Role of progesterone receptors during postpartum estrus in rats

We studied the role of progesterone receptor (PR) in the display of female sexual behavior during postpartum estrus in rats. Adult female rats were treated with the PR antagonist, RU486 (1.25 and 5 mg), 3 h after parturition and sexual behavior was evaluated throughout the first postpartum day. Estradiol and progesterone serum levels changed during the first 24 h postpartum. The highest estradiol and progesterone levels were found at 9 and 12 h postpartum, respectively. The predominant PR isoform in the hypothalamus and the preoptic area was PR-A during postpartum day. The content of PR-A increased at 6 h postpartum in the hypothalamus and the preoptic area, and decreased in both regions at 9 h. PR-B content only increased in the preoptic area at 12 h postpartum. The highest display of lordotic and proceptive behaviors were found at 12 h postpartum. The treatment with 1.25 and 5 mg of RU486 respectively reduced lordosis by 61% and 92% at 12 h postpartum. These results suggest that PR is essential in the display of postpartum estrus in rats.     

The inhibition of female rabbit sexual behavior by progesterone: progesterone receptor-dependent and-independent effects

In the pregnant domestic rabbit, scent marking (“chinning”) and sexual behavior are inhibited by ovarian-derived progesterone (P). In order to distinguish behavioral effects of P that are PR-dependent from those mediated by its ring A reduced metabolites, we administered P, P+RU486 (PR antagonist), chlormadinone acetate (CA, synthetic progestin that does not form ring A reduced metabolites), or vehicle to ovariectomized (ovx) estradiol-benzoate (EB)-treated female rabbits, via sc injection, on experimental day 0. Chinning was quantified daily, and mating tests were done on days -1, 1, 3, 5, and 7. On day 1, chinning was significantly decreased, and the latency to be mounted by the male was significantly increased (indicating decreased sexual attractivity of the female) in P-treated females. The effect of P on chinning, but not its effect on sexual attractivity, was completely blocked by RU486 and replicated by CA. Although CA had no effect on attractivity on day 1, it decreased both sexual receptivity and attractivity on day 3. In a preference test in which the male could interact with either an ovx EB-treated female or an ovx female that had received one of the above hormone treatments 24 h earlier, P decreased sexual attractivity and increased aggression. The effect of P on aggression, but not its effect on attractivity, was blocked by RU486 and replicated by CA. These results indicate that both PR-dependent and PR-independent mechanisms decrease sexual attractivity, whereas PR activation is necessary for the inhibition of chinning and sexual receptivity, and for the stimulation of aggression.     

Sexual dimorphism in the content of progesterone and estrogen receptors, and their cofactors in the lung of adult rats

Progesterone and estradiol play an important role in the regulation of lung functions such as ventilation and vasoconstriction. The genomic actions of progesterone and estradiol are mediated by their nuclear receptors: progesterone receptors (PR) and estrogen receptors (ER). These actions are linked to interactions between steroid receptors and some cofactors that function as coactivators or corepressors. In this work we determined the content of PR isoforms, ER-beta, one coactivator (SRC-1), and one corepressor (SMRT) in the lung of both female rats during the estrous cycle and intact males by Western blot. The rat lung presented a higher content of PR-A than that of PR-B during the estrous cycle. The highest content of both PR isoforms was observed on the day of proestrus whereas the lowest one was found on the day of estrus. In contrast, the content of ER-beta was the lowest on the day of proestrus and it increased at estrus. The content of SRC-1 and SMRT increased on the day of diestrus. SRC-1 content decreased at proestrus and estrus, while SMRT content decreased at proestrus and increased again at estrus. In the lung of adult male rats the content of PR isoforms, ER-beta and SMRT was lower than in that of females, whereas the content of SRC-1 was similar in both sexes. Our results suggest a sexual dimorphism in the content of PR, ER-beta, and SMRT in the rat lung as well as a relation of their content to the physiological levels of progesterone and estradiol.     

Estrogen and progesterone receptors in carpal tunnel syndrome

Carpal tunnel syndrome (CTS) is a compression median nerve neuropathy common in women at menopausal age. The aim of this work was to study immunohistochemically the expression of estrogen (ER) and progesterone (PR) receptors in CTS and control specimens. Biopsies of transverse carpal ligament (TCL) and flexor tendon synovitis were collected from 23 women and from 7 men undergoing surgery for median nerve decompression at the wrist for CTS. In TCL and synovial tissue, cells expressed ER and PR with statistically significant differences related to the age and sex of patients. Immunoreactivity was observed in fibroblasts of TCL, and in lining cells and fibroblasts of synovial tissue. In women, the number of ER-positive cells in the TCL and synovial tissue increased with the age, peaking at 55-70 years, and then decreasing. PR-immunoreactivity was observed only in fibroblasts of TCL and its expression decreased with age, while no immunolabeling was found in the synovial tissue. In TCL samples, the number of ER- and PR-positive cells in non-CTS patients was significantly lower than in CTS patients. These results demonstrate that ER and PR are present in TCL and flexor tendon synovitis, suggesting a role for sex steroid hormones in the pathogenesis of CTS disease.     

