Estrous Responsiveness Research Articles

Antisense oligonucleotide blocks progesterone-induced lordosis behavior in ovariectomized rats

Antisense oligonucleotides were utilized to interfere with the synthesis of progesterone receptor. A specific aODN was shown to decrease significantly the levels of PR in the T47D cell line known to express the PR gene. The molecule described was proved to be effective in hindering the effect of progesterone on target gene expression in T47D cells. The aODN was then utilized in an in vivo study to test its efficacy on the female rat sex behavior. Its injection in the ventromedial hypotalamus significantly inhibited the estrous responsiveness induced by progesterone. This study provides a clear demonstration of the full involvement of PR in the manifestation of lordosis behavior and demonstrates the activity of an aODN as antiprogestative.

Effects of non-competitive NMDA receptor antagonists on reproductive and motor behaviors in female rats

MK-801 and dextrorphan, selective non-competitive antagonists atN-methyl- d-aspartate (NMDA) receptors, were used to evaluate the effect of NMDA receptor blockade on sexual and motor behaviors in female rats. Ovariectomized rats were treated with estradiol benzoate (EB) for 48 or 72 h followed by progesterone (P) 3.5–4 h before testing the animals for sexual receptivity. After testing for estrous responsiveness, the effect of NMDA antagonists on several motor behaviors was also assessed. Lordosis frequency and intensity were inhibited in animals that received 0.5 mg/kg MK-801 30 min before EB; the same dose of MK-801 was relatively ineffective when administered 24 h after EB. In neither case did MK-801-treated females differ from controls when motor behaviors were assessed after mating tests. When 30 mg/kg dextrorphan, a short-acting NMDA antagonist, was administered 15 min before P, sexual behavior was not blocked. However, both 0.05 mg/kg MK-801 and 30 mg/kg dextrorphan suppressed ongoing female sexual behavior within 30 min in animals made receptive with EB and P. These deficits in sexual behavior were associated with changes in motor performance. MK-801 (0.1 mg/kg) and dextrorphan (30 mg/kg) abolished movement in the vertical dimension (e.g. jumping and rearing). By contrast, the drugs increased movement in the longitudinal (locomotion) and lateral (circling) dimensions. At 0.2 mg/kg, MK-801 blocked movement in both the vertical and longitudinal dimensions; however, it failed to block circling. Only at 0.4 mg/kg did MK-801 inhibit lateral movements and righting reflexes. It is likely that the acute inhibition of sexual behavior by NMDA antagonists is related to changes in sensorimotor processing and/or motor performance.

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 μg 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.

Progesterone versus estrogen facilitation of female sexual behavior by intracranial administration to female rats

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 role of ovarian sympathetic innervation in the control of estrous responsiveness in the rat

This study examined the contribution of the superior ovarian nerve (SON) to estrous responsiveness and ovarian function in cycling rats. Section of the SON was carried out at 1100 on proestrus, and lordotic responsiveness was measured at 1500, 1700, and 2100 on that day and at 0900, 1200, and 1500 on the day of estrus. SON section decreased lordosis intensity significantly at 1500 and 1700 on proestrus and at 0900 on estrus. Pacing of sexual contacts with males was decreased at 2100 in nerve-sectioned (Nervx) animals when compared with sham-operated controls (SHAM). Serum progesterone (P) concentrations were significantly lower in Nervx animals than in Sham animals 30 min after surgery, but were not different between groups at 4.5 hr. Serum estradiol (E 2) concentrations did not differ between groups at either time. In addition, Nervx and Sham groups did not differ on measures of pregnancy/pseudopregnancy initiation or on measures of ovarian function 10 days after surgery. These data suggest that the integrity of the SON is necessary for the display of full estrous responsiveness in cycling rats, and suggest that the acute decreases in serum P occurring as a consequence of SON section may be responsible for the deficits seen in Nervx animals.

Temporal aspects of ventromedial hypothalamic progesterone action in the facilitation of estrous behavior in the female rat.

