 micrograms P. In a third experiment, we assessed the effects of anisomycin application to the VMH or POA of female rats receiving estradiol (E; diluted 1:250 with cholesterol) implants in the VMH and systemic P. Treatment of the VMH with anisomycin prior to E in the VMH suppressed lordotic responding, whereas anisomycin application to the POA prior to E in the VMH had no effect on lordosis. The results of these experiments suggest that reducing protein synthesis in the region of the VMH disrupts the action of estrogen on the VMH, and that the facilitative action of anisomycin in the POA of female rats requires more estrogen treatment than threshold stimulation of the VMH alone.     

Facilitation of Sexual Receptivity by Ventromedial Hypothalamic Implants of the Antiprogestin RU 486

RU 486 is known primarily as an antagonist to progestins and glucocorticoids. However, RU 486 has also been shown to have agonistic progestational properties in biochemical and behavioral studies. In the current study, RU 486 was implanted directly into tim ventromedial hypothalamus (VMH) to test for facilitative action on the receptive behavior of female ovariectomized Long-Evans rats primed with 5 μg of estradiol benzoate. Cannulae containing RU 486, progesterone (P), or empty cannulae were implanted 48 hr after estrogen priming. The lordosis quotient and the lordosis score were assessed 4 hr after the cannulae were lowered by a standardized test consisting of 10 mounts by a stimulus male. P and RU 486 significantly facilitated receptivity compared to blank implants in terms of lordosis quotient and lordosis score, with no significant difference between the hormone treatments. While only a single dose of each treatment was given in the current study, RU 486 facilitated lordosis when implanted to the VMH as well as progesterone in contrast to our previous results where the steroids were administered systemically.     

Antagonism of central estrogen action by intracerebral implants of tamoxifen

In order to determine the neural site(s) of estradiol (E2) priming of receptive behavior in female Long-Evans rats, we attempted to inhibit the behavioral effects of peripheral injections of E2 by administering the E2 antagonist tamoxifen (TX) to particular brain regions. Crystalline TX was administered unilaterally or bilaterally via 28-gauge cannulae into the ventromedial hypothalamic nucleus (VMN), the preoptic area (POA), or the interpeduncular region (IP) 1 hr prior to the first of three daily E2 benzoate injections. Subjects were tested for the presence or absence of behavioral estrus 5 hr after a 200-micrograms progesterone injection given 4 days after the initial hormone treatment. Results of this experiment showed that TX inhibits lordosis when directed toward the VMN, but not when directed toward the POA or IP. The quality of the lordosis response and the proportion of subjects showing solicitation behavior were both lower in VMN subjects treated with TX than in POA or IP subjects given the same treatment. Unilateral implants were as effective as bilateral implants in inhibiting the behavior of VMN subjects. A second experiment measured uptake of radiolabeled E2 by nuclei of hypothalamic (HYP) and POA tissue following bilateral TX administration to the VMN or POA. TX was capable of inhibiting uptake of [3H]E2 into nuclei of cells located near the implant site. Most subjects which showed behavioral inhibition also showed a reduction in uptake of [3H]E2 by HYP tissue. These data support the hypothesis that exposure of the VMN to E2 is necessary for the priming of estrous behavior in the female rat.     

Mu-, delta-, and kappa-opioid receptor agonists selectively modulate sexual behaviors in the female rat: differential dependence on progesterone.

Previous studies suggested that opioid receptor agonists infused into the lateral ventricles can inhibit (through mu receptors) or facilitate (through delta receptors) the lordosis behavior of ovariectomized (OVX) rats treated with estrogen and a low dose of progesterone. The present study investigated the behavioral and hormonal specificity of those effects using more selective opioid receptor agonists. Sexually experienced OVX rats were implanted stereotaxically with guide cannulae aimed at the right lateral ventricle. One group of rats was treated with estradiol benzoate (EB, 10 micrograms) 48 hr and progesterone (P, 250 micrograms) 4 hr before testing, whereas the other group was treated with EB alone. Rats were infused with different doses of the selective mu-receptor agonist DAMGO, the selective delta-receptor agonist DPDPE, or the selective kappa-receptor agonist U50-488. The females were placed with a sexually vigorous male in a bilevel chamber (Mendelson and Gorzalka, 1987) for three tests of sexual behavior, beginning 15, 30, and 60 min after each infusion. DAMGO reduced lordosis quotients and magnitudes significantly in rats treated with EB and P, but not in rats treated with EB alone. In contrast, DPDPE and U50-488H increased lordosis quotients and magnitudes significantly in both steroid-treatment groups. Surprisingly, measures of proceptivity, rejection responses, and level changes were not affected significantly by mu or kappa agonists, although proceptivity and rejection responses were affected by DPDPE treatment. These results suggest that the effects of lateral ventricular infusions of opioid receptor agonists on the sexual behavior of female rats are relatively specific to lordosis behavior. Moreover, the facilitation of lordosis behavior by delta- or kappa-receptor agonists is independent of progesterone treatment, whereas the inhibitory effect of mu-receptor agonists on lordosis behavior may require the presence of progesterone.     