The role of progestin receptors and the mitogen-activated protein kinase pathway in delta opioid receptor facilitation of female reproductive behaviors

The present study investigated the role of the progestin receptor (PR) and the mitogen-activated protein kinase (MAPK) pathway in the facilitation of lordosis behavior by the delta opioid receptor agonist [D-Pen(2), D-Pen(5)]-enkephalin (DPDPE). Ovariectomized, estrogen-primed rats were treated with the PR antagonist RU486 or the MAPK inhibitor PD98059 prior to intraventricular (icv) infusion of DPDPE. Both RU486 and PD98059 blocked receptive and proceptive behaviors induced by DPDPE at 60 min, and RU486 continued to inhibit estrous behavior at 90 min. Because delta opioid receptors can activate the p42/44 MAPKs, extracellular signal regulated kinases (ERK), we determined the effects of DPDPE on ERK phosphorylation. Icv infusion of DPDPE increased the levels of phosphorylated ERK in the hypothalamus and preoptic area of female rats, assessed by immunoblotting. These results support the participation of the PR and the MAPK pathway in the facilitation of lordosis behavior by delta opioid receptors.     

Immunolocalization of hepatic estrogen and progesterone receptors in the female lizard Uromastyx acanthinura

The hormonal regulation of hepatic synthesis of vitellogenin during the annual reproductive cycle was performed for the first time in the deserticole, oviparous, diurnal and herbivorous Uromastyx acanthinura, a lizard belonging to the Agamidae family. In order to elucidate what kind of estrogen receptor is involved in this process, an immunohistochemical study was performed. Changes were obtained in the labeling and cellular distribution of the estrogen and progesterone receptors according to the period of the reproductive cycle and the experimental administration of 17β-estradiol. Only the ERβ subtype was present; it was found in all phases of the cycle with a variable localization: nuclear and cytosolic during vitellogenesis, mainly cytosolic in the female with egg retention (luteal phase) and strictly cytosolic in females at sexual rest. The progesterone receptors were present only at the luteal phase and during sexual rest and disappeared completely from females after 17β-estradiol treatment in sexual rest. Our data suggested that mediation of action of the 17β-estradiol in the vitellogenin synthesis in the lizard U. acanthinura occured via ERβ. PRA and PRB could both be necessary for the negative effect of progesterone on the hepatic synthesis of vitellogenin.     

Medroxyprogesterone acetate acutely facilitates and sequentially inhibits sexual behavior in female rats

Medroxyprogesterone acetate (MPA), a synthetic progestin commonly used in contraception and hormone replacement therapy, appears to inhibit libido in women, but little is known about the mechanisms through which it may exert this effect. We compared the acute and sequential actions of MPA and natural progesterone (P4) on sexual behavior in female rats to test the hypothesis that MPA inhibits sexual behavior, at least in part, by acting as a potent progesterone receptor (PR) agonist. Ovariectomized females were placed in one of three dose groups (high, mid, or low), and each subject was tested under three different conditions (MPA, P4, and vehicle). The order of progestin treatment was balanced among subjects, and within each dose group equimolar quantities of MPA and P4 were administered. During each trial, females were injected with estradiol benzoate (EB, 4 mug) followed by one of three progestin treatments (MPA, P4, or vehicle) at +44 h, and behavioral testing at +48 h. On the next day, all females were given a standard 500-microg injection of P4 at +68 h and were tested again for sexual behavior at +72 h. On the first day of behavioral testing, both MPA and P4 induced a pronounced rise in receptive and proceptive behavior at the mid and high doses, but at the lowest dose MPA had a much greater effect in comparison to P4. On the second day of behavioral testing, MPA attenuated the expression of proceptive and receptive behavior at both the mid and high doses, whereas P4 only attenuated the expression of lordosis and only did so at the highest dose. These findings illustrate that MPA and P4 have a similar impact on sexual behavior in female rats and suggest that the inhibitory effects of MPA may be attributable, at least in part, to its potent effects at the progesterone receptor.     