Progesterone (P) action following estrogen priming is required normally for the facilitation of estrous behavior in female rats. Although mechanisms by which P exerts its influence on estrous responsiveness have not been elucidated, the primary site of action of P has been shown to be the ventromedial nucleus of the hypothalamus (VMN). The objective of the present series of experiments was to describe the temporal parameters of P action in the VMN in the facilitation of estrous behavior in estrogen-primed female rats. Subjects were female Long-Evans rats stereotaxically outfitted with 23-ga. guide cannulae directed towards the VMN. Crystalline P was applied directly to the brain tissue via bilateral 28-ga. insert cannulae, which could be inserted and removed easily through the guide cannulae. Animals were ovariectomized and estrogen primed with 5% estradiol Silastic capsules. They received a counterbalanced series of two experimental tests: one involving a manipulation with a P-filled implant, and another with a blank implant. In the first experiment, a significant increase in estrous responsiveness occurred only after 2 hr exposure of the VMN to P, whereas 4 hr were required for a full display of estrous behavior, including solicitation. In Experiment 2, P was lowered into the brain for either 1, 2, or 4 hr, and testing took place 4 hr after the lowering of the implant. It was found that 2 hr of P exposure was sufficient to facilitate full estrous responsiveness at 4 hr. In Experiment 3, it was revealed that the duration of estrous responsiveness was directly related to the time the P implant remained in the brain. In the fourth experiment, the time course of P retention in brain tissue, revealed by determination of 3H-progesterone levels in hypothalamus, agreed with the behavioral findings. Progesterone levels in the region of the VMN remained high while a P implant was in place, but declined rapidly after removal. A dual mechanistic hypothesis for P action in the facilitation of estrous behavior is presented.

Actions of progestins on estrous behavior in female rats

The present study evaluated the facilitative actions of progesterone and the synthetic progestin R 5020 on estrous responsiveness in ovariectomized, estrogen-primed female rats. The dose-response and time-response characteristics of the behavior facilitating actions of both progesterone and R 5020 were measured. The threshold doses for the facilitation of estrous behavior in estrogen-primed female rats were 1 μg of R 5020 and 100 μg of progesterone. These doses of progestins facilitated estrous responsiveness with a similar time course that approached maximum at one hour. To examine the possible mechanism(s) of action of each progestin the synthetic progestin antagonist RU 38486 was used. The inhibitory effects of RU 38486 on estrous behavior facilitated by a threshold dose of progesterone or R 5020 were found to be almost identical. RU 38486 (5 mg) administered 1 hr prior to progesterone or R 5020 suppressed lordosis behavior by 44% and 47% respectively. These results suggest that progesterone and R 5020 facilitate estrous responsiveness through the same mechanism.

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.

Intrahypothalamic effects of progestin agonists on estrous behavior and progestin receptor binding

To investigate whether metabolism of progesterone (P) to other progestins is necessary for the facilitation of estrous behavior in estrogen-primed rats, we evaluated the behavioral effectiveness of intrahypothalamic implants of two P metabolites, 5α-dihydroprogesterone (DHP) and 20α-hydroxyprogesterone (20HP), and of desoxycorticosterone (DOC). We also determined whether the progestin receptor binding capacity of the steroids correlated with their behavioral efficacy. Implants of DHP and 20HP into the ventromedial hypothalamus were considerably less effective than P in activating estrous behavior; in contrast, the mineralocorticoid DOC was nearly as effective as P. Binding studies showed that P had the highest affinity for brain progestin receptors followed by DHP, DOC and 20HP. Thus there was a poor correlation between the behavioral efficacy and the progestin receptor binding properties of the steroids tested. These data suggest (1) that neither 5α-reduction nor 20α-hydroxylation are necessary for P activation of estrous responsiveness and (2) that the structural features required for the behavior promoting effects of P may not be identical to those required for binding to brain progestin receptors.

Progesterone in the ventromedial hypothalamus of ovariectomized, estrogen-primed rats inhibits subsequent facilitation of estrous behavior by systemic progesterone

The putative neural target sites of progesterone's (P) inhibitory on estrous behavior were re-examined utilizing intracranial hormone implants. Subjects were estrogen-primed ovariectomized, Long-Evans rats, and all were outfitted with permanent indwelling guide cannulae aimed for the ventromedial hypothalamus (VMH), preoptic area (POA), or midbrain. In a series of 4 weekly testing paradigms, the ability of blank or P-filled implants to both facilitate estrous responsiveness and to interfere with an animal's ability to exhibit estrous behavior following a systemic injection of P 24 hours later was assessed. P-filled implants placed bilaterally into the VMH inhibited the subsequent facilitation of estrous behavior by systemic P administration. Neither P-filled implants in other brain regions nor blank implants lowered into the VMH had this same effect. Seven of the females that exhibited progesterone-induced behavioral refractoriness 24 h after P stimulation of the VMH exhibited facilitation of estrous responsiveness in behavioral tests given 4.5 h after cannulae placement suggesting that progesterone may exert its facilitative and inhibitory actions on estrous responsiveness at the same neuronatomical locus.

Behavioral effects of progestin in the brain.

