Actions of RU 38486 on progesterone facilitation and sequential inhibition of rat estrous behavior: correlation with neural progestin receptor levels.

The present study examined the actions of the antiprogestin RU 38486 on progesterone (P)-induced facilitation and sequential inhibition of estrous behavior and on brain cytosol progestin receptor (PR) levels in ovariectomized, estrogen-primed (0.5 micrograms of estradiol benzoate for 3 days) female rats. RU 38486 suppressed P-facilitated receptive and proceptive responses in a dose-dependent fashion when administered 1 hr prior to P. RU 38486 did not, however, block the sequential inhibitory effect of P. Indeed, when it was administered alone at a dose of 1 mg, animals were rendered behaviorally unresponsive to a P treatment 25 hr later. It is unclear whether RU 38486 is an agonist for P sequential inhibition of estrous behavior or if the apparent agonist action of RU 38486 actually results from a long-term antagonist effect. Estrogen-induced PRs remain elevated (35-55% above basal) in the hypothalamus (HYP) and preoptic area (POA) at 24 and 48 hr following the last estrogen injection. Thus P facilitation of estrous behavior is correlated with increased levels of cytosol PRs. RU 38486 reduced cytosol PRs in both brain regions to basal levels within 2 hr, and the levels remained suppressed 25 hr following the treatment. Hence there is a strong correlation between behavioral inhibition and suppressed cytosol PRs at both short (5 hr) and long (25 hr) intervals after RU 38486 administration. A P treatment which produced sequential inhibition of estrous responsiveness 24 hr later did not reduce the estrogen-induced level of cytosol PRs in either brain region. These results suggest that mechanisms in addition to induction of PRs may be necessary to ensure successful mating.

A simple method of drying virus on inanimate objects for virucidal testing.

A simple method for drying virus on inanimate objects (cover slips) under vacuum in the cold is described. Following this procedure virus maintains high titers (10(6-7)) for periods of 1-3 wk at -70 degrees C depending on the virus. For virucidal assay of disinfectants, cover slips are exposed to medium simulating the disinfectant (virus control) or disinfectant in an upright position in an Ultra-Vu cuvette. Cover slips are readily removed and placed in tissue culture medium for dilution of virus and determination of virus titer. Cytotoxicity of disinfectant is determined by exposing cover slip without virus to disinfectant, then placing it in medium, diluting the medium and incubating with the indicator cells. The use of this technique results in high titers of virus on cover slips, which are inanimate objects requiring minimal manipulation. The titration of virus or cytotoxicity in microplates is cell, medium, serum, and labware economical.

Antagonism of female sexual behavior with intracerebral implants of antiprogestin RU 38486: correlation with binding to neural progestin receptors.

The steroidal antiprogestin 17 beta-hydroxyl-11 beta-(4-dimethylaminophenyl)-17 alpha-(1-propynl)estra-4,9-dien-3-one (RU 38486) was administered systemically or was implanted into the ventromedial hypothalamus and other brain regions (habenula, preoptic area, interpeduncular region, in order to determine whether the compound could antagonize progesterone (P) activation of estrous responsiveness and whether the compound would exert its behavioral effects at the presumed site of P action and/or at other neural sites implicated in the regulation of female sexual behavior. RU 38486 (5 mg) administered sc 1 h before 200 micrograms P inhibited P facilitation of lordosis behavior in estrogen-primed rats. Intracerebral application of RU 38486 to the ventromedial hypothalamus reduced lordosis responses in 14 of the 25 animals tested. Similar implants in the habenula also inhibited lordosis in 5 of the 14 animals tested. Antiprogestin implants in the interpeduncular region and preoptic area were virtually without effect (1 of 7 inhibited in each group). Interactions of RU 38486 with steroid binding sites in the hypothalamus-preoptic area (HPOA) were also assessed. RU 38486 appeared to be a competitive inhibitor of progestin ([3H] R5020) binding in HPOA cytosols. Scatchard analysis of [3H]RU 38486 binding showed that when unlabeled P was used as the competitor to assess nonspecific binding, the antiprogestin bound with high affinity [dissociation constant Kd = 8.4 nm] to brain cytosols. In addition, the number of [3H]RU 38486 binding sites in HPOA cytosol increased by approximately 50% in estrogen-primed female rats. Competition studies indicated that unlabeled RU 38486 was the most effective competitor for [3H]RU 38486 binding but that P and R5020 were nearly as effective. Corticosterone, hydrocortisone, deoxycorticosterone, and triamcinolone also competed for [3H]RU 38486 binding but were somewhat less effective than the progestins. Testosterone and estradiol did not displace [3H]RU 38486 except at very high molar excesses. Thus RU 38486 appears to bind with highest affinity to HPOA progestin receptors, but it also binds to glucocorticoid receptors. These data are consistent with the interpretation that inhibition of estrous responsiveness by RU 38486 is associated with the antagonist's interference with brain progestin binding.

Induction of estrous behavior in ovariectomized rats by sequential replacement of estrogen and progesterone to the ventromedial hypothalamus.

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.

Priming of estrous responsiveness by implants of 17 beta-estradiol in the ventromedial hypothalamic nucleus of female rats.

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.

Activation of masculine sexual behavior by intracranial estradiol benzoate implants in male rats.

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.

Activation of feminine sexual behavior in castrated male rats by intrahypothalamic implants of estradiol benzoate.

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.

Autoradiographic localization of androgen-concentrating cells in the brain of the male domestic fowl.

Cells in the male fowl brain which accumulate radioactivity following 3H testosterone (T) administration were identified by autoradiography. Labelled cells were found principally in hypothalamic, limbic and midbrain structures. Marked uptake was observed in the preoptic area (POA) and in the anterior and posterior hypothalamus. There was also a significant amount of labelling in the archistriatum (ARCH), particularly in the nucleus taeniae (Tn), and in the lateral septum. In the midbrain, substantial uptake of labelled hormone was found in the nucleus intercollicularis (ICo). The pattern of accumulation of T in the male fowl was comparable to that for sex hormone uptake in vertebrates in general. Furthermore, accumulation was generally found in areas known to be concerned with sex hormone-dependent functions.

Sexual behavior: Extreme reduction of postejaculatory refractory period by midbrain lesions in male rats.

The refractory period that characteristically follows ejaculation was abolished or significantly reduced by rostral midbrain lesions in male rats. The postejaculatory vocalization was also abolished or reduced, but other aspects of copulatory performance were unaffected. The results were attributed to disruption of biogenic amine pathways that pass from the ventral part of the rostral midbrain into the posterior hypothalamus.