Regulation of cytosol and nuclear progesterone receptors in rabbit uterus by estrogen,antiestrogen and progesterone administration

A synthetic progestin, 16α-ethyl-21-hydroxy-19-nor-4-pregnene-3,20-dione (ORG 2058), was utilized to measure progesterone receptors from the rabbit uterus. This steroid has a high affinity for both cytosol and nuclear receptors, with K_D values of 1.2 nM (at 0–4°C) and 2.3 nM (at 15°C), respectively. Administration of estradiol-17β or a non-steroidal antiestrogen, tamoxifen, for 5 days to estrous rabbits led to a progressive rise in the cytosol receptor levels: from 34 000 to 120 000 (estradiol-17β) and 80 000 (tamoxifen) receptors/ cell, without any major influence on the nuclear receptor content. A single intravenous injection of progesterone (5 mg/kg) elicited a 3-fold increase in the mean nuclear receptor content at 30 min after injection (from 18 000 to 48 000 receptors/nucleus). Nuclear receptor accumulation was short-lived and returned to control levels within 4 h after treatment. A second dose of progesterone given 24 h later doubled the nuclear receptor level (from 18 000 to 35 000 receptor/nucleus). The concomitant decline in the cytosol receptor content was twice that accounted for by the nuclear receptor accumulation (70 000 vs. 30 000, and 40 000 vs. 17 000 receptors/cell, after the first and second progesterone injection, respectively). Following progesterone administration, the cytosol receptor level reached a nadir by 30 min, exhibited minimal replenishment within the ensuing 24 h, and remained at approx. 50% of the pretreatment values. After a single dose or two consecutive doses of progesterone, total uterine progesterone receptor content declined to about 60% of the level prior to each dose, a nadir being reached at 2 h after treatment.     

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.     

Relevance of ovarian signaling for the early behavioral transition from estrus to pregnancy in the female rabbit

During estrus, the female domestic rabbit (Oryctolagus cuniculus) displays scent marking behavior (chinning), which is immediately inhibited after mating, temporarily recovers, and then declines and remains inhibited across pregnancy. Chinning is inhibited by progesterone (P) and the activation of the progesterone receptor (PR), but it is unlikely that P participates in the “acute” (immediate) or “early” inhibition of chinning (24 to 96 h post-mating, before plasma P levels rise). Since PR is activated in a ligand-independent manner by a variety of signaling molecules, some of which (e.g., GnRH) are also associated with reflexive ovulation in this species, we hypothesized that neurochemical/neuroendocrine signals associated with mating activate PR, resulting in the inhibition of chinning. In Experiment 1, we tested whether the PR antagonist, RU486 (20 mg, injected s.c. at − 1 h, or at − 7 h and + 3 h relative to mating) prevented the post-mating inhibition of chinning in intact females. RU486 did not prevent the post-mating decline in chinning, indicating that PR activation associated with mating is not necessary for this effect. In Experiment 2, we used ovariectomized (OVX), estradiol benzoate (EB)-treated females to test the hypothesis that ovarian signaling is necessary for the post-mating inhibition of chinning. The acute inhibition of chinning occurred in OVX females, but the early inhibition was absent. We conclude that ovarian signaling is necessary for the early, but not acute, post-mating inhibition of chinning. The PR seems not to participate in either of these phases.     

Involvement of the corticoadrenal hormones in the pheromonal restoration of reproductive activity in aging rats

The involvement of the adrenal progesterone and corticosterone in the early gonadotropin secretion associated with the pheromonal restoration of ovarian cyclic activity (PRCA) in aging female rats is studied. PRCA is induced by male urinary pheromones and is preceded by an alpha-adrenergic-mediated release of the hypothalamic decapeptide luteinizing hormone-releasing hormone and plasma increases of estradiol, progesterone and the gonadotropins luteinizing hormone and follicle stimulating hormone. Aging reproductive Wistar female rats were used to study the effects of bilateral adrenalectomy and of a subcutaneous injection of the antisteroid RU486 on plasma levels of corticosterone, progesterone and gonadotropins in rats stimulated with nasal spraying of male urine (MU) or saline. The results demonstrate that progesterone and corticosterone released by MU are from adrenal origin, and that these adrenal secretory products are critical for MU-induced increase of gonadotropins. This suggests that olfactory stimulation of ACTH release stimulates adrenal release of progesterone and corticosterone, and both trigger the events that initiate the activation of the hypothalamus-pituitary-ovarian axis that leads to PRCA.     

