Interactions of progesterone and dihydroprogesterone with dihydrotestosterone on estrogen activated sexual receptivity in female mice

Lordosis behavior in response to a mounting stimulation was quantified in ovariectomized female CD-1 and SW mice primed with estradiol benzoate and either progesterone or dihydroprogesterone. Both progestins were effective in stimulating receptivity, in CD-1 females, while only progesterone was effective in SW females. Dihydrotestosterone given concurrently with both the estrogen and progestin injections was found to have no effect on progesterone stimulated receptivity, but it produced a dose related inhibition of receptivity in the dihydroprogesterone treated females.     

Activation and inhibition of isolation induced inter-male fighting behavior in castrate male CD-1 mice treated with steroidal hormones

Intermale fighting behavior between castrate male CD-1 mice living in isolation and castrate male CD-1 mice living in groups of 10 was activated by treating the isolated males with either testosterone or testosterone propionate. Fighting behavior was not activated by treating isolated males with androstenedione or dihydrotesterone even though these androgens were active in maintaining seminal vesicle weight at levels equal to, or greater than, those observed in gonadally intact males. Neither fighting behavior nor seminal vesicle weight was stimulated by treatment with progesterone, estradiol benzoate, or estradiol benzoate plus progesterone. Concurrent administration of progesterone inhibited fighting behavior activated by low, but not moderate to high doses of testosterone propionate. These results suggest that the hormone requirements for activation of fighting behavior in the male CD-1 mouse are more restrictive than those for maintenance of peripheral target tissues and that progesterone acts in the brain to competitively inhibit androgen-activated fighting.     

Induction of lordosis behavior in female rats by intravenous administration of progestins

Progesterone is rapidly metabolized by neural cells in the rat. Progesterone could, therefore, act as a “prohormone,” stimulating lordosis behavior in estrogen-primed rats only after metabolic conversion. Were such the case, one might expect one or more of the naturally occurring metabolites of progesterone to be more potent than the parent compound. Estradiol benzoate-primed rats were therefore administered intravenously 200 μg of progesterone or one of five immediate metabolites of progesterone. The steroid 20α-dihydroprogesterone was found to be more potent than progesterone. Both 20α-hydroxy-5α-pregnan-3-one and 3α-hydroxy-5α-pregnan-20-one were less potent than progesterone, but more potent than the vehicle propylene glycol. Neither 5α-pregnane-3α, 20α-diol nor 5α-pregnane-3,20-dione (dihydroprogesterone, DHP) differed from the vehicle in potency. The data suggest that 20α-dihydroprogesterone, which is secreted at high levels during the estrous cycle, could play a role in the regulation of sexual receptivity. The data also suggest that 5α-reduction is probably not crucial for progesterone's action.     

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.     

Orphanin FQ in the mediobasal hypothalamus facilitates sexual receptivity through the deactivation of medial preoptic nucleus mu-opioid receptors

Sexual receptivity, lordosis, can be induced by sequential estradiol and progesterone or extended exposure to high levels of estradiol in the female rat. In both cases estradiol initially inhibits lordosis through activation of β-endorphin (β-END) neurons of the arcuate nucleus of the hypothalamus (ARH) that activate μ-opioid receptors (MOP) in the medial preoptic nucleus (MPN). Subsequent progesterone or extended estradiol exposure deactivates MPN MOP to facilitate lordosis. Opioid receptor-like receptor-1 (ORL-1) is expressed in ARH and ventromedial hypothalamus (VMH). Infusions of its endogenous ligand, orphanin FQ (OFQ/N, aka nociceptin), into VMH–ARH region facilitate lordosis. Whether OFQ/N acts in ARH and/or VMH and whether OFQ/N is necessary for steroid facilitation of lordosis are unclear. In Exp I, OFQ/N infusions in VMH and ARH that facilitated lordosis also deactivated MPN MOP indicating that OFQ/N facilitation of lordosis requires deactivation of ascending ARH-MPN projections by directly inhibiting ARH β-END neurons and/or through inhibition of excitatory VMH–ARH pathways to proopiomelanocortin neurons. It is unclear whether OFQ/N activates the VMH output motor pathways directly or via the deactivation of MPN MOP. In Exp II we tested whether ORL-1 activation is necessary for estradiol-only or estradiol + progesterone lordosis facilitation. Blocking ORL-1 with UFP-101 inhibited estradiol-only lordosis and MPN MOP deactivation but had no effect on estradiol + progesterone facilitation of lordosis and MOP deactivation. In conclusion, steroid facilitation of lordosis inhibits ARH β-END neurons to deactivate MPN MOP, but estradiol-only and estradiol + progesterone treatments appear to use different neurotransmitter systems to inhibit ARH-MPN signaling.     