In this article we review research on the role of progestins in the regulation of estrous responsiveness in female rats. Estrous responsiveness normally results from a synergistic action of estradiol (E2) and progesterone (P). E2 primes the system but normally does not result in estrous behavior. The full expression of estrous responsiveness results from the action of P on the E2-primed system. It has been demonstrated with implants of dilute E2 (1 part E2: 250 parts cholesterol) that the site of E2 priming is the ventromedial nucleus of the hypothalamus (VMN). In females primed with systemically administered E2, P also acts on the VMN to facilitate full estrous responsiveness. It has been shown in addition that estrous responsiveness results from sequential application of E2 and P to the VMN but not to other areas of the brain. The VMN is also the site at which P produces sequential inhibition of estrous responsiveness. The time course of P action in facilitating full estrous responsiveness is about two hours, regardless of whether the hormone is administered intracerebrally or intravenously. The duration of estrous responsiveness is directly correlated with the length of time P is in contact with brain tissue. Experiments with the protein synthesis inhibitor anisomycin are consistent with the view that P acts in the VMN by way of a protein synthetic mechanism to facilitate estrous behavior; however, other mechanisms must be considered as alternatives. Finally, we address the question of whether estrogenic priming depends upon induction of progestin receptors in the VMN. Results indicate that estrogenic priming of estrous responsiveness may occur without concomitant induction of progestin receptors.

Blockade of progesterone-activated estrous behavior in rats by intracerebral anisomycin is site specific.

The hypothesis that progesterone (P) activates estrous behavior in estrogen-primed female rats via a protein synthetic mechanism was examined. The protein synthesis inhibitor anisomycin was applied intracerebrally via 28-gauge bilateral implants to neural sites implicated in the mediation of estrous responsiveness. Results showed that anisomycin blockade of P-activated estrous behavior was neural site specific. Animals with anisomycin placed in the ventromedial hypothalamus (VMH) (n = 27) showed low levels of lordosis and solicitation behavior 4 h after the subcutaneous administration of 500 micrograms P, whereas animals with anisomycin implants in the preoptic area (n = 11) or the midbrain area in the region of the interpeduncular nucleus (n = 11) displayed high levels of estrous behavior. No deficits in open-field activity were observed following localized anisomycin treatment, and all animals appeared to be healthy. Results of the present study are consistent with the hypothesis that P acts to promote estrous behavior via a receptor-mediated genomic protein synthetic mechanism, and provide additional evidence that the VMH is the primary site of P action.

Progesterone in the ventromedial hypothalamus facilitates estrous behavior in ovariectomized, estrogen-primed rats.

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.

Effects of an anti-androgen and 5α-reductase inhibitors on estrus duration in the cycling female rat

Five experiments examined the role of circulating androgens in the control of sexual behavior (lordosis) in the intact cycling rat. The androgen receptor blocker, flutamide (FLU), was administered daily to cycling rats beginning on the day of estrus, and lordotic responsiveness was measured on the 2nd subsequent proestrus day and on the day of estrus. FLU-treated females showed significantly higher levels of lordosis throughout the end of the period of estrus than controls (Experiment 1). Neither the maximal levels of lordosis seen on the evening of proestrus nor the time of onset of estrous responsiveness during the preceeding afternoon were affected by FLU (Experiment 2). Serum estradiol concentrations seen on the morning of proestrus (Experiment 3) did not differ between FLU- and vehicle-treated animals. The weak 5α-reductase inhibitor, testosterone-17β-carboxylic acid (17βC), prolonged slightly, but did not significantly lengthen, the period of estrus (Experiment 4), while the highly potent steroidal 5α-reductase inhibitor, 4 MA, significantly increased the rate at which estrous behavior declined on the day of estrus (Experiment 5). Circulating androgens do not appear to affect the maximal level of sexual receptivity displayed nor the time of estrus onset; however, they may govern the duration of the period of estrus by influencing the rate of estrus termination.

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.

Dose-response and time-response relationships between progesterone and the display of patterns of receptive and proceptive behavior in the female rat

The relationship between administration of progesterone and the display of patterns of receptive (response to the male) and preceptive (female initiated) sexual behavior was examined in ovariectomized, estrogen-primed female rats in a “restrained male” test situation. It was found that the degree of receptivity and proceptivity displayed was directly proportional to progesterone dose and time from progesterone injection (up to 4.5 hr). Higher progesterone doses and longer period of time from progesterone injection (up to 4.5 hr) were both associated with shorter latencies to return to the male following intromission and ejaculation. Receptivity could be induced with estrogen alone but progesterone was required for the display of proceptivity and higher doses of progesterone were needed to effect increases in proceptivity relative to receptivity. Proceptive behavior also occurred in a narrower time range than did receptive behavior. Receptivity alone is characterized as the lowest degree, and receptivity plus proceptivity as the highest degree, of expression of the total behavior pattern of the estrous female rat. Receptivity and proceptivity together constitute a continuum of estrous responsiveness. Increasing the progesterone dose from 0 to 200 μg, and increasing the latency from progesterone injection from 0 to 4.5 hr, were associated with increasing degree of expression of the total behavioral continuum.