Estrogen- and progesterone receptors in normal cycling endometrium as studied by end-point titration

A thorough knowledge of the normal physiological fluctuations in estrogen-(ER) and progesterone receptors (PgR) is essential to characterize the changes in ER and PgR in the abnormal endometrium. We investigated the distribution of ER and PgR in frozen human cycling endometrial tissue using the commercially available ER-and PgR-ICA kits. Two-fold end-point titration (EPT) of ER and PgR antibodies was implemented to semi-quantitate more accurately ER and PgR. Semiquantitation of ER and PgR using EPT was significantly correlated to results obtained using either simple scoring or enzyme-immunoassay (EIA) methods. ER and PgR staining fluctuated in relation to the menstrual cycle. In most subphases PgR exceeded ER in both epithelial and stromal cells. Highest levels of ER and PgR were demonstrated in the glands of the functionalis in mid-to-late proliferative phases, whereas both receptors were almost undetectable by immunohistology in the glands of mid-to-late secretory phases. Endometrial stromal cells had high and nearly constant EPT values for PgR, but low values for ER througout the menstrual cycle. EPT values for ER and PgR were generally higher in the basalis than in the functionalis but showed similar cyclic fluctuations. Our results further substantiate the view that the response to hormonal stimulation is cell-type specific, and suggest differences in steroid metabolism according to cell type and layer.     

Induction of abortion with aglepristone significantly changed the expression of progesterone and estrogen receptors in canine endometrial stromal cells

In the present study, resorption/abortion was induced between days 25 and 45 of gestation with aglepristone (group IRA, n=10). The aim was to observe the change in the distribution of progesterone (PR) and estrogen receptors (ER), in comparison to a group of spontaneous resorptions/abortions (group SRA, n=5), and a further group of normal healthy pregnant animals, ovariohysterectomized between days 25 and 45 of gestation (controls, n=7). The receptors were assessed by means of immunohistochemistry (IHC) and RT-PCR, at the placental and interplacental sites of the uterine horn as well as in the corpus uteri. Significant differences were observed between the controls on one side and the groups of resorption/abortion on the other side. The total scores of the progesterone receptors (TPR) in the placental and interplacental part of the uterine horn, was significantly lower in the endometrial stromal cells (ESC) of the control group than in those of the SRA- and IRA-group, respectively (placenta: 5.8 vs. 6.5 and 6.7, p<0.01; interplacental sites: 5.6 vs. 6.6 and 6.6, p<0.05). In contrary, the total scores of the estrogen receptors (TER) at interplacental sites and the corpus uteri, respectively, was significantly higher in the myometrial smooth muscle cells (MSMC) and the ESC (p<0.05) of the controls. We therefore conclude, that the here observed differences between groups point to an up-regulation of TPR- and a down-regulation of TER-scores in endometrial stromal cells at different uterine sites during resorption/abortion, which indicates a special role of these cells.     

Medroxyprogesterone acetate attenuates estrogen-induced nitric oxide production in human umbilical vein endothelial cells

We report the novel observation that medroxyprogesterone acetate (MPA) attenuates the induction by 17beta estradiol (E2) of both nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) activity in human umbilical vein endothelial cells. Although MPA had no effect on basal NO production or basal eNOS phosphorylation or activity, it attenuated the E2-induced NO production and eNOS phosphorylation and activity. Moreover, we examined the mechanism by which MPA attenuated the E2-induced NO production and eNOS phosphorylation. MPA attenuated the E2-induced phosphorylation of Akt, a kinase that phosphorylates eNOS. Treatment with pure progesterone receptor (PR) antagonist RU486 completely abolished the inhibitory effect of MPA on E2-induced Akt phosphorylation and eNOS phosphorylation. In addition, the effects of actinomycin D were tested to rule out the influence of genomic events mediated by nuclear PRs. Actinomycin D did not affect the inhibitory effect of MPA on E2-induced Akt phosphorylation. Furthermore, the potential roles of PRA and PRB were evaluated. In COS cells transfected with either PRA or PRB, MPA attenuated E2-induced Akt phosphorylation. These results indicate that MPA attenuated E2-induced NO production via an Akt cascade through PRA or PRB in a non-genomic manner.     