The effect of estrogen treatment on scopolamine inhibition of lordosis.

Previous evidence indicates that the cholinergic muscarinic antagonist, scopolamine, inhibits lordosis in female rats. In the experiments reported here, the effects of various doses and repeated administrations of estrogen on the scopolamine inhibition of lordosis were examined. In the first experiment, intraperitoneal injections of scopolamine (1 mg/rat) completely inhibited lordosis in ovariectomized rats primed with low doses of estradiol benzoate (0.25 or 0.5 micrograms for 3 days) and progesterone (500 micrograms). However, scopolamine was significantly less effective in inhibiting lordosis in females primed with a higher dose of estradiol benzoate (25 micrograms for 3 days) and progesterone (500 micrograms). When hormone priming was repeated on subsequent weeks, scopolamine continued to inhibit lordosis in females that received 0.25 micrograms estradiol benzoate but was less effective in females primed with 0.5 micrograms. Scopolamine failed to inhibit lordosis in females treated with 25 micrograms estradiol benzoate on these later tests. In the second experiment, various doses of scopolamine (1, 2, or 4 mg/rat) were administered intraperitoneally to females primed with the highest dose of estradiol benzoate (25 micrograms) and progesterone (500 micrograms). Lordosis was inhibited equally by all scopolamine doses during the first week. As in the first experiment, scopolamine failed to inhibit lordosis at all doses on subsequent weeks of testing. These results indicate that the ability of scopolamine to inhibit lordosis is reduced by increasing the dose or the number of estrogen exposures. Because higher doses of scopolamine failed to restore its inhibitory effect on lordosis an upregulation of muscarinic receptors by estrogen cannot account for the reduced effectiveness of scopolamine.     

Orphanin FQ in the mediobasal hypothalamus facilitates sexual receptivity through the deactivation of medial preoptic nucleus mu-opioid receptors

Sexual receptivity, lordosis, can be induced by sequential estradiol and progesterone or extended exposure to high levels of estradiol in the female rat. In both cases estradiol initially inhibits lordosis through activation of β-endorphin (β-END) neurons of the arcuate nucleus of the hypothalamus (ARH) that activate μ-opioid receptors (MOP) in the medial preoptic nucleus (MPN). Subsequent progesterone or extended estradiol exposure deactivates MPN MOP to facilitate lordosis. Opioid receptor-like receptor-1 (ORL-1) is expressed in ARH and ventromedial hypothalamus (VMH). Infusions of its endogenous ligand, orphanin FQ (OFQ/N, aka nociceptin), into VMH–ARH region facilitate lordosis. Whether OFQ/N acts in ARH and/or VMH and whether OFQ/N is necessary for steroid facilitation of lordosis are unclear. In Exp I, OFQ/N infusions in VMH and ARH that facilitated lordosis also deactivated MPN MOP indicating that OFQ/N facilitation of lordosis requires deactivation of ascending ARH-MPN projections by directly inhibiting ARH β-END neurons and/or through inhibition of excitatory VMH–ARH pathways to proopiomelanocortin neurons. It is unclear whether OFQ/N activates the VMH output motor pathways directly or via the deactivation of MPN MOP. In Exp II we tested whether ORL-1 activation is necessary for estradiol-only or estradiol + progesterone lordosis facilitation. Blocking ORL-1 with UFP-101 inhibited estradiol-only lordosis and MPN MOP deactivation but had no effect on estradiol + progesterone facilitation of lordosis and MOP deactivation. In conclusion, steroid facilitation of lordosis inhibits ARH β-END neurons to deactivate MPN MOP, but estradiol-only and estradiol + progesterone treatments appear to use different neurotransmitter systems to inhibit ARH-MPN signaling.     

Induction of lordosis in female rats: two modes of estrogen action and the effect of adrenalectomy.

In ovariectomized female rats, progesterone treatment alone does not induce lordosis, but following estrogen treatment by an appropriate interval it greatly enhances the performance of lordosis compared to that with estrogen alone. This “facilitating” effect of progesterone is thought to act synergistically with the initial “priming” effect of estrogen. In the present experiments a second estrogen treatment given to estrogen-primed ovariectomized rats in place of progesterone was found to facilitate lordosis. Latency of the facilitation of lordosis following this second estrogen treatment was similar to that of progesterone and was much shorter than that required for the usual “priming” effect, but higher doses were needed for the “facilitatory” effect. Experiments with adrenalectomized-ovariectomized female rats showed that this short latency effect of second estrogen treatment need not be mediated by the adrenals. These results raise the possibility that estrogen acts on the central nervous system in more than one way to induce lordosis.     

