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. 2009 May 7;150(8):3833–3844. doi: 10.1210/en.2008-0774

Progesterone Receptor A (PRA) and PRB-Independent Effects of Progesterone on Gonadotropin-Releasing Hormone Release

Nicole Sleiter 1, Yefei Pang 1, Cheryl Park 1, Teresa H Horton 1, Jing Dong 1, Peter Thomas 1, Jon E Levine 1
PMCID: PMC2717864  PMID: 19423765

Abstract

Progesterone’s (P4) negative feedback actions in the female reproductive axis are exerted in part by suppression of hypothalamic GnRH release. Here we show that P4 can inhibit GnRH release by a mechanism independent of a nuclear P4 receptor (PRA/B). Injections of P4, but not vehicle, allopregnanolone, or dexamethasone, acutely suppressed LH levels in both wild-type and P4 receptor knockout ovariectomized mice; pituitary responsiveness to GnRH was retained during P4 treatment, indicating a hypothalamic action. Superfusion of GnRH-producing GT1-7 cells with medium containing 10−7 m P4 produced a rapid reduction in GnRH release. Incubation with P4 (10−9 to 10−7 m) inhibited forskolin-stimulated cAMP accumulation; cotreatment with pertussis toxin prevented this effect. Treatment of GT1-7 cell membranes with P4 caused activation of an inhibitory G protein (Gi), as shown by immunoprecipitation with a Gi antibody of most of the increase in membrane-bound [35S]GTPγ-S. Saturation binding analyses demonstrated the presence of a high affinity (Kd 5.85 nm), limited capacity (Bmax 62.2 nm) binding site for P4. RT-PCR analysis revealed the presence of mRNAs encoding both isoforms of the membrane P4 receptors, mPRα and mPRβ. Western blotting, immunocytochemistry, and flow cytometry experiments similarly revealed expression of mPR proteins in the plasma membranes of GT1-7 cells. Treatment with mPRα siRNA attenuated specific P4 binding to GT1-7 cell membranes and reversed the P4 inhibition of cAMP accumulation. Taken together, our results suggest that negative feedback actions of P4 include rapid PRA/B-independent effects on GnRH release that may in part be mediated by mPRs.

Progesterone suppresses GnRH release through a mechanism that is independent of nuclear progesterone receptor activation but may in part be mediated by membrane progesterone receptors

Ovarian progesterone (P4) secretions are key regulatory signals that control virtually all aspects of female reproduction. The actions of P4 are critically important in the regulation of mammary gland development and ovulation as well as blastocyst implantation, epithelial cell proliferation, and contractility in the uterus (1,2,3). In neuroendocrine tissues, P4 regulates reproductive behaviors (4) and exerts feedback effects on pituitary gonadotropin secretions (5,6,7,8,9,10,11) and hypothalamic GnRH release (12,13,14). The latter actions include homeostatic feedback suppression of pulsatile GnRH neurosecretion (14,15,16,17) as well as the inhibition of ovulatory GnRH and gonadotropin surges (18,19,20,21,22,23,24). It is likely that the negative feedback actions of P4 are integral to the efficacy of P4-based contraceptive preparations. Moreover, disturbances in P4 feedback have been implicated in the pathogenesis of infertilities associated with hyperandrogenemia, such as polycystic ovarian syndrome (25,26). Despite their physiological and clinical importance, the cellular mechanisms by which P4 exerts negative feedback effects on GnRH neurosecretion have remained incompletely understood.

The majority of P4’s actions are currently believed to be dependent upon the binding and activation of its cognate cytoplasmic or nuclear P4 receptors (nPRs) (2,3,27). Bound nPRs recruit coactivator proteins and function as ligand-activated transcription factors that regulate transcription of target genes. These genomic effects of P4 generally require hours to be manifest as physiological responses, owing to the time intervals required for translation of the regulated proteins. Physiological analyses of mice bearing deletion mutations of nPRs [PR knockout (PRKO) mice] have revealed overlapping but nonidentical roles for the PRB and the N-terminally truncated PRA in mammary gland development and uterine function (28,29). Similarly, neuroendocrine assessments of PRKO mice have demonstrated a relatively unambiguous involvement of nPRs in P4’s facilitatory effects on female sexual behavior (30,31) as well as in the generation of preovulatory gonadotropin surges (32,33). The negative feedback actions of P4, however, have not been investigated in animals devoid of nPRs. Although a previous report from this laboratory demonstrated modestly higher LH levels in ovary-intact PRKO vs. wild-type (WT) mice (32), the LH responses of both genotypes to exogenous P4 have not yet been assessed. Pharmacological antagonism of nPRs in ewes was previously found to block the effects of P4 on GnRH release (14), suggesting that P4’s inhibitory effects are largely mediated by nPRs in that species. However, relatively rapid effects of P4 on GnRH pulse generator activity in monkeys (13) and gonadotropin secretion in humans (34) and cattle (35,36) are suggestive of additional mechanisms that do not involve alterations in gene transcription.

Rapid effects of P4, each potentially mediated by one of or more of several nonclassical signaling mechanisms, have been documented in a variety of tissues. Some of the rapid actions of P4 have been attributed to the ability of bound PRA and PRB to interact with the Src tyrosine kinase localized to the plasma membrane, which in turn prompts cellular responses via activation of the Src/Ras/Raf-1/MAPK signaling pathway (37,38,39). At least three PRA/B-independent pathways have also been identified that may mediate the effects of P4 in a variety of tissues and cell types. P4 is known to be rapidly metabolized in the brain to several neurosteroids, including allopregnanolone [3α-hydroxy-5α-pregnan-20-one (3α5αTHP)], which has been shown to modulate γ-aminobutyric acidA (GABAA) receptors in the brain (40). Evidence suggests that this metabolite may interact with GABAA receptors to modulate GnRH (41) and LH (42) secretion. Second, a putative P4 membrane binding protein, called P4 receptor membrane component 1 (PGRMC1), has been suggested to mediate the ability of P4 to activate protein kinase G or other rapid signaling mechanisms in certain cells (43,44).

Recently, a family of G protein-coupled receptor-like proteins (GPCR) was discovered that specifically bind P4 and mediate rapid cellular responses to the steroid, despite the fact that they bear no structural similarities to nPRs (45). Originally identified in the ovaries of the spotted seatrout, homologs of these putative membrane P4 receptors (mPRs) were subsequently identified in lower vertebrates and several mammalian species, including humans (46,47). Three separate genes have been identified encoding three closely related proteins, mPRα, mPRβ, and mPRγ, that appear to function as plasma membrane-bound GPCRs mediating rapid actions of P4 via activation of an inhibitory G protein (Gi) and suppression of adenylyl cyclase activity and cAMP production (46,47). An ovine mPR homolog has also been identified that appears to be localized at least in part to the endoplasmic reticulum and coupled to the mobilization of intracellular Ca2+ (48). Heterologous expression of human mPRα, mPRβ, and mPRγ in yeast has confirmed their ability to function as membrane P4 receptors (49).

