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Two experiments examined the role of the steroid hormones, estradiol (E2), progesterone (P), and testosterone (T), in activating scent marking and 50-kHz ultrasonic vocalizations in ovariectomized Long-Evans rats in response to a devocalized male rat. In Experiment 1, females received, in a counterbalanced order, a series of six hormone treatments consisting of two injections (48-54 and 4 hr before behavioral tests). The six treatments were 8 micrograms E2 followed by 500 micrograms P or oil, 2 micrograms E2 followed by 500 micrograms P or oil, and oil followed by 500 micrograms P or oil. Injections of either the high or low dose of E2 followed by P resulted in high levels of vocalizations. Neither E2 by itself or P by itself were very effective. Surprisingly, none of the hormone treatments were effective in activating marking above the level seen when the females received control injections of oil. Four other hormone treatments were examined in Experiment 2: daily injections of 500 micrograms T, daily injections of 50 micrograms E2, implantation of silastic capsules of E2 (5% E2, 5 mm length) followed by 500-micrograms P injections before behavioral tests, and implantation of silastic capsules of E2 followed by oil injections. Animals receiving E2 by silastic capsule followed by P injection displayed the highest levels of marking and vocalizations across the five weekly tests. These results suggest that while E2 and P synergize for the display of female-typical behaviors similar to the hormonal regulation of lordosis, the mechanism of E2 action may be different for the two signaling behaviors. Scent marking appears to be responsive to the tonic levels of E2 released from silastic capsules.

The effects of testosterone (T) and its major metabolites, estradiol (E2) and dihydrotestosterone (DHT), on the restoration of sebaceous scent marking and 50-kHz ultrasonic vocalizations in male rats were measured in two studies (Experiments 1 and 2) employing different hormone levels. Silastic capsule administration of high and low doses of T (10 and 20 mm) or E2 (5%; 5 and 10 mm) completely restored marking to precastration levels. Both doses of DHT (30 and 40 mm) or no hormone replacement were without effect when tested in presence of estrous odor cues. In our testing paradigm, males appeared to mark glass objects with a sebaceous secretion rather than urine. Only the high dose of T (20 mm) restored vocalizations to intact levels, while animals receiving the high or low dose of E2 or DHT showed no restoration of behavior. In Experiment 3, habituation to estrous odor cues over weekly tests was not observed for marking or vocalizations. These results are discussed with respect to similarities and differences in the hormonal control of signaling behaviors by androgen metabolites in male rodents.

The progestin receptor antagonist RU 38486 (henceforth referred to as RU 486) was tested for facilitative effects on female receptive behavior in ovariectomized Long-Evans rats primed with 2 micrograms estradiol benzoate (EB). RU 486 (0, 0.5, 1.6, or 5.0 mg) was administered 48 hr after estrogen priming. The lordosis quotient (LQ) and lordosis score (LS) were assessed 4 hr after RU 486 administration in a standardized test consisting of a 10-mount test by a stimulus male. A significant dose effect was found by both LQ and LS, with those subjects receiving 5 mg of RU 486 being significantly more receptive than vehicle control animals. Thus RU 486 acted as a weak progestin agonist under testing conditions typical for assessment of progestin facilitation of female sexual behavior in rats. Low levels of proceptive behavior (hops and darts) were seen in a minority of the tests, and did not vary systematically as a function of the dose of RU 486 administered. We also examined the effects of RU 486 given before progesterone (P) on receptivity in a blocking paradigm and confirmed previous reports that the antagonist significantly attenuates facilitation of sexual behavior when given in combination with P. A progestin receptor assay of the cytosols of the hypothalamus-preoptic area in estrogen-primed female rats treated with 5 mg RU 486 revealed a significantly greater depletion of available cytosolic P receptors than when rats were treated with a similarly facilitating dose of P (100 micrograms). The results suggest a possible dual mode of action for RU 486--a weak, receptor-mediated agonistic effect on sexual behavior when given alone to estrogen-primed rats, and a competitive blocking effect on receptivity when administered with P.