Mechanisms responsible for progesterone's protection against lordosis-inhibiting effects of restraint I. Role of progesterone receptors

Progestins and antiprogestins are widely used therapeutic agents in humans. In many cases, these are indicated for the treatment of reproductive activities. However, progesterone has widespread physiological effects including a reduction of the response to stress. We have reported that 5 min of restraint reduced lordosis behavior of ovariectomized rats hormonally primed with estradiol benzoate. When ovariectomized rats received both estradiol benzoate and progesterone priming, restraint had minimal effects on lordosis. Progesterone influences behavior through classical intracellular progesterone receptor-mediated nuclear events as well as extranuclear events. How these multiple events contribute to the response to stress is unclear. The current project was designed to initiate examination of the mechanisms responsible for progesterone's ability to protect against the effects of the restraint. In the first experiment, ovariectomized rats, primed with 10 μg estradiol benzoate, received 500 μg progesterone 4 h, 1 h, or 30 min before restraint. When progesterone was injected 4 h before restraint, progesterone eliminated the effects of restraint. In contrast, progesterone 30 min before restraint offered no protection. Effects of progesterone 1 h before restraint were equivocal allowing the suggestion that less than 4 h of progesterone priming might be sufficient. In the second experiment, the synthetic progestin, medroxyprogesterone, was shown to mimic effects of progesterone in preventing effects of restraint. Finally, the progesterone receptor antagonist, RU486, attenuated progesterone's protection against restraint. These findings offer evidence that ligand-activated progesterone receptor mechanisms contribute to the maintenance of lordosis behavior in the presence of mild stress.     

Actions of progesterone and its 5alpha-reduced metabolites on the major proteins of the myelin of the peripheral nervous system

The sciatic nerve, and the Schwann cells in particular, are able to synthesize progesterone and possess the enzymes forming the 5alpha-reduced and the 3alpha-5alpha-reduced derivatives of progesterone: dihydroprogesterone and tetrahydroprogesterone. Moreover, the progesterone receptor (PR) is present in the sciatic nerve and in Schwann cell cultures. These facts suggest that progesterone and its derivatives might play a role in the control of the synthesis of the two major proteins of the peripheral nervous system (PNS): the glycoprotein Po (Po) and peripheral myelin protein 22 (PMP22). We have shown that: (a) dihydroprogesterone enhances the low mRNA levels of Po in the sciatic nerve of aged male rats; (b) progesterone and its derivatives stimulate the gene expression of Po in the sciatic nerve of adult rats and in Schwann cell cultures; (c) tetrahydroprogesterone increases PMP22 gene expression in the sciatic nerve of adult rats and in Schwann cell cultures. In additional experiments, utilizing agonists and antagonists of PR and GABAA receptor, we have observed that progesterone and its derivatives control Po gene expression via the PR, while tetrahydroprogesterone modulates the expression of PMP22 through the GABAA receptor.     

Potential contribution of progesterone receptors to the development of sexual behavior in male and female mice

We previously showed that estradiol can have both defeminizing and feminizing effects on the developing mouse brain. Pre- and early postnatal estradiol defeminized the ability to show lordosis in adulthood, whereas prepubertal estradiol feminized this ability. Furthermore, we found that estradiol upregulates progesterone receptors (PR) during development, inducing both a male-and female-typical pattern of PR expression in the mouse hypothalamus. In the present study, we took advantage of a newly developed PR antagonist (ZK 137316) to determine whether PR contributes to either male- or female-typical sexual differentiation. Thus groups of male and female C57Bl/6j mice were treated with ZK 137316 or OIL as control: males were treated neonatally (P0–P10), during the critical period for male sexual differentiation, and females were treated prepubertally (P15–P25), during the critical period for female sexual differentiation. In adulthood, mice were tested for sexual behavior. In males, some minor effects of neonatal ZK treatment on sexual behavior were observed: latencies to the first mount, intromission and ejaculation were decreased in neonatally ZK treated males; however, this effect disappeared by the second mating test. By contrast, female mice treated with ZK during the prepubertal period showed significantly less lordosis than OIL-treated females. Mate preferences were not affected in either males or females treated with ZK during development. Taken together, these results suggest a role for PR and thus perhaps progesterone in the development of lordosis behavior in female mice. By contrast, no obvious role for PR can be discerned in the development of male sexual behavior.     