We performed parallel in vivo and in vitro studies to determine the extent to which rapid inhibitory effects of P4 on GnRH release are mediated by PRA/B-dependent vs. PRA/B-independent signaling mechanisms. Our experiments demonstrate that P4 can exert inhibitory effects on GnRH release that are manifest in the absence of nPRs and that these effects may be mediated by mPRs coupled to Gi protein and the inhibition of intracellular cAMP formation.

Materials and Methods

Animals

Adult female PRKO mice (129SvEv/C57BL/6 hybrid background) were generated by breeding homozygous PRKO male and heterozygous PRKO female mice at the Northwestern University animal facility. Isogenic WT mice were generated by mating heterozygous littermates. No more than two litters from each pair were used to control for any possible changes in allelic composition from the PRKO breeding colony. The animals were housed five to a cage under a 12-h light, 12-h dark cycle with lights on from 0600–1800 h. The mice were fed standard rodent chow and had access to both food and water ad libitum. All animal and surgical procedures were reviewed and approved by the Northwestern University Institutional Animal Care and Use Committee.

Surgical procedures and experimental treatments

The effects of P4 and other treatments were assessed in ovariectomized (OVX) mice. In preliminary experiments, attempts were made to examine the effects of P4 in OVX, estrogen-primed animals. However, we could find no regimen of estrogen treatment that would partially suppress LH levels, so as to allow for an analysis of additional inhibitory effects of P4. Moreover, initial experiments revealed an inhibitory effect of P4 in OVX animals that were not primed with estrogen, and thus all in vivo experiments were conducted in OVX, unprimed animals. Mice at least 8 wk of age were anesthetized with 100 mg/kg ketamine (Ketaset; Fort Dodge Animal Health, Fort Dodge, IA), and 10 mg/kg xylazine (Phoenix Scientific, Inc., St. Joseph, MO) and bilaterally OVX. On d 7 after OVX, animals were anesthetized with halothane, and a 25-gauge needle was used to withdraw 100 μl blood after cardiac puncture. Immediately after the first blood sample was obtained, the mice were injected sc with 0.1 ml sesame oil vehicle or 0.1 ml sesame oil containing 400 μg P4, 16 μg allopregnanolone, or 400 μg dexamethasone. These doses were selected on the basis of results of previous studies of the behavioral effects of P4 in mice (50) and hormonal effects of allopregnanolone (42) in rats. The relatively high dose of dexamethasone was intentionally chosen to exceed a comparable dose on a per body weight basis in rats (51). In a subset of animals where the responsiveness to GnRH was tested, the animals were injected with 0.1 ml saline containing 200 ng/kg GnRH 10 min before the second blood sample. A terminal blood sample was obtained 4 h after the first blood sample by cardiac puncture; the mice were reanesthetized, the heart was exposed, and a 25-gauge needle was inserted into the right ventricle of the heart, allowing withdrawal of 1–2 ml blood. All blood samples were centrifuged, and plasma was stored at −20 C until assayed for LH levels.

GT1-7 cell perifusions

The GnRH release rate over time from immortalized GT1-7 cells (kindly provided by Dr. Pamela Mellon, University of California-San Diego, San Diego, CA) was determined using a cell perifusion system (52). Cells were grown on Cytodex 3 beads (Amersham Pharmacia, Uppsala, Sweden) for 4–7 d in DMEM containing 10% fetal calf serum on nonadherent petri dishes (Fisher Scientific, Houston, TX). The medium was replaced every 48 h. The GT1-7 cells on beads were loaded into perifusion columns (1.0 ml volume) to a height of 0.75 cm. DMEM, aerated with 95% O2/5% CO2 and kept in a 37 C water bath, was perifused through temperature-controlled columns at a flow rate of 0.1 ml/min. After a 60-min equilibration period, 0.5-ml fractions were collected every 5 min for 4 h. Fractions were frozen and stored at −80 C until RIA.

GT1-7 cell culture and small interfering RNA (siRNA) transfections

The GT1-7 cells were cultured (American Type Culture Collection, Manassas, VA) in DMEM/Ham’s F-12 medium without phenol red supplemented with 10% fetal bovine serum and 100 μg/ml gentamicin, with changes of medium every 1–2 d. The cells became approximately 80% confluent after 4–5 d in culture, at which time the medium was replaced with fresh medium containing charcoal-stripped 5% fetal bovine serum, and the cells were cultured for an additional 16–18 h before use in experiments. Cells were treated with 10 μm forskolin for 30 min before challenges with steroids. A subset of cells was also treated with pertussis toxin during the same period. Cell cultures were treated in triplicate for 5 min with ethanol (EtOH)-DMEM vehicle, P4, allopregnanolone, or dexamethasone. After the 5-min steroid treatments, the medium was removed and cells were lysed for 20 min in 150 μl 0.1 N HCl at 4 C. The lysates were collected, lyophilized, and reconstituted in 250 μl cAMP assay buffer (Biomedical Technologies, Stoughton, MA). They were then stored at −20 C until assayed for cAMP content. In some experiments, the GT1-7 cells were transiently transfected with mouse mPRα or mPRβ siRNA oligonucleotides (siGENOME SMARTpool for mPR), or nonspecific, presynthesized siRNA (control: siCONTROL Non-Targeting siRNA #1; Dharmacon, Chicago, IL) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) at 25 C at a final concentration of 100 nm, after the manufacturer’s procedures (Dharmacon). The medium was replaced with fresh medium after 36 h incubation, and cells were cultured for an additional 24 h before use in experiments. Untransfected GT1-7 cells and cells transfected with mouse mPRα or mPRβ siRNA or CTL siRNA were treated with 100 nm P4 dissolved in 10 μl EtOH (1% of total volume) or 10 μl vehicle for 20 min at 37 C. At the end of the incubation period, the cells were lysed and the cAMP concentration in the cells was measured.