Inhibition of estrous behavior in rats by intrahypothalamic application of agents that disrupt nuclear binding of estrogen-receptor complexes

This study tested the hypothesis that estrogen facilitation of reproductive behavior in female rats requires the binding of estrogen-receptor complexes to the genomic components of hypothalamic cell nuclei. Female rats were implanted stereotaxically with bilateral guide cannulae aimed at the ventromedial nucleus of the hypothalamus (VMH). Animals were ovariectomized following recovery from the implant surgery and randomly assigned to receive one of four drug treatments: actinomycin-D, ethidium bromide, netropsin, or 4', 6-diamidino-2-phenylindole. Each female received at least two tests for estrous behavior 48 hr after estrogen priming. On one test, drug-filled cannulae were lowered into the VMH 1 hr prior to a subcutaneous injection of 2-3 micrograms of estradiol benzoate (EB); on the other test blank cannulae were inserted 1 hr prior to EB treatment. Intracranial administration of all four compounds, which disrupt estrogen-receptor binding to hypothalamic nuclei, inhibited both the quantity and the quality of lordosis responses to systemic injections of EB. The results support the hypothesis that specific receptor interactions with the genome of hypothalamic cells mediate estrogen facilitation of estrous behavior in female rats.     

Inhibition of estrogen facilitation of sexual behavior by the intracerebral infusion of actinomycin-D

It has been shown previously that intracerebral actinomycin-D (Act-D) pellets inhibit estrogen facilitated female sexual behavior, but it was not possible to test the reversibility of this effect. In the present study an attempt was made to distinguish between the possible temporary interruption by Act-D of the biochemical action of estrogen which facilitates sexual receptivity and permanent toxic effects of the drug. Act-D in saline was infused into the third ventricle or the preoptic area (POA) to determine whether a reversible suppression of sexual behavior as measured by the lordosis quotient (LQ) could be produced. Ovariectomized rats were implanted with midline guide tubes entering the third ventricle (eight rats) or with bilateral tubes extending to the corpus callosum above the POA (67 rats). Each animal served as its own control since pretest and Act-D and recovery tests were performed 10–14 days apart in most subjects. For each behavioral test implanted subjects were primed with 3μg estradiol benzoate (EB) and 0.5 mg progesterone (P) 48 hr later. Behavioral tests, each involving 50 mounts, were performed 4–6 hr after P. Following the pretest the animals were retested under experimental conditions. Inner cannulae were inserted into the POA through the guide tubes and 0.11 μg Act-D infused 24 or 12 hr before, simultaneously with, or 6, 12, 18, or 26 hr after EB. A recovery test was performed 10–14 days later with no intracerebral infusion. The control procedure (infusion of of saline either simultaneously with or 12 hr after EB) did not alter the LQ. Act-D infusion produced a reversible suppression of lordosis which was dependent upon the time of administration of Act-D. Intraventricular infusion of Act-D 6 hr after EB reversibly inhibited lordosis behavior and no lesions were produced. Act-D infused into the POA simultaneously with EB or 6 hr later reversibly suppressed the LQ. In the 6 hr group, for example, the LQ fell from 78.3 to 35.7, but 10–14 days later reached 74.3. Although brain lesions of varying extent were produced by Act-D, the marked but reversible suppression of lordosis behavior is consistent with the view that Act-D inhibits estrogen facilitation of lordosis behavior by means of a biochemical rather than cytotoxic action.     