Progesterone facilitation of lordosis in male and female Sprague-Dawley rats following priming with estradiol pulses

Adult male Sprague-Dawley rats rarely exhibit progesterone-facilitated lordosis following steroid treatments which are effective in females. In contrast, progesterone-facilitated lordosis has been observed following priming with estradiol pulses in another strain. The aim of this study was to compare progesterone-facilitated feminine sexual behavior in adult male and female Sprague-Dawley rats following priming with estradiol benzoate (EB) or estradiol pulses. Female sexual behavior was measured in adult, gonadectomized males and females treated as follows: Two pulses of estradiol followed by progesterone or oil the next day; EB (two doses) for 3 days, and progesterone or oil the next day. These protocols were repeated at 4- or 6-day intervals, respectively. Progesterone-facilitated lordosis was observed consistently in both sexes treated with estradiol pulses. By the fifth test, lordosis quotients did not differ between the sexes, but the lordosis ratings in progesterone-treated males remained lower than those observed in females. Proceptivity (hop-darting) was facilitated by progesterone in females, but was never observed in males. Lordosis was induced in both sexes by 15 micrograms EB, but was not reliably facilitated by progesterone. Treatment with the lower dose of EB (1.5 micrograms) induced high levels of receptivity in females (occasionally facilitated by progesterone), but not in males regardless of subsequent treatment (i.e, progesterone or oil). These data suggest that progesterone-facilitated lordosis can be induced in male Sprague-Dawley rats, if a regimen of estradiol pulses is used. Thus, the brain of the adult male is not inflexibly differentiated with regard to progesterone facilitation of feminine receptive behavior.     

Ovarian hormones,copulatory stimuli,and female sexual behavior in the Mongolian gerbil

The effects of repeated estradiol benzoate (1.0 or 3.3 μg) and progesterone (0.5 mg) injections and mating experiences (10 min or 4 hr) were examined in ovariec-tomized female Mongolian gerbils (Meriones unguiculatus) paired with sexually vigorous but unfamiliar males. Estradiol benzoate alone for 2 days did not elicit female sexual responses. Estradiol benzoate for 2 days followed by progesterone facilitated lordosis in the female. In females injected daily with 1.0 μg estradiol benzoate, progesterone exposure for approximately 1 day produced a significant inhibition in subsequent lordosis, although these females continued to respond to the male with lordosis quotients of approximately 60. A 4-hr mating experience produced a nearly complete elimination of sexual activity in tests observed 24 hr later with a fresh male. Footstomping was positively associated with sexual activity in both sexes, although females footstomped more often in the 1.0 rather than the 3.3-μg estradiol benzoate condition. Fighting was most frequent in tests in which lordosis quotients were low. Interactions among copulatory experiences and inhibitory effects of progesterone summed to terminate female sexual receptivity and alter other social interactions in the gerbil.     

Progesterone in high doses may overcome progesterone's desensitization effect on lordosis by translocation of hypothalamic progestin receptors

The cellular mechanism by which progesterone desensitizes the central nervous sytem to further facilitation of lordosis by progesterone was studied. In the first experiment guinea pigs were injected with doses of estradiol benzoate and progesterone that result in refractoriness to further stimulation of lordosis by moderate doses of progesterone. Confirming previous reports, a large dose of progesterone was effective in overcoming this desensitization, resulting in lordosis responses. A lower dose of progesterone was ineffective in this regard. In subsequent experiments, cytosol and nuclear progestin receptors were measured in the hypothalamus-preoptic area and cerebral cortex with an in vitro exchange assay using [³H]R 5020 as the ligand. A high, effective dose of progesterone resulted in 46–48% higher levels of nuclear progestin receptors assayed in the hypothalamus-preoptic area than did a lower, ineffective dose. These results are consistent with the idea that progesterone injection in estradiol-primed rodents causes a subsequent desensitization to itself by decreasing the concentration of cytoplasmic progestin receptors in hypothalamus-preoptic area. A subsequent progesterone injection then results in a lower level of nuclear progestin receptors. Large doses of progesterone may overcome this desensitization by causing accumulation of levels of nuclear progestin receptors sufficient to facilitate lordosis.     