The CNS sites of action for progesterone facilitation of female sexual behavior are disputed. Among the areas most often cited are the ventromedial hypothalamus and the ventral midbrain. There is also a controversy about whether estradiol may substitute for progesterone for the facilitation of receptive behavior when given systemically or intracranially. We tested VMH and ventral midbrain applications of estradiol versus progesterone for the facilitation of female sexual behavior in estrogen-primed, ovariectomized female rats. Subjects were implanted with bilateral guide tubes aimed at ventral hypothalamic or midbrain sites. Estrogen-primed rats received either 28-gauge insert cannulae filled at the lumen with pure progesterone, estradiol, or cholesterol, or empty tubes, and were tested for receptivity with intact, experienced stud males just before, and 1 and 4 hr after, intracranial hormone administration. Significant estrous responsiveness was seen only in the 4-hr test after progesterone was implanted in the VMN in the first intracranial cannula test. We conclude, in contrast to some previous reports, that administration of progesterone to the VMN is more effective in the facilitation of female sexual behavior than when it is implanted in the ventral midbrain, and that administration of estradiol to either site is ineffective.

The present experiment was designed to determine whether sequential replacement of estrogen and progesterone to the ventromedial hypothalamus (VMH) would be sufficient to induce estrous behavior in ovariectomized rats. Bilateral cannulae containing 17 beta-estradiol (E2) diluted with cholesterol (1:250) were lowered into the VMH, preoptic area or midbrain and left in place for 4 days. On day 5, the E2 inserts were removed and P-filled cannulae were lowered into half of the subjects. The remaining females received systemic progesterone (500 micrograms). This steroid regimen was repeated 2 weeks later with the mode of progesterone administration reversed. All subjects were tested for estrous behavior twice after progesterone treatment. In a second experiment, 3H-P:P-filled cannulae were lowered into the VMH of estrogen-primed females in order to estimate the extent of hormone spread from full-strength P-filled cannulae. Results indicated that estrogen and progesterone stimulation of the VMH is sufficient to activate estrous behavior in spayed female rats, however, precise localization of the hormone implants within the VMH is essential. 9 of the 11 females with both cannulae located within or at the border of the ventromedial nucleus (VMN) exhibited estrous behavior whereas only half of the females with only one implant resting in the VMN exhibited estrous responsiveness. Subjects with neither cannula located within or at the border of the VMN did not exhibit the behavior. The facilitative effects of P appeared to result from hormonal stimulation of the VMH and not from leakage of the steroid into other brain regions or into the systemic circulation. Following placement of tritiated progesterone implants into the VMH, high levels of radioactivity were recovered only from the mediobasal hypothalamus. The low levels of radioactivity measured in other brain regions, pituitary, uterus and blood indicate that relatively little if any hormone reached these tissues.

Ultrasonic vocalizations were measured when male rats were placed with ovariectomized females that had experienced various hormonal and behavioral treatments. In Experiment 1, 18 males were tested with females in each of the following conditions: nonestrous (OVX), estrogen treated (E), estrogen and progesterone treated (EP), and estrogen and progesterone treated and given two intromissions from a stud male prior to testing (EPI). Control conditions included clean cage (CL) and cage soiled by an estrous female (SOI). The treatments differed in effect on rate and maintenance of vocalization: EP greater than E greater than EPI = OVX greater than SOI greater than CL. In tests in which males produced a high rate of vocalization, some males with short intromission latencies shifted from the normal 50-kHz pulse to a 22-kHz pulse. In Experiment 2, the effect of the female's vocalization and movement on the rate of and latency to vocalization was measured. Twenty-one males were presented with each of the following stimulus conditions: estrous female with red light (EP), estrous female without red light (EP dark), estrous anesthetized female (EP anes), and nonestrous anesthetized female (OVX anes). Effects on vocalization of various treatments were as follows: EP = EP dark greater than EP anes greater than OVX anes. These data suggest that the 50-kHz vocalizations constitute a graded response influenced by the female's hormonal and sexual condition.