Effect of preoptic region implants of dilute estradiol on the maternal behavior of ovariectomized, nulliparous rats

Estrogen implanted directly into the medial preoptic region of pregnant Charles River Sprague-Dawley rats hysterectomized and ovariectomized on Day 16 of gestation mimics the effects of systemic estrogen treatment at this time by reducing the latency to respond to foster pups with maternal behavior (Numan, Rosenblatt, and Komisaruk, 1977). The present report describes the pup-directed responses of ovariectomized, nulliparous Zivic-Miller Sprague-Dawley rats that received bilateral medial preoptic implants of either cholesterol (n = 11) or estradiol diluted 1:10 with cholesterol (n = 11). Two days after treatment these animals were housed with three foster pups: their responsivity to pups and quality of nests built were then assessed, at first hourly and then daily. Rats receiving intracranial estradiol required significantly shorter exposures to pups than did cholesterol-treated animals before initiating carrying and grouping of 3 dispersed pups in a maternal nest during a 15-min test. On other measures, however, the groups did not differ (e.g., proportion grouping pups overnight, time required to complete retrieval of pups to the nest, time required to rebuild a disrupted nest). Animals treated with cholesterol and animals with estradiol implants did not differ in uterine weight at the time of sacrifice, suggesting that estrogen did not leak, even from this well-vascularized implant site, into the circulation. Thus, as in the pregnant animal, the facilitating effects of estrogen on maternal behavior can be mediated through the medial preoptic region; however, the effects were evident only when a test requiring retrieval of several pups within an arbitrarily short interval was given.     

Effects of intracranial implants of testosterone propionate on intermale aggression in the castrated male mouse

Three experiments were conducted in order to assess the effects of intracranial implants of testosterone propionate (TP) on intermale aggression in the castrate male CF-1 mouse. In Experiment 1, seven groups received bilateral implants containing a total of 27 μg crystalline TP into the septum, neocortex, lateral ventricles, preoptic-anterior hypothalamus, hippocampus, medial reticular formation, and subcutaneously, and were tested 2 and 4 days after treatment. An eighth group received blank pellets in the brain. Animals receiving septal or lateral ventricle implants fought significantly more than other groups on trial 1. This difference had disappeared by trial 2, indicating diffusion of the hormone. The diffusion was corroborated by significant seminal vesicle growth. In Experiment 2 animals received bilateral implants of a total of 4.5 μg TP in paraffin or a blank pellet into the septum, preoptic-anterior hypothalamus, cortex, amygdala, olfactory bulbs, medial reticular formation, or subcutaneously. None of these treatments proved effective for activating aggression. Experiment 3 explored the activational effects of 10 μg of TP implanted bilaterally into the same areas as in Experiment 2, excluding the olfactory bulbs and cortex. Implants into the septum were followed by significantly increased fighting. Implants into the preoptic area were only marginally effective whereas the remaining two areas were not responsive to hormone treatment and did not differ from control animals. No seminal vesicle growth was detected as a result of the hormone treatments. These results would indicate that the septum is important in the control of androgen-dependent, intermale aggression in the male CF-1 mouse.     

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.     

Facilitation of female sexual behavior in male rats by septal lesions: an interaction with estrogen.

Although destruction of the septal region markedly facilitates the lordosis behavior of female rats in response to estrogen priming, comparable lesions were found to be ineffective in facilitating the lordotic behavior of estrogen primed male rats. Neither the age at the time of septal destruction nor castration influenced the lordosis behavior of males. However, if prepubertal castrated males were given subcutaneous ovarian grafts or injected daily with 2 μgm estradiol benzoate (EB) during the 30 day period following septal destruction, a prolonged facilitation of the activational effects of EB on lordosis behavior was observed. Male rats subjected to septal destruction alone, chronic exposure to EB alone, exposure to ovarian grafts for 30 days prior to septal destruction, or chronic treatment with EB started 6 mo after septal lesioning, failed to show an increase in behavioral responsiveness to estrogen. Thus, in order for septal lesions to facilitate lordosis behavior of male rats, exposure to EB or ovarian tissue must occur within an apparent critical period following septal destruction. Adult male rats were found to be more responsive to this interaction of septal lesions and EB exposure than pubertal animals. It is suggested that the prolonged facilitation of lordosis behavior which follows septal destruction and estrogen exposure in the male rat may be due to hormonal modifications of the recovery process following brain damage.     