Preparation of plasma membranes and other subcellular fractions

Plasma membranes and other subcellular fractions were prepared from GT1-7 cells after homogenization and centrifugation procedures described previously (53). The cells were scraped from the culture dish and homogenized by sonication in assay buffer for 10–15 sec. The homogenate was centrifuged at 1000 × g for 7 min to remove the nuclei and heavy mitochondria (nuclear fraction), and the supernatant was centrifuged at 20,000 × g for 20 min to obtain a crude plasma membrane fraction. A more purified plasma membrane fraction was obtained by an additional centrifugation step at 6500 × g for 45 min with a 1.2 m sucrose pad. The supernatant from the 20,000 × g spin was centrifuged at 100,000 × g for 60 min to obtain the microsomal pellet and the cytosolic fractions (46).

P4 receptor binding assays

Binding of radiolabeled P4 to plasma membrane preparations of GT1-7 cells was measured as described previously (53). Cell membrane fractions were incubated for 30 min at 4 C with [2,4,6,7-3H]progesterone ([3H]P4,102.1 Ci/mmol) alone (total binding) and in the presence of 100-fold excess non-radiolabeled P4 (nonspecific binding). The reaction was stopped by filtration through glass fiber filters (Whatman GF/B filters). The filters were washed several times with wash buffer, and the radioactivity bound to the membranes was measured by liquid scintillation counting. Specific [3H]P4 binding was calculated by subtracting nonspecific binding from total binding. Competition of R5020 (1 μm) for [3H]P4 binding to plasma membranes was examined using a single-point assay.

Activation of G proteins

G protein activation was determined by measuring the increase in [35S]GTPγ-S binding to plasma membranes (∼50 μg protein) following procedures described previously (53). Plasma membranes of GT1-7 cells were incubated at 25 C for 15 min with 100 nm P4 together with 10 μm GDP and 0.5 nm [35S]GTPγ-S (∼12,000 cpm, 1.0 Ci/mol) in Tris buffer (total binding) and in the presence of 1 μm GTPγ-S (nonspecific binding). An equal volume of 100 μm GTPγS/GDP solution was added to stop the reaction. Aliquots of the reaction mixture were filtered through Whatman GF/B glass fiber filters, followed by several washes and subsequent scintillation counting.

Identification of activated G proteins

The identity of the G proteins activated by P4 was determined by immunoprecipitation of the membrane-bound [35S]GTPγ-S with specific antibodies to G protein α-subunits (53). Plasma membranes were incubated with 1 μm P4 in the presence of 4 nm [35S]GTPγ-S, 10 μm GDP, and protease inhibitors at 25 C, and after 30 min, the reaction was stopped by the addition of ice-cold buffer containing 100 μm GDP and 100 μm unlabeled GTPγ-S, followed by centrifugation. The pellet was resuspended in buffer containing Triton X-100, sodium dodecyl sulfate (SDS), and protease inhibitors and incubated at 4 C for 6 h with G protein Gi, Go, and Gs α-subunit antisera (1:300; Santa Cruz Biotechnology, Santa Cruz, CA). Protein A-Sepharose beads were added, and after an overnight incubation, the immunoprecipitates were pelleted by centrifugation, washed, and boiled in SDS, and the radioactivity in the immunoprecipitated [35S]GTPγ-S-labeled G protein α-subunits counted.

Hormone and cAMP immunoassays

Reagents for LH RIA, including the RP-3 LH standard, were provided by the National Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, MD). The lower limit of detection of the LH assay was 0.2 ng/ml, and the intraassay coefficient of variance was 3.42%, whereas the interassay coefficient of variance was 17.3%. Serum LH values are plotted as means ± sem. P4 levels were measured in some mouse serum samples using an RIA kit purchased from ICN Pharmaceuticals, Inc., Diagnostics Division, Costa Mesa, CA. The sensitivity of the P4 RIA was 0.15 ng/ml, and the intraassay coefficient of variance was 7.7%. GnRH levels in superfusion media were measured using the R1245 GnRH antibody obtained from Dr. Terry Nett, Colorado State University, Fort Collins, CO. The sensitivity of the assay was 0.1 pg/ml, and the intraassay and interassay coefficients of variation were 4.26 and 14.4%, respectively. The cAMP concentration in the GT1-7 cell lysates was measured in initial experiments using a RIA kit from Biomedical Technologies (Stoughton, MA) and in siRNA experiments using an enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI) following the manufacturers’ instructions.

Flow cytometry

Localization of the mPRα protein on the surface of GT1-7 cells was investigated by flow cytometry using the N-terminal mPRα antibody as described previously (53). Washed intact cells were preincubated in blocking solution and incubated for 1 h at room temperature with the mPRα antibody (1:1000) or control rabbit serum in blocking solution. The cells were washed again before incubation with AlexaFluor 488 goat antirabbit IgG antibody (Alexa 488; Molecular Probes, Eugene, OR) in blocking solution for 30 min at room temperature in the dark. After final washes with blocking solution, the cells were resuspended in PBS and analyzed on a flow cytometer (FACSCalibur; Becton Dickinson, Franklin Lakes, NJ). Data were analyzed with CellQuest Pro software (BD Biosciences, San Diego, CA).

Western blot analysis

Plasma membrane proteins and other subcellular fractions from GT1-7 cells were solubilized in gel loading buffer, resolved on 12% SDS-PAGE gels, and transferred to nitrocellulose membranes for Western blot analysis of mPRα and mPRβ proteins as described previously (53). A human mPRα polyclonal antibody generated against an N-terminal 15-amino-acid peptide of human mPRα with a sequence differing at only one position (underlined) to the corresponding region of mouse mPRα (TVDRAEVPPLFWKPC) (1:2500) was incubated with the membranes overnight. Similarly, membranes were incubated overnight with a mPRβ polyclonal antibody (1:2500) generated to a common N-terminal amino acid region of human and mouse mPRβs differing at only one position (KILEDGLPKMPCTVC). The blotted membranes were blocked with 5% nonfat milk in a TBST (50 mm Tris; 100 mm NaCl; 0.1% Tween 20, pH 7.4) buffer for 1 h before incubation with the human mPRα or mPRβ antibody. The membranes were subsequently washed several times and then incubated for 1 h at room temperature with horseradish peroxidase conjugated to goat antirabbit antibody (Cell Signaling, Danvers, MA) and visualized by treatment with enhanced chemiluminescence substrate (SuperSignal; Pierce Biotechnology, Rockford, IL).