Ejaculatory patterns were observed in normally reared, postpuberally castrated male and female rats treated with sex hormones and electrical shock in adulthood. In a preliminary experiment, 3 females treated with 50 microgram estradiol benzoate (EB) and 500 microgram progesterone (P) showed intromission and ejaculatory behavior when subjected to shock. Refractory periods were abnormally short and there was almost no postejaculatory vocalization. In Experiment 1, males and females were tested with electrical shock following daily treatment with 32 microgram EB with and without 500 microgram P on the test day. There was no difference between males and females in preejaculatory behavior, but females displayed abbreviated refractory periods and no postejaculatory vocalization. Progesterone had no observed effect. In Experiment 2 castrated males and females were subjected to shock after treatment with 8 microgram EB per day for 3 weeks. No P was given. Again females showed drastically reduced refractory periods and little vocalization. In Experiment 3, males and females treated with TP and shock displayed ejaculatory patterns, normal refractory periods and vocalization. Results show that female rats are capable of exhibiting the ejaculatory response without sex hormone treatment in perinatal life or androgen treatment in adulthood. It was also demonstrated that there is a sex difference in the postejaculatory behavior shown by estrogen-treated male and female rats.

Five groups of rats were observed for sexual behavior as intact subjects and in the 3 weeks following castration. Particular emphasis was placed upon ultrasonic vocalization recorded during the refractory period (RP) following ejaculation. All rats received daily injections (100 mug test steroid + 100 mug dihydrotestosterone propionate, DHTP) from the day of castration. Treatment groups were: Testosterone propionate (TP) + DHTP, N = 5; 19-hydroxytestosterone propionate (19HTP) + DHTP, N = 6; Oestradiol dipropionate (OP) + DHTP, N = 5; DHTP (200 mug/day), N = 6; Vehicle only, N = 7. Ejaculation and normal RPs were maintained with TP, 19HTP and OP treatments. In rats treated with DHTP alone or vehicle only ejaculatory ability declined and RPs increased in length. Ultrasonic vocalization was normal in all rats except those treated with OP, where it was either reduced or absent. In Week 4 treatments in TP + DHTP and OP + DHTP groups were reversed. During Weeks 5-7 vocalization was gradually restored in rats that previously received OP and partially inhibited in animals previously treated with TP. These results indicate that; (1) vocalization may not be dependent on the presence of testicular androgens and, (2) OP has a gradual inhibitory effect on vocalization when given at high doses and this effect is reversible.

The putative neural target sites of progesterone (P) action for the facilitation of estrous behavior in female rats were reexamined using the hormone implant technique. Subjects were ovariectomized, estrogen-primed Long-Evans females. All were outfitted with bilateral double barreled cannulae assemblies consisting of 23-gauge guide cannulae and 28-gauge inserts. Subjects in Exp 1 received sc injections of individually determined threshold priming doses of estradiol benzoate. Estrogenic priming for subjects in Exp 2 was provided by sc placed Silastic capsules (5 mm) filled with 17 beta-estradiol-cholesterol (1:20). Each subject was tested for estrous behavior with a male after P-filled cannulae and after blank inserts were lowered into the brain. Behavioral tests were conducted 1 and 4.5 h postcannulae placement. Thirty-five of 40 females with P in the ventromedial hypothalamus exhibited high levels of lordosis behavior during the 4.5-h test. Moreover, 29 of the 35 exhibited solicitation behavior as well. Estrous behavior was not exhibited by these animals during the 1-h test. Implants in other regions of the brain (i.e. mesencephalic reticular formation, preoptic area, central grey, caudate putamen, and hippocampus; n = 80) did not consistently facilitate estrous responsiveness. In Exp 3, levels of estrous responsiveness were similar before and after adrenalectomy (n = 10); thus, facilitation of estrous behavior by P-filled implants in the ventromedial hypothalamus was not dependent upon activation of the hypothalamic-pituitary-adrenal axis.