The comparative potency of various steroids to complete the priming process for lordosis in guinea pigs

Ovariectomized adult guinea pigs were treated with a regimen of estradiol benzoate (0.2 μg/animal estradiol benzoate at hr 0 and 19) that was shown to be minimally effective for the induction of lordosis. They were then treated with 10, 20, or 80 mg of enclomiphene, 5, 20, 40, or 100 μg of estradiol, or testosterone, cortisol, estrone, estriol, diethylstilbestrol, catechol estradiol, or catechol estrone (all at a dose equivalent to 5 μg of estradiol) at hr 28. At hr 39 all females were given 0.5 mg progesterone, and subsequently tested for lordosis behavior. Of the various agents injected at hr 28 only estradiol (at all doses given), estrone, estriol, and diethylstilbestrol were effective in supporting display of lordosis behavior. The results indicate that the antiestrogen enclomiphene, the catechol estrogens, and at least some C₁₉ and C₂₁ steroids are weaker than E₂ or ineffective in facilitating lordosis behavior when given late in the priming period. Because previous work had shown that enclomiphene has partial estrogenic effects on lordosis behavior when administered early in the priming period (i.e., at hr 0, 19), it is suggested the early and late phases of the priming process induced by E₂ entail qualitatively different neural processes.     

A contributory role for midbrain progesterone in the facilitation of female sexual behavior in rats

Progesterone (P) facilitation of sexual receptivity in rodents has been achieved by intracranial administration to the ventral hypothalamus; the preoptic area; and midbrain areas such as central gray, mesencephalic reticular formation, and ventral tegmental nucleus. In our laboratory, by far the most effective site in rats has been the ventromedial nucleus of the hypothalamus (VMN). However, several reports of sensitivity to P in the midbrain of rats and other rodent species led us to investigate whether stimulation of the ventral midbrain of female rats might contribute to facilitation of sexual receptivity. Ovariectomized Long-Evans rats received one cannula aimed at the VMN, and another aimed at the contralateral ventral mesencephalon. P in both cannulae, following a priming dose of estradiol, caused significantly higher lordosis quotients (LQ) than blank tubes. Controls with bilateral cannulae in the VMN responded when both tubes were filled with P, but did not respond to unilateral VMN P stimulation. P in the VMN and contralateral anterior preoptic area did not result in a greater degree of receptivity than did the empty tubes. These studies indicate that although progesterone stimulation in the midbrain alone is not sufficient to facilitate receptivity in female rats with our methods, the midbrain may play an auxiliary role. P implants in the midbrain appear to facilitate receptivity in the case of VMN implant treatments that are subthreshold for stimulating lordosis. The results are discussed in light of similar studies in other rodent species, and in the context that more than one brain site may be important in the natural stimulation of sexual receptivity by gonadal hormones.     

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).     

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.     

Effects of serotonin agonists on lordosis,myoclonus, and cytoplasmic progestin receptors in guinea pigs

Peripheral treatment with the serotonin releaser fenfluramine or the serotonin agonist quipazine abolished lordosis behavior in ovariectomized estradiol and progesterone-primed female guinea pigs. Quipazine was also effective when administered into a lateral cerebroventricle. The lowest dose of fenfluramine that induced myoclonus (10 mg/kg) was higher than the dose needed to inhibit lordosis (5 mg/kg). Therefore, it appears that myoclonus and lordosis are differentially sensitive to serotonin agonists. The effects of quipazine on lordosis were time dependent. Quipazine had no effect on lordosis when given prior to the onset of sexual receptivity. These data suggest that serotonin agonists might be effective only when progesterone has had sufficient time to induce sexual receptivity. Quipazine did not affect cytoplasmic progestin receptors in brain areas involved in steroid hormone effects on lordosis. This finding, and the finding that quipazine had no effect on lordosis when given prior to the onset of sexual receptivity, suggest increased serotonin transmission does not interfere with estrogen priming or sensitivity of hypothalamic cells to progesterone.     

Induction of CCK mRNA levels in the limbic-hypothalamic circuit: Time course and site-specific effects of estrogen