Immunocytochemistry

Transfected cells were grown on coverslips for immunocytochemical analysis. Immunocytochemistry of GT1-7 cells was conducted as described previously with few modifications (53). GT1-7 cells, grown on coverslips, were fixed with 2% paraformaldehyde and 0.25% glutaraldehyde in PBS for 15 min at 4 C, rinsed with PBS, and incubated for 10 min with 13 mm NaBH4 in PBS at 4 C to reduce autofluorescence, followed by several washes. The cells were blocked in 2% BSA in PBS for 1.5 h at 4 C and rinsed with PBS. The cells were then incubated overnight at 4 C with the mPRα or mPRβ primary antibodies (dilution 1:1000) in 2% BSA, followed by several rinses of PBS. Cadherin was visualized using a monoclonal anticadherin antibody purchased from Abcam, Inc. (Cambridge, MA) at a dilution of 1:1000. The specificity of the immunoreactions was confirmed by preabsorbing the antisera with peptide antigens (0.02 mg peptide/1 ml antibody) overnight at 4 C. The cells were subsequently incubated with AlexaFluor 488 goat antirabbit secondary antibody (dilution 1:2000) (Molecular Probes). The coverslips were wet-mounted to slides using 80% glycerol in PBS and the presence of fluorescent-labeled mPRα proteins in the cells visualized using a Nikon fluorescence microscope.

Biotinylation of surface proteins

The biotinylation of surface proteins was performed as described previously (54). Briefly, GT1-7 cells were incubated in 25-cm2 flasks until they were 90% confluent, washed with PBS (pH 8), and then incubated with 1 mg/ml sulfo-NHS-LC biotin (Pierce) for 30 min at 4 C to label the cell surface proteins. The reaction was stopped by washing the cells with ice-cold PBS containing 50 mm Tris-HCl (pH 7.5), and the cells were removed from the plates with lysis buffer containing 150 mm NaCl, 10 mm Tris-HCl (pH 7.5), 1% Nonidet P-40 (USB, Cleveland, OH), and 0.1% protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) and solubilized by shaking for 30 min at 4 C followed by centrifugation for 10 min at 20,000 × g to remove the cell debris. An immobilized streptavidin-agarose slurry (Pierce Biotechnology) was added to the supernatants, and the mixture was incubated at 4 C overnight with continuous shaking to absorb the biotinylated proteins. The mixture was washed with buffer (0.1% Nonidet P-40, 150 mm NaCl, and 20 mm Tris-HCl, pH 7.5), and the biotinylated proteins were eluted by boiling the slurry for 10 min in 100 μl 2× SDS sample buffer. Final samples were centrifuged, and the supernatants were electrophoresed followed by Western blot analysis.

RT-PCR of mPRα and mPRβ

The following RT-PCR protocol was used for initial identification of mPRα and mPRβ mRNAs in mouse tissues and GT1-7 cells. Total RNA was extracted with Tri-reagent (Sigma-Aldrich), and reverse transcription was performed by following standard procedures (3). The forward primer for mPRβ was 5′-TGACGACTGCCATCCTAGAGCG-3′, and the reverse primer was 5′-CAATGCCCCTGCCTCCACAAAG-3′, whereas the forward primer for mPRα was 5′-CAGAAGCCTCCGCAACCAGAAC-3′, and the reverse primer was 5′-GAGCCACAGCACTGAACGAGAG-3′. These primer sets generated products of 305 and 310 bp for mPRβ and mPRα, respectively. Five microliters of the RT reaction were combined with 10 ng of each of the specific primers, 100 nm dNTPs, 1× PCR buffer, and 2 mm MgCl2 in a PCR. This reaction was incubated for an initial denaturation step at 94 C for 2 min, a cycle denaturation step at 94 C for one min, a cycle annealing step at 61 C for one min, a cycle extension step at 72 C for 1 min, and a final extension step at 72 C for 10 min. Expected PCR products were confirmed by DNA sequence analysis.

Expression of mouse mPRα mRNA in GT1-7 cells after transfection with mPRα siRNA was determined by a similar but modified RT-PCR protocol. The PCR was conducted in 30 μl PCR SuperMix (Invitrogen) consisting of 0.5 μl of the RT reaction and 0.2 μm of each of the primers. Gene-specific primers for mouse mPRα (sense, 5′-CCGTGTACCAGTTTGGCAG-3′, and antisense, 5′-CGGGCCTGATAATCCAGTG; mPRβ: sense, 5′-TCGTCCATCACTTACCTCACC-3′, and antisense, 5′-GCCCACAATGTCACAGGAAC-3′) were designed according to the mouse mPRβ sequence (GenBank accession nos. NM_027995 and AF 313617). After an initial denaturation for 5 min at 94 C, the PCR was performed on an Eppendorf Mastercycler for 35 cycles with the cycling profile of 30 sec at 94 C, 30 sec at 55 C, and 1 min at 72 C followed by a 10-min extension at 72 C. The PCR (5 μl) was electrophoresed on an agarose gel (1%) containing ethidium bromide to visualize the products. For semiquantitative RT-PCR, 25 cycles of PCR were performed (linear portion of cycle/product curve).

Statistical analysis

The effects of P4, genotype, and GnRH on LH secretion in vivo were analyzed by three-way ANOVA with repeated measures. Planned comparisons within treatment groups and genotypes were carried out to compare the effects of P4 on initial vs. terminal blood samples using paired t tests. A response was considered significant if P < 0.05. The effects of P4 on GnRH release from GT1-7 cells were analyzed by two-way ANOVA with repeated measures. To assess the effects of treatments on cAMP levels in GT1-7 cell cultures, one-way ANOVAs were performed with post hoc comparisons by Neuman-Keuls test. Linear and nonlinear regression analyses for all receptor binding assays and calculations of Kd and binding capacity were performed using GraphPad Prism for Windows (version 3.02; Graph Pad Software, San Diego, CA). Three-way ANOVAs were carried out using NCSS version 2004 (Number Cruncher Statistical Systems, Kaysville, UT).

Results

P4 suppresses LH levels in both WT and PRKO mice

The administration of 400 μg P4 (Fig. 1B), but not oil (Fig. 1A), to WT mice (n = 6 per group) produced a significant suppression of LH levels 4 h later (mean ± se of LH levels before and after P4 treatment: 3.1 ± 0.5 and 1.84 ± 0.13 ng/ml, P < 0.05; before and after oil vehicle treatment: 2.64 ± 0.47 and 4.60 ± 0.76 ng/ml, P > 0.05). Similarly, the administration of P4 (n = 5; Fig. 1D), but not oil (n = 4; Fig. 1C), to PRKO animals also suppressed LH levels (LH levels before and after P4 treatment: 1.74 ± 0.23 and 0.40 ± 0.09 ng/ml, P < 0.01; before and after oil vehicle treatment: 1.44 ± 0.17 ng/ml and 1.27 ± 0.44 ng/ml, P > 0.05). Mean serum P4 levels at 4 h after injection were 112.5 ± 35 ng/ml.