In an attempt to obtain a more precise localization of the neural target site(s) of estrogen-priming action in the activation of estrous behavior, 30-gauge cannulae containing a preparation of 17 beta-estradiol diluted 1:250 with cholesterol were implanted bilaterally into the brains of ovariectomized rats. Subjects were tested for sexual behavior with intact males at 4-day intervals for 3 weeks beginning 2-3 days after stereotaxic surgery. Animals received a systemic injection of 0.5 mg progesterone 4-6 h before all but 1 test. After this treatment, estrous behavior was observed in 19 of the 20 animals judged to have both cannulae resting in or within 0.25 mm of the ventromedial nucleus of the hypothalamus (VMN). Virtually no estrous behavior was observed without the administration of progesterone. Behavioral scores decreased as a function of the distance of the cannulae tips from the VMN. Cholesterol implants in the same region (n = 8) were without effect. Implants in other regions of the brain (e.g. preoptic area, diagonal band of Broca, lateral habenula, amygdala, or cortex; n = 52) were ineffective. These results indicate that estrogenic stimulation of the region of the VMN alone is sufficient to prime the activation of estrous behavior in the ovariectomized rat.

The ventromedial nucleus of the hypothalamus (VMN) is thought to constitute an essential neural substrate for hormonal induction of female sexual behavior. In previous work, implants of dilute (0.4%) estradiol (E2) have been found to prime progesterone-facilitated female sexual behavior in rats only when they are within or very close to the VMN. In other studies, induction of cytosolic progestin receptors (PRc) in the hypothalamus has been correlated with systemic E2 priming of receptive behavior. We combined dilute E2 implants with a Palkovits punch microassay for PRc in the an effort to examine the relationship between estrous behavior and estrogen-induced PRc in the VMN of individual female rats. Ovariectomized rats were given bilateral guide cannulae aimed at the VMN and were primed for 3 days with 1) blank implants plus oil sc, 2) blank implants plus 0.5 micrograms E2 benzoate sc, 3) implants of 0.4% E2 in cholesterol, or 4) implants of 2.0% E2. On day 4 all rats received progesterone (P) sc and were tested for receptive behavior. The following week all subjects received the same estrogen-priming treatment but were killed on day 4 without P treatment. The VMN was punch dissected and assayed for PRc. High levels of lordosis were seen in the E2 benzoate-treated and 2.0% E2-implanted animals, while little behavior was seen in the oil-treated animals. Among 0.4% E2-implanted animals, higher levels of lordosis were observed in subjects with implant placements in the rostral than in the caudal VMN; however, lordosis quotients were not correlated with PRc induction measured in the entire VMN of individual animals. In general, our findings suggest that local estrogenic stimulation of target cells in the VMN is capable of both priming the estrous response and induction of P receptors. Whether these two effects are causally related awaits future study.

The effects of intracerebral estradiol benzoate (EB) implants upon lordosis behavior in castrated male rats were investigated. 27 guage EB-filled or Blank implants were placed unilaterally in either the preoptic area (POA) or the region of the ventromedial hypothalamic nucleus (VMH). When 0.5 microgram of EB was administered subcutaneously for eight days, males with EB implants had significantly higher lordosis quotients (LQ) than males with Blank implants. EB implants located in the region of the VMH were significantly more effective in stimulating lordosis responses than implants in the POA. These results indicate that, in male rats as in females, the VMH region is a primary site for the hormonal activation of lordosis behavior.

Intracranial implants of estradiol benzoate (EB) were used to explore sites of action for the activation of masculine copulatory behavior in castrated male rats. In experiment 1, 27 or 30 gauge implants were placed unilaterally throughout the hypothalamus and preoptic area. Dihydrotestosterone (DHT) was administered subcutaneously (s.c.) to each male. 25 of 42 males with EB implants located in the medial anterior hypothalamic-preoptic area (AHPOA) exhibited ejaculatory patterns on at least 40% of the post-implantation tests. In contrast, only 4 of 18 of the males with implants located posterior to the anterior hypothalamic area displayed this level of response. This difference in the proportion of animals responding for each implant group was statistically significant. Seminal vesicle weights were not correlated with the occurrence of copulation. In experiment 2, either EB-filled or empty cannulae were placed unilaterally in the AHPOA. Half of the males of each group also received DHT s.c. Intracranial EB in conjunction with DHT s.c. resulted in a significantly greater frequency of ejaculatory response than any other treatment. Despite significant differences among the groups in seminal vesicle weights, there was no correlation with occurrence of ejaculatory behavior. These results are consistent with the hypothesis that testosterone (T) exerts its effects upon masculine sexual behavior via conversion to estradiol (E2); and that the principal neural site of action is the AHPOA.