Relevance of mating-associated stimuli, ovulation, and the progesterone receptor for the post-coital inhibition of estrous behavior in the female rabbit

Estrous female domestic rabbits (Oryctolagus cuniculus) display scent marking (“chinning”) and sexual receptivity. Mating induces ovulation, which occurs approximately 12 h later, and also decreases chinning and receptivity. In the present study, we explored the participation of mating-associated stimuli, ovulation, and the progesterone receptor (PR) in mediating such behavioral effects. We found that copulatory stimuli were not necessary, and that ovulation alone was sufficient, as these behavioral changes were replicated in unmated females by intravenous administration of human chorionic gonadotropin (hCG). The post-mating administration (s.c.) of 5 μg/day estradiol benzoate (EB), prevented the decline in chinning and receptivity. A lower dose of EB (1 μg/day) had no effect, nor did the antiprogestin RU486 (20 mg, s.c., administered 3 h before mating). However, the combination of a single pre-mating administration of RU486 plus the post-mating administration of 1 μg/day EB completely blocked the decline in estrous behavior. We propose that PR activation around the time of mating and a post-mating decline in ovarian estradiol secretion and/or estradiol responsiveness act in parallel to terminate estrus in this species.     

Estrogen regulates the expression of N-methyl-d-aspartate (NMDA) receptor subunit epsilon 4 (Grin2d), that is essential for the normal sexual behavior in female mice

Estrogen plays important roles in the reproductive behavior of animals. In the present study, we found that the Grin2d gene of mouse possessed half-sites of the estrogen-responsive element (ERE) in the 3′-untranslated region (UTR). Quantitative PCR analysis showed that the reduced Grin2d mRNA expression in the hypothalamus of the ovariectomized mice was restored by estrogen administration. Downregulation of Grin2d mRNA expression was also detected in the hypothalamus of estrogen receptor alpha-knockout female mice. Moreover, estrogen-induced lordosis response was decreased in Grin2d-knockout mice. These results suggest that estrogen regulates lordosis behavior through the regulation of Grin2d expression in the hypothalamus of female mice.     

Lipid peroxidation in liver tissue of ovariectomized and pinealectomized rats: effect of estradiol and progesterone supplementation

The present study aimed to determine the effect of estradiol-progesterone supplementation and pinealectomy on lipid peroxidation of liver tissue in ovariectomized rats. The study was carried out on 36 adult Sprague-Dawley female rats, which weighed 200-250 g. The rats were divided into 6 groups: Group 1: Sham Ovariectomy (Sham-Ovx), Group 2: Ovariectomy (Ovx), Group 3: Ovx + Estradiol-Progesterone supplementation (Ovx + H), Group 4: Sham Pinealectomy and Ovx (Sham Pnx -Ovx), Group 5: Ovx -Pnx, Group 6: Ovx -Pnx + H. Malondialdehyde (MDA), reduced form of glutathione (GSH) and glutathione peroxidase (GSH-Px) levels were determined in liver tissue of rats. The highest MDA levels and the lowest GSH-Px levels were determined in the ovariectomized-pinealectomized group, whereas the lowest MDA was in the Sham-Ovx group, and the highest GSH-Px levels were found in the Sham-Ovx and Ovx + Hormone supplemented group. Furthermore, the highest GSH levels were in group 1 and lowest levels were in group 5. The findings of this study demonstrate that ovariectomy led to lipid peroxidation in liver tissues of rats. Pinealectomy in addition to ovariectomy, increases lipid peroxidation, but, estradiol and progesterone supplementations to the ovariectomized-pinealectomized rats protect against lipid peroxidation to a significant extent.     

Estrogenic effects of zearalenone on the expression of progestin receptors and sexual behavior in female rats

Zearalenone is a resorcylic acid lactone compound that is produced by fungal infection of edible grains and is believed to influence reproduction by binding to estrogen receptors. In order to study the potential estrogenic effects of this compound in the brain, we examined the effects of zearalenone on the expression of neuronal progestin receptors and feminine sexual behavior in female rats. Ovariectomized rats were treated with zearalenone (0.2, 1.0, or 2.0 mg), estradiol benzoate, or vehicle daily for 3 days. They were then either perfused, and progestin receptors visualized by immunocytochemistry, or injected with progesterone and tested for sexual receptivity with male rats. Progestin receptor-containing cells were counted in the medial preoptic area and ventromedial hypothalamus. The two highest doses of zearalenone increased the concentration of neuronal progestin receptors, as did 10 microg of estradiol. The highest dose of zearalenone (2 mg) also induced progestin receptor staining density comparable to that of 10 microg of estradiol benzoate. In behavioral tests, ovariectomized animals treated with 2 mg of zearalenone followed by progesterone showed levels of sexual receptivity comparable to females treated daily with estradiol benzoate (2 microg) followed by progesterone. These studies suggest that, although structurally distinct and less potent than estradiol, zearalenone can act as an estrogen agonist in the rat brain.