Figure 1.

Figure 1

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P4 injections suppress serum LH levels in both WT and PRKO mice. Blood samples were obtained from OVX WT (A and B) and PRKO (C and D) mice just before and 4 h after injection of 0.1 ml oil vehicle (A and C) or 400 μg P4 (B and D) in 0.1 ml oil. Depicted are LH levels measured in individual animals before and after injections connected by solid lines. P4, but not oil, injections produced significant reductions in serum LH levels in animals of both genotypes. *, P < 0.05 for postinjection compared with preinjection LH values.

Allopregnanolone and dexamethasone do not suppress LH levels

Some rapid effects of P4 have been linked to the heterologous activation of GABAA chloride channels, wherein P4 is rapidly metabolized in the brain to 3α5αTHP (or allopregnanolone), which in turn activates the channels. We tested the possibility that P4’s effects on LH secretion in vivo may be mediated by its metabolism to allopregnanolone and its subsequent actions via this mechanism. Injection of allopregnanolone produced no significant change in LH levels in WT mice (n = 6, Fig. 2A), suggesting that P4 metabolites are not responsible for the response to P4 that we observed (LH levels before and after allopregnanolone in WT mice: 4.02 ± 0.76 and 3.66 ± 0.95 ng/ml, P > 0.05).

Figure 2.

Figure 2

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Allopregnanolone and dexamethasone injections do not alter serum LH levels in OVX WT mice. Blood samples were obtained from mice just before and 4 h after injection of 16 μg allopregnanolone (A) or 400 μg dexamethasone (B). Depicted are LH levels measured in individual animals before and after injections connected by solid lines. Neither compound produced any significant effect on serum LH levels for postinjection compared with preinjection LH values (P > 0.05). (See Fig. 1 for responses to oil injections, all groups collected simultaneously).

P4 is also known to activate glucocorticoid receptors (GRs) under certain experimental circumstances. We assessed whether this mechanism may account for the effects of P4 on LH secretion by testing the ability of dexamethasone, a specific GR agonist, to suppress LH secretion. In WT mice (n = 7, Fig. 2B), dexamethasone did not alter LH levels (LH levels in WT mice before and after dexamethasone: 3.31 ± 0.37 and 4.71 ± 0.68 ng/ml, P > 0.05).

P4 does not impair pituitary responsiveness to GnRH

The suppression of LH by P4 may result from a reduction in GnRH secretion or through direct modulation of LH release from gonadotropes. To assess the latter possibility, WT mice were administered either saline vehicle or GnRH (200 ng/kg, sc) 10 min before obtaining the terminal blood sample. This amount of GnRH was previously used to study pituitary responsiveness and the GnRH self-priming mechanism in WT and PRKO animals (33). Although saline injections were without effect, the administration of GnRH produced 2- to 3-fold increases in LH levels in both oil- and P4-treated mice (Fig. 3). Administration of P4 did not blunt the response to GnRH compared with corresponding oil-treated controls; rather, responses to GnRH tended to be greatest in the P4-treated WT animals, although these were not statistically different from those observed in the oil-treated WT animals. Treatment with P4 did not blunt the response to GnRH in the PRKO mice, although the tendency to exhibit exaggerated responses to GnRH, as noted in the WT mice, was not evident in the PRKO mice.

Figure 3.

Figure 3

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P4 does not impair pituitary responsiveness to GnRH stimulation in either OVX WT (A) or OVX PRKO (B) females. Blood samples were obtained from OVX mice of both genotypes just before and 4 h after injection of 0.1 ml oil or 400 μg P4 in 0.1 ml oil. Ten minutes before the second sample was obtained, an injection of 0.9% saline vehicle or 200 ng/kg GnRH was given sc. Saline injections were without effect on LH levels, whereas GnRH injections stimulated LH secretion in both oil- and P4-treated animals of both genotypes (repeated-measures ANOVA, P < 0.04). Responses to GnRH tended to be greater, rather than reduced, in WT P4-treated animals (A), an effect not seen in the PRKO animals (B) as reflected in the significant interaction between genotype, P4, and the response to the GnRH injection (repeated-measures ANOVA, P = 0.04). *, P < 0.05, paired t test, one-tailed test.

P4 suppresses GnRH release from superfused GT1-7 cells

The effects of P4 on GnRH release from superfused GT1-7 cells are depicted in Fig. 4. Shown are the mean GnRH release rates ± se at each consecutive 10-min collection period before, during, and after superfusion with EtOH vehicle (A) or P4 (10−7 m) (B) added to the medium. Statistical analysis of mean GnRH release rates collapsed over 40-min time bins revealed a significant suppressive effect of P4 (Fig. 4.D) but not EtOH vehicle on mean GnRH release (Fig. 4B).

Figure 4.

Figure 4

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P4 suppresses GnRH release from superfused immortalized GT1-7 cells. GnRH release was measured in consecutive 10-min superfusion fractions before, during, and after exposure to medium containing EtOH vehicle (A) or P4 (10−7 m). (C) When data were collapsed over 40-min bins, a significant inhibitory effect of P4 (D) on GnRH release during the 120 min of P4 exposure was observed compared with baseline values, whereas EtOH vehicle was without effect (B). *, P < 0.05.

P4 suppresses forskolin-stimulated cAMP accumulation in GT1-7 cells

The GT1-7 cell cultures were pretreated for 30 min with 10 μm forskolin and 1 μm 3-isobuytl-1-methylxanthine and then treated for 5 min with EtOH vehicle or 10−8, 10−7, or 10−6 m P4. Figure 5A demonstrates that all three concentrations of P4, but not EtOH vehicle, produced a significant suppression of cAMP accumulation. The effects of P4 were abolished by pretreatment with pertussis toxin (Fig. 5B), indicating that these suppressive effects are mediated by activation of Gi. Neither allopregnanolone nor dexamethasone exposure at concentrations of 10−8, 10−7, or 10−6 m produced any significant effect on forskolin-stimulated cAMP accumulation (data not shown).

Figure 5.

Figure 5

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P4 rapidly suppresses forskolin-stimulated cAMP accumulation. A, Bars denote forskolin-stimulated cAMP levels in GT1-7 cells incubated with medium containing EtOH or one of three concentrations of P4 (10−8 to 10−6 m), revealing a significant suppression by P4 at all three levels compared with vehicle (veh). B, The inhibitory effects of P4 were abolished by pretreatment with pertussis toxin (PTX), indicating that the effects of P4 are mediated by activation of Gi. **, P < 0.001 compared with vehicle control; ***, P < 0.0001 compared with vehicle control.