Estrogen and progesterone dose-dependently reduce disruptive effects of restraint on lordosis behavior

Ovariectomized rats were hormonally primed with various doses of estradiol benzoate (EB; 0.5-10 microg) in combination with various doses of progesterone (2.5-500 microg) to induce sexual receptivity. Females were then subjected to 5 min restraint and the effect on lordosis behavior was monitored for the next 30 min. Such mild stress has been previously shown to transiently reduce lordosis behavior of ovariectomized females hormonally primed only with 10 microg EB. In the current study, doses of progesterone of 25 microg or more in combination with 10 microg EB reduced the effects of restraint. Also priming doses of EB from 4.0 to 10 microg in combination with 250 microg progesterone prevented the lordosis-inhibiting effects of restraint. These findings reinforce prior observations of the dose-dependency of both estrogen and progesterone in the facilitation of lordosis behavior and introduce the female's lordosis response to mild restraint as a potentially useful index of the female's response to stress.     

Progesterone reduces the effect of the serotonin 1B/1D receptor antagonist, GR 127935, on lordosis behavior

Ovariectomized rats were hormonally primed with 10 μg estradiol benzoate or with estradiol benzoate plus 500 μg progesterone. Rats received a bilateral infusion with 200 ng of the 5-HT_1B/1D receptor antagonist, N-[4-methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2′-methyl-4′-(5-methyl-1,2,4-oxadiazol-3-yl)-1-1′-biphenyl-4-carboxamide hydrochloride (GR 127935), into the ventromedial nucleus of the hypothalamus (VMN), followed by a 5 min restraint or home cage experience. In estrogen-primed females that had experienced minimal handling between ovariectomy and use in the experiment, infusion with the water vehicle transiently inhibited lordosis behavior, and the 5-HT_1B/1D receptor antagonist amplified this inhibition. There were no effects in rats hormonally primed with estrogen and progesterone. Handling for two days before the experiment reduced the effects of the infusions in estrogen-primed rats. However, when a 5 min restraint experience followed infusion with GR 127935, there was a significant decline in lordosis behavior that persisted for 10 to 15 min after the experience. Regardless of the prior experience or type of infusion, the addition of progesterone to the hormonal priming completely prevented the lordosis inhibition. These findings are consistent with prior evidence that progesterone protects against the inhibitory effects of a 5 min restraint experience on lordosis behavior. Moreover, these are the first experiments to demonstrate an inhibitory effect of a selective 5-HT_1B/1D receptor antagonist in the VMN on lordosis behavior of estrogen primed, but not estrogen and progesterone primed, ovariectomized rats.     

Immunological basis for susceptibility and resistance to pulmonary blastomycosis in mouse strains

The immunological basis for a >10-fold resistance of outbred CD-1 mice compared to inbred BALB/c mice to pulmonary blastomycosis was investigated. Bronchoalveolar macrophages (BAM) from CD-1 mice killed yeast cells of Blastomyces dermatitidis (Bd) by 25% and this increased to 59% when activated by IFN-gamma. In contrast, BAM from BALB/c mice lacked significant killing (5%) of Bd but could be activated by IFN-gamma for enhanced killing (19%). Peritoneal macrophages (PM) from CD-1 mice had significant fungicidal activity for Bd (43%) and this increased to 63% with IFN-gamma treatment. By contrast, PM from BALB/c mice did not significantly kill Bd (14%) but were activated by IFN-gamma for significant killing (24%). Fungicidal activity of peripheral blood polymorphonuclear neutrophils (PMN) from CD-1 (87%) was greater than that of BALB/c (75%) (P<0.05). Macrophage inflammatory protein-1alpha (MIP-1alpha) production by BAM from BALB/c was significantly less than that from CD-1 in response to co-culture with Bd. IFN-gamma production by CD-1 spleen cells in response to concanavalin A (Con A, 1microg/ml) was 8-fold greater than that by BALB/c spleen cells. In contrast, BAM and PM from BALB/c mice in co-culture with Bd secreted several-fold more TNFalpha than BAM or PM from CD-1 mice. IL-2 production by BALB/c spleen cells in response to Con A was 3- to 4-fold greater than that by CD-1 spleen cells. Depressed IL-2 production by Con A stimulated CD-1 spleen cells correlated with depressed proliferative responses. Resistance of CD-1 mice to pulmonary blastomycosis correlates with enhanced fungicidal activity of BAM, PM, PMN, and IFN-gamma production by Con A stimulated spleen cells, compared to BALB/c mice. Consistent with the in vitro enhancement of effector cell function by IFN-gamma, in vivo therapy with IFN-gamma significantly (P<0.0001) improved survival of BALB/c mice with pulmonary blastomycosis.