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, 150 (8), 3833-44

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

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

Nicole Sleiter et al. Endocrinology.

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 (PR(A/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 (G(i)), as shown by immunoprecipitation with a G(i) antibody of most of the increase in membrane-bound [(35)S]GTPgamma-S. Saturation binding analyses demonstrated the presence of a high affinity (K(d) 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, mPRalpha and mPRbeta. Western blotting, immunocytochemistry, and flow cytometry experiments similarly revealed expression of mPR proteins in the plasma membranes of GT1-7 cells. Treatment with mPRalpha 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 PR(A/B)-independent effects on GnRH release that may in part be mediated by mPRs.

Figures

Figure 1
Figure 1
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.
Figure 2
Figure 2
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).
Figure 3
Figure 3
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.
Figure 4
Figure 4
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.
Figure 5
Figure 5
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.
Figure 6
Figure 6
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.
Figure 7
Figure 7
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.
Figure 8
Figure 8
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.
Figure 9
Figure 9
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.
Figure 10
Figure 10
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
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
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.
Figure 13
Figure 13
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
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.

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