Immunoprecipitation experiments were conducted to assess the degree to which P4 may specifically activate Gi. Treatment of isolated plasma membranes in vitro with 100 nm P4 caused a significant increase in specific binding of [35S]GTPγ-S, indicating P4 activates G proteins in GT1-7 cells (Fig. 6A). Immunoprecipitation of the activated G protein α-subunits bound to [35S]GTPγ-S with specific G protein α-subunit antibodies showed that a rabbit Gi antibody precipitated almost all of the total radioactive GTPγ-S activated by P4 (1 μm), whereas negligible radioactivity was precipitated with a specific α-Gs antibody and control rabbit serum (Fig. 6B). These results demonstrating that P4 causes activation of an inhibitory G protein are consistent with the previous results showing cAMP production is decreased in these cells via a pertussis toxin-sensitive pathway after P4 treatment.

Figure 6.

Figure 6

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Activation of G proteins in plasma membranes of GT1-7 cells by P4. A, Treatment with 100 nm P4 increases specific binding of [35S]GTPγ-S to plasma membrane preparations relative to vehicle or cortisol (Cort). *, P < 0.05 compared with vehicle treatment; n = 4. B, Treatment of GT1-7 cells with 1 μm P4 increases immunoprecipitation of [35S]GTPγ-S bound to G protein α-subunits by an antiserum specific for Gαi (Gi) but not for Gαs (Gs). For control rabbit serum (CTL) or G protein antibodies, n = 4. Veh, Vehicle. **, P < 0.001 compared with vehicle control.

This signaling pathway is presumably mediated through a specific G protein-coupled P4 receptor. Saturation and Scatchard analyses of [3H]P4 binding to the plasma membranes of GT1-7 cells show the presence of a high-affinity (Kd 5.85 nm), limited-capacity (Bmax 62.24 nm) single binding site for P4 (Fig. 7A), characteristics typical of steroid membrane receptors. A single-point competitive binding assay showed that the PR agonist R5020 displayed negligible binding to plasma membranes compared with that of P4 (Fig. 7B) and did not significantly alter forskolin-stimulated cAMP accumulation in GT1-7 cells (Fig. 7C), indicating that the action of P4 is not mediated by PR.

Figure 7.

Figure 7

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A, Representative saturation analysis and Scatchard plot of specific [3H]P4 binding to plasma membranes prepared from GT1-7 cells. The binding assay was repeated three times with different batches of cells, and similar results were obtained on each occasion. B, Single-point competition assay of binding of R5020 (1 μm) and P4 (100 nm and 1 μm) to plasma membranes of GT1-7 cells. C, Effects of 100 nm R5020 and P4 whole cell forskolin-stimulated cAMP levels accumulation. Veh, Vehicle. **, P < 0.001 compared with control.

Expression of mPRα and mPRβ in GT1-7 cells

Members of the novel membrane P4 receptor family, mPRα and mPRβ, activate inhibitory G proteins and, therefore, are candidates for the P4 receptors mediating these effects in the mouse hypothalamus and in GT1-7 cells. RT-PCR shows that both mPRα and mPRβ mRNAs are expressed in the mouse preoptic area (POA) and in GT1-7 cells (Fig. 8). PCR products of the expected sizes for both mPRs were also found with mRNA from uterus and kidney, whereas no bands could be seen in any of the RT-minus lanes, indicating a lack of genomic DNA contamination.

Figure 8.

Figure 8

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RT-PCR analyses reveal that both mPRβ (A) and mPRα (B) mRNAs are expressed in the mouse preoptic area (POA) of the mediobasal hypothalamus (POA-MBH) and GT1-7 cells. mRNA was also detected in pituitary (P), uterus (U), and kidney (K) tissues.

Western blot analysis of plasma membranes prepared from mouse POA and GT1-7cells using N-terminal antibodies for mPRα and mPRβ showed that both receptor proteins are expressed, with immunoreactive bands at approximately 40 and 80 kDa, corresponding to the expected sizes of mPR monomers and dimers, respectively (Fig. 9, A and B). Both intracellular and cell surface expression of mPRα was also observed in GT1-7 cells by Western blot analysis with the mPRα antibody. Strong immunoreactive bands around 40 kDa were present in the microsomal fraction as well as in the plasma membrane fractions, whereas no mPRα protein could be detected in the cytoplasmic and nuclear fractions (Fig. 9C).

Figure 9.

Figure 9

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A and B, mPR protein expression in plasma membrane fractions of GT1-7 cells. The mPRα (A) and mPRβ (B) proteins were detected in GT1-7 cell membranes by Western blot analyses. Mkr, Molecular weight protein standards; pep, blocked by preincubation with peptide antigen. C, mPR protein expression in subcellular fractions of GT1-7 cells. The mPRα protein was detected in subcellular fractions of GT1-7 cells by Western blot analysis. Cyt, Cytosolic; Ms, microsomal; Mem, plasma membrane; Mem (sp), plasma membrane purified with sucrose pad; Nu, nuclear.

Cell surface localization of mPRα was confirmed in nonpermeabilized GT1-7 cells by flow cytometry. Incubation of the cells with the IgG fraction of the N-terminal mPRα antibody resulted in a marked increase in fluorescence compared with that observed with a control rabbit serum IgG fraction (Fig. 10A). These results also indicate that mouse mPRα is orientated in GT1-7 cells with the N-terminal on the outside of the cell. A 40-kDa band, which was absent after preincubation with the mPRβ peptide antigen (Fig. 10B), was detected with the mPRβ antibody on Western blots of immunoprecipitated biotinylated surface proteins, indicating that mPRβ is also expressed on the surface of GT1-7 cells. Localization of the mPRα and mPRβ proteins in the plasma membranes of GT1-7 cells was confirmed by immunocytochemistry. A large proportion of mPRα and mPRβ immunoreactivity was concentrated near the cell membrane and colocalized with cadherin, a specific plasma membrane marker (Figs. 11 and 12).

Figure 10.

Figure 10

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Localization of mPRs on the surface of GT1-7 cells. A, Flow cytometry of mPRα expression on nonpermeabilized cells using the human mPRα antibody (hmPRα-IgG, bottom). Fluorescence intensity (Alexa 488) is compared with that obtained with control rabbit IgG (CTL-rabbit IgG, top). B, Western blot analysis of mPRβ (biot) after biotin surface labeling and immunoprecipitation of cell-surface proteins. pep/biot, Blocked by preincubation with peptide antigen.

Figure 11.