Estrogenic regulation of cholecystokinin (CCK) and its receptors is correlated with the initiation and termination of lordosis behavior. To understand the effect of circulating estrogen concentration on the temporal aspects of CCK mRNA expression in the posterodorsal medial amygdaloid nucleus (MeApd) and the central part of the medial preoptic nucleus (MPNc) of the limbic-hypothalamic circuit, ovariectomized female rats were treated with a 10 mm Silastic™ capsule filled with estradiol, a bolus injection of 50 μg estradiol benzoate or 2 μg estradiol benzoate every 4 days for five “cycles.” In situ hybridization was used to compare the relative changes of CCK mRNA levels at 0 h to levels measured at 6, 12, 24, 48, 72, or 96 h after estrogen administration. In the MPNc and the MeApd, the 10-mm capsule significantly increased and maintained CCK mRNA levels from 6 to 96 h. The range of the increase was 3.0–5.1-fold in the MPNc and 2.8–5.0 in the MeApd. The 50-μg injections significantly increased and maintained CCK mRNA levels in the MPNc from 12 to 96 h (range of the increase 2.4–4.1-fold) and in the MeApd from 24 to 96 h (range of the increase 2.2–2.8-fold). The repeated administration of 2 μg estrogen induced a significant increase of message levels in the MPNc at 12 and 24 h that were 4.2- and 4.7-fold, respectively. In the MeApd this estrogen treatment did not significantly increase CCK mRNA. These studies demonstrate that small doses (2 μg) of estrogen that mimic the pattern and circulating levels of estrogen dramatically stimulate CCK mRNA levels in the limbic-hypothalamic circuit. To further study this steroid stimulation, ovariectomized female rats were implanted with estradiol-filled cannulae into the bed nucleus of the stria terminalis or MeA. Estrogen elevated CCK mRNA levels locally in each nucleus. Implants in the bed nucleus also elevated CCK mRNA levels in the MeApd indicating that physiologic estrogen stimulation of CCK in the MeApd is the result of both local and distal transsynaptic elevation of CCK mRNA levels. The site-specific induction of CCK mRNA levels within the limbic-hypothalamic nuclei provides another important facet of estrogenic modulation of CCK induction. © 1996 John Wiley & Sons, Inc.     

Antiestrogens: Effects of tamoxifen,nafoxidine, and CI-628 on sexual behavior,cytoplasmic receptors,and nuclear binding of estrogen

These experiments utilized the estrogen antagonists CI-628, nafoxidine, and tamoxifen as tools to investigate potential molecular mechanisms of estrogen activation of female rat sexual behavior. Adult female rats, ovariectomized 4–7 days previously and matched for body weight, were administered single sc injections of one of the three antiestrogens, and the ability of the antagonists to block estrogen-induced sexual behavior, to deplete and replenish hypothalamic estrogen receptors, and to inhibit the binding of estradiol by hypothalamic nuclei 2 hr, or 1, 2, 4, or 7 days later was assessed. All three compounds produced a dose- and time-dependent inhibition of estrogen-activated lordosis, with tamoxifen being the most potent inhibitor. The three antiestrogens also caused prolonged depletion of hypothalamic estrogen receptors, but there was no correlation between receptor levels and the degree of inhibition of lordosis behavior at any time point following antiestrogen treatment. Rats showed high levels of sexual receptivity when antiestrogens were injected 2, 4, or 7 days before estrogen; however, hypothalamic estrogen receptors were still markedly (up to 70%) reduced at some of these time points. In contrast, there was a large (r = 0.67), significant correlation between the ability of all three agents to reduce [³H]estradiol binding by brain cell nuclei and their ability to reduce the display of estrogen-induced female sexual behavior. Antiestrogen injections which inhibited lordosis always decreased the level of specific estradiol binding by hypothalamic nuclei. These data indicate that delayed receptor replenishment does not adequately explain the antagonism of lordosis behavior by antiestrogens. The results presented here strongly point to the cell nucleus as the critical locus of receptor-mediated interactions which underlie estrogen and antiestrogen regulation of female sexual behavior.     

Effect of forebrain implants of testosterone or estradiol on scent-marking and sexual behavior in male and female rabbits

Chinning consists of rubbing the chin against an object, thereby depositing secretions from the submandibular glands. As mating, chinning is stimulated in male and female rabbits by testosterone and estradiol, respectively. To investigate the brain sites where steroids act to stimulate chinning and mating we implanted into the ventromedial hypothalamus (VMH) or the medial preoptic area (MPOA) of gonadectomized male and female rabbits testosterone propionate (TP; males) or estradiol benzoate (EB; females) and quantified chinning and sexual behavior. EB implants into the VMH or MPOA reliably stimulated chinning in females. Most of those implanted into the VMH and around half of the ones receiving EB into MPOA or diagonal band of Broca (DBB) showed lordosis. Chinning, but not sexual behavior, was stimulated in males by TP implants into the MPOA or DBB. Neither chinning nor mounting were reliably displayed by males following TP implants into the VMH. Results indicate that, in females, the VMH is an estrogen-sensitive brain area that stimulates both chinning and lordosis while the MPOA seems to contain subpopulations of neurons involved in either behavior. In males, androgen-sensitive neurons of the MPOA, but not the VMH, are involved in chinning stimulation but it is unclear if these areas also participate in the regulation of copulatory behavior.