Figure 11

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Immunocytochemical staining of GT1-7 cells with mPRα and antibodies for the transmembrane protein cadherin. Peptide block indicates preabsorption with peptide antigen; 2nd Ab only, incubation with second antibody only.

Figure 12.

Figure 12

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Immunocytochemical staining of GT1-7 cells with mPRβ and antibodies for the transmembrane protein cadherin. Peptide block indicates preabsorption with peptide antigen; 2nd Ab only, incubation with second antibody only.

Treatment of GT1-7 cells with mPRα siRNA caused an approximately 60% decrease in expression of mPRα mRNA (Fig. 13A) and protein (Fig. 13B), which was accompanied by a 50% decrease in specific [3H]P4 binding (Fig. 13C). This treatment also blocked the P4-induced decrease in cAMP content in forskolin-stimulated GT1-7 cells (Fig. 13D). In contrast, although treatment of GT1-7 cells with mPRβ siRNA caused significant decreases in mPRβ mRNA and protein expression (Fig. 14, A and B) and specific [3H]P4 binding (Fig. 14C), the P4-induced decrease in cAMP content in forskolin-stimulated GT1-7 cells was not blocked (Fig. 14D). The results of the siRNA experiments suggest that the specific [3H]P4 receptor binding and down-regulation of adenylyl cyclase by P4 in GT1-7 cells are mediated by mPRα.

Figure 13.

Figure 13

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Effects of transfection with 100 nm mPRα siRNA (mPRα siRNA) on mPRα mRNA expression (A), protein expression (B), specific [3H]P4 binding to cell membranes (C), and cAMP production by membranes in response to 100 nm P4 treatment (D) 18 h later. Cad, Cadherin loading control; CTL, nonspecific control siRNA. n = 6. *, P < 0.05 compared with control siRNA or vehicle control. si, siRNA.

Figure 14.

Figure 14

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Effects of transfection with 100 nm mPRβ siRNA (mPRβ siRNA) on mPRβ mRNA expression (A), protein expression (B), specific [3H]P4 binding to cell membranes (C) and cAMP production by membranes in response to 100 nm P4 treatment (D) 18 h later. Cad, Cadherin loading control; CTL, nonspecific control siRNA. n = 6. *, P < 0.05 compared with control siRNA or vehicle control. si, siRNA.

Discussion

Ovarian P4 secretion is critically important in the physiological regulation of GnRH and LH pulsatility. Surprisingly, relatively few studies have focused on the receptors that may convey these actions. One pharmacological study in sheep provided evidence that the intracellular P4 receptors, PRA/PRB, are important in mediating P4’s suppressive effects on pulsatile GnRH neurosecretion in that species (14). Until the present studies, however, this issue had not been directly addressed by determining the extent to which P4’s actions may be altered in PR-deficient animals. We have unexpectedly found that acute P4 treatment can exert a significant inhibitory effect on LH release in PRKO mice, suggesting that alternative pathways exist that mediate at least some of P4’s inhibitory actions on GnRH and LH secretion. Our parallel in vitro studies in immortalized GT1-7 cells have identified at least one plausible mechanism that may explain P4’s PRA/PRB-independent effects on GnRH release in the mouse, a rapid inhibitory effect of P4 on cAMP accumulation mediated by the activation of the recently discovered G protein-coupled mPRs (45,46). Taken together, these in vivo and in vitro studies reveal the existence of PRA/PRB-independent actions of P4 that may contribute to its physiological actions in neuroendocrine systems.

P4 is the principal ovarian steroid hormone in the circulation during the ovulatory cycle and pregnancy. In primates (13), sheep (9), and other animals that exhibit a distinct luteal phase of the cycle (35), the luteal rise in P4 serves to retard the GnRH pulse generator and thereby restrain LH secretion. In rodents, the major release of P4 occurs on the afternoon and evening of proestrus and is responsible for the suppression of both GnRH pulsatility (11) and release of successive GnRH surges on subsequent days (24). The elevation of P4 secretion during pregnancy in female mammals also contributes to the suppression of basal GnRH pulsatility (55) and prevents release of preovulatory LH surges (56), presumably by blocking neurosecretion of GnRH surges (57,58). Ovarian P4 additionally appears to inhibit GnRH neurosecretion during suckling in rats (59).

It is not known whether a common mechanism mediates all of the foregoing inhibitory actions of P4 on GnRH release and whether any single suppressive effect may be exerted through the combined activation of multiple pathways. It has been largely assumed, nonetheless, that most of P4’s inhibitory effects on GnRH release depend upon activation of the nuclear PRA/B receptors in GnRH neurons (60) and/or the afferent circuitries that govern GnRH release (57,58). By using PR antagonists to block the effects of endogenous or exogenous hormone, it has been possible to demonstrate that at least some of the effects of P4 are mediated by PRA/B. A role for PRA/B has been most firmly established in ewes, where RU486 was shown to block the inhibitory effects of P4 on GnRH and LH pulse frequency (14). In proestrous rats, administration of RU486 has been shown to evoke increases in LH pulsatility and continued release of LH surges on estrus (11). Similarly, treatment of lactating rats with RU486 accelerates the onset of LH pulsatility after pup removal (59). In all of these studies, however, the ability of RU486 to block P4’s inhibitory actions was assessed under circumstances in which P4 circulates in the low- to mid-physiological range of values. To our knowledge, there have been no studies of PRA/B mediation of P4’s actions when the steroid is acutely elevated to high physiological levels, such as those reached during the proestrous P4 surge or during pregnancy. The present studies demonstrate that the administration of P4, at a dose that produces high physiological plasma P4 levels, can suppress LH secretion in vivo, even in the absence of nuclear PR expression (61). Likewise, the exposure of immortalized GnRH neurons to a similar concentration (10−7 m) of P4 in vitro was found to rapidly suppress GnRH release.

P4 has previously been found to exert robust effects on cells that do not express appreciable numbers of nuclear PRs, such as T lymphocytes (62) and the corpus luteum (63). At least some of these have been shown to be mediated by relatively rapid, nongenomic PRA/B-independent signaling pathways (64). We have previously determined that the GT1-7 cells express little or no mRNA encoding nuclear PRs in the absence of estrogen priming (unpublished observation), and in the present studies, we have found that GT1-7 cell plasma membranes possess high-affinity binding sites for P4. We therefore pursued the hypothesis that P4 can activate membrane receptors that have previously been shown to mediate P4 effects in other cells. One of the best characterized of these cellular pathways in the brain involves the heterologous activation of GABAA receptors. P4 is rapidly metabolized in the brain to 3α5αTHP, which in turn can act as a potent barbiturate-like modulator of GABAA receptors (40). Because previous studies suggested that the neural and/or pituitary actions of 3α5αTHP may be involved in the stimulatory actions of P4 on LH secretion (41,42), we assessed the ability of this metabolite to mimic the suppressive effects of P4. However, a role for 3α5αTHP in the suppression of GnRH and LH secretion was not supported by the present studies because the in vivo and in vitro administration of the metabolite failed to alter release of either hormone.

Recently a novel cDNA was discovered in the spotted seatrout by Zhu et al. (45,46) that encodes a protein with all of the major characteristics of a membrane P4 receptor. Three putative mPRs (mPRα, mPRβ, and mPRγ) were identified that have the structural and functional attributes of GPCRs and belong to a broader family of highly conserved progestin and adiponectin receptors (53). The mPRs have been shown to couple to inhibitory G proteins, producing a reduction in cAMP levels upon activation in a variety of reproductive tissues (53). A member of the mPR/adiponectin receptor family has also been found to be localized to the endoplasmic reticulum in ovine luteal cells and to produce an elevation of intracellular Ca2+ upon activation by P4 (48). In the present studies, we have found that the mammalian mPRα and mPRβ mRNAs, as well as their corresponding proteins, are expressed in GT1-7 cells.

We have obtained several pieces of evidence that support the hypothesis that P4 can activate Gi-coupled receptors in GT1-7 cells and that mouse mPRα comprises at least a portion of this P4-responsive receptor population. P4 suppressed forskolin-stimulated cAMP accumulation, and this action was prevented by coadministration of pertussis toxin. Treatment of GT1-7 cell plasma membranes with P4 increased specific binding of [35S]GTPγ-S, indicating P4 activates G proteins in GT1-7 cells, and immunoprecipitation of activated G protein α-subunits bound to [35S]GTPγ-S with specific G protein α-subunit antibodies revealed that the Gi antibody precipitated almost all of the total radioactive GTPγ-S activated by P4. The demonstration that both mPRα and mPRβ are expressed on the cell membranes of GT1-7 cells is in agreement with previous studies using other cell types and is consistent with the proposed mechanism of action through coupling to and activation of G proteins in the plasma membrane (53,65). Evidence that mPRα may specifically mediate the activation of Gi by P4 was provided by the findings that mPRα siRNA treatment of GT1-7 cells caused similar decreases in both mPRα and specific [3H]P4 binding and a reversal of the inhibitory effect of P4 on forskolin-stimulated cAMP accumulation. In contrast, although mPRβ siRNA treatments caused a modest reduction in [3H]P4 binding, they did not alter the cAMP response to P4, demonstrating that mPRβ may contribute to the total binding of P4 in GT1-7 cells, yet it does not mediate the rapid effects of P4 on cAMP accumulation. Collectively, these experiments reveal that P4 can activate mPRα and thereby prompt Gi-mediated suppression of cAMP formation. Although technical limitations precluded the assessment of siRNA effects on GnRH release in superfused GT1-7 cells, it would appear reasonable to infer that the activation of mPRα and rapid suppression of cAMP accumulation can mediate at least some of the inhibitory effects of P4 on GnRH release.

Our studies have revealed a PRA/B-independent, inhibitory effect of P4 on GnRH release in vivo and in GT1-7 cells; however, we do not know the extent to which these in vivo and in vitro effects are manifest through a common mechanism. Nevertheless, there are common features of these effects that suggest that the mechanisms may overlap. Both the in vivo and in vitro effects observed in these studies were not mimicked by 3α5αTHP or dexamethasone, making it unlikely that GABAA receptor or heterologous GR activation contributes to either effect. Moreover, the inhibitory effects of P4 in vivo and in vitro were observed in the absence of cotreatment with estradiol-17β. It is well known that the inhibitory effects of P4 on LH secretion in rats are usually dependent upon prior or cotreatment with estradiol-17β. Thus, treatment of OVX, estradiol-17β-primed female rats with P4 implants that recapitulate low- or mid-physiological range steroid levels results in an inhibition of pulsatile LH secretion (10). The induction of nuclear PRs by estradiol-17β appears to be the key determinant of these P4 actions. Some studies, however, have shown that P4’s inhibitory actions can be observed after ovariectomy, in the absence of cotreatment with estradiol-17β (17). Higher levels of P4, such as those reached during pregnancy (>40 ng/ml in serum), appear to retard LH pulsatility in the absence of estrogen priming (66).

It is possible that several cellular mechanisms function in a parallel, integrated, and/or redundant manner to mediate the negative feedback actions of P4 on GnRH release. Apart from PRA/B- and mPR-mediated mechanisms, these pathways may include some mediated by the activation of the membrane-associated P4 binding protein, P4 receptor membrane component 1 (PGRMC1), which has been shown to be involved in the female reproductive behaviors (67). It is also possible that these different putative signaling pathways may be differentially activated under different physiological circumstances and thereby play distinct physiological roles in the regulation of the reproductive axis. For example, the mPRs may be recruited to suppress GnRH release under conditions of elevated serum P4, such as on the late afternoon of proestrus. The physiological function of this action may be to rapidly terminate the neurosecretion of the primary GnRH and LH surges, a possibility that remains to be tested.

P4 is one of several steroid hormones that have been shown to exert their actions through a heterogeneous set of signaling pathways. These include genomic and nongenomic mechanisms, intracellular and plasma membrane receptors, and rapid vs. delayed effects. The present in vivo and in vitro findings demonstrate that P4, like estrogen (68), testosterone (69,70), glucocorticoids (71,72), and mineralocorticoids (64,73) can exert physiologically relevant actions that are independent of the activation of classic nPRs. The extent to which these PRA/B-independent effects are mediated by activation of mPRs in GnRH neurons or their afferent circuitries in vivo remains to be determined and is the subject of current studies.

Footnotes

This work was supported by National Institutes of Health Grants ESO12961 (to P.T.) and U54 HD041859.

Disclosure Summary: The authors have nothing to declare.

First Published Online May 7, 2009

Abbreviations: EtOH, Ethanol; GABAA, γ-aminobutyric acidA; Gi, inhibitory G protein; GPCR, G protein-coupled receptor-like protein; GR, glucocorticoid receptor; mPR, membrane P4 receptor; nPR, nuclear P4 receptor; OVX, ovariectomized; P4, progesterone; POA, preoptic area; PRKO, PR knockout; SDS, sodium dodecyl sulfate; siRNA, small interfering RNA; 3α5αTHP, 3α-hydroxy-5α-pregnan-20-one; WT, wild type.

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