Non-genomic Actions Of Progesterone Research Articles

Membrane progesterone receptor beta (mPR\u03b2/Paqr8) promotes progesterone-dependent neurite outgrowth in PC12 neuronal cells via non-G protein-coupled receptor (GPCR) signaling

Recently, sex steroid membrane receptors garnered world-wide attention because they may be related to sex hormone-mediated unknown rapid non-genomic action that cannot be currently explained by their genomic action via nuclear receptors. Progesterone affects cell proliferation and survival via non-genomic effects. In this process, membrane progesterone receptors (mPRα, mPRβ, mPRγ, mPRδ, and mPRε) were identified as putative G protein-coupled receptors (GPCRs) for progesterone. However, the structure, intracellular signaling, and physiological functions of these progesterone receptors are still unclear. Here, we identify a molecular mechanism by which progesterone promotes neurite outgrowth through mPRβ (Paqr8) activation. Mouse mPRβ mRNA was specifically expressed in the central nervous system. It has an incomplete GPCR topology, presenting 6 transmembrane domains and did not exhibit typical GPCR signaling. Progesterone-dependent neurite outgrowth was exhibited by the promotion of ERK phosphorylation via mPRβ, but not via other progesterone receptors such as progesterone membrane receptor 1 (PGRMC-1) and nuclear progesterone receptor in nerve growth factor-induced neuronal PC12 cells. These findings provide new insights of regarding the non-genomic action of progesterone in the central nervous system.

Membrane Associated Progesterone Receptors: Promiscuous Proteins with Pleiotropic Functions - Focus on Interactions with Cytochromes P450.

Membrane-associated progesterone receptors (MAPR) are a group of four rather small, partially homologous proteins, which share a similar non-covalent heme-binding domain that is related to cytochrome b5, a well-known functional interaction partner of microsomal cytochrome P450 (CYP) monooxygenase systems. Apart from their structural similarities the four proteins progesterone membrane component 1 (PGRMC1, also referred to as IZA, sigma-2 receptor, Dap1), PGRMC2, neudesin (NENF) and neuferricin (CYB5D2) display surprisingly divergent and multifunctional physiological properties related to cholesterol/steroid biosynthesis, drug metabolism and response, iron homeostasis, heme trafficking, energy metabolism, autophagy, apoptosis, cell cycle regulation, cell migration, neural functions, and tumorigenesis and cancer progression. The purpose of this mini-review is to briefly summarize the structural and functional properties of MAPRs with particular focus on their interactions with the CYP system. For PGRMC1, originally identified as a non-canonical progesterone-binding protein that mediates some immediate non-genomic actions of progesterone, available evidence indicates mainly activating interactions with steroidogenic CYPs including CYP11A1, CYP21A2, CYP17, CYP19, CYP51A1, and CYP61A1, while interactions with drug metabolizing CYPs including CYP2C2, CYP2C8, CYP2C9, CYP2E1, and CYP3A4 were either ineffective or slightly inhibitory. For the other MAPRs the evidence is so far less conclusive. We also point out that experimental limitations question some of the previous conclusions. Use of appropriate model systems should help to further clarify the true impact of these proteins on CYP-mediated metabolic pathways.

Sperm gamma-aminobutyric acid type A receptor delta subunit (GABRD) and its interaction with purinergic P2X2 receptors in progesterone-induced acrosome reaction and male fertility.

The mechanism underlying the non-genomic action of progesterone in sperm functions and related Ca2+ mobilisation remains elusive. Herein we report the expression of gamma-aminobutyric acid type A receptor delta subunit (GABRD) in human and rodent sperm and its involvement in mediating the progesterone-induced acrosome reaction. GABRD was localised in the sperm head/neck region. A δ(392-422)-specific inhibitory peptide against GABRD blocked the progesterone-induced acrosome reaction and the associated increase in intracellular Ca2+. Similarly, an inhibitory effect against both progesterone-induced Ca2+ influx and the acrosome reaction was observed with a P2X2 receptor antagonist. The lack of synergism between the GABRD and P2X2 inhibitors suggests that these two receptors are playing a role in the same pathway. Furthermore, a co-immunoprecipitation experiment demonstrated that GABRD could undergo protein-protein interactions with the Ca2+-conducting P2X2 receptor. This interaction between the receptors could be reduced following progesterone (10μM) inducement. Significantly reduced GABRD expression was observed in spermatozoa from infertile patients with reduced acrosome reaction capacity, suggesting that normal expression of GABRD is critical for the sperm acrosome reaction and thus male fertility. The results of the present study indicate that GABRD represents a novel progesterone receptor or modulator in spermatozoa that is responsible for the progesterone-induced Ca2+ influx required for the acrosome reaction through its interaction with the P2X2 receptor.

Nongenomic actions of progesterone and 17β-estradiol on the chloride conductance of skeletal muscle

Myotonia congenita, caused by mutations in ClC-1, tends to be more severe in men and is often exacerbated by pregnancy. We performed whole-cell patch clamp of mouse muscle chloride currents in the absence/presence of 100 μM progesterone or 17β-estradiol. 100 μM progesterone rapidly and reversibly shifted the ClC-1 activation curve of mouse skeletal muscle (V50 changed from -52.6 ± 9.3 to +35.5 ± 6.7; P < 0.01) and markedly reduced chloride currents at depolarized potentials. 17β-estradiol at the same concentration had a similar but smaller effect (V50 change from -57.2 ± 7.6 to -40.5 ± 9.8; P < 0.05). 1 μM progesterone produced no significant effect. Although the data support the existence of a nongenomic mechanism in mammalian skeletal muscle through which sex hormones at high concentration can rapidly modulate ClC-1, the influence of hormones on muscle excitability in vivo remains an open question.

Novel actions of progesterone: what we know today and what will be the scenario in the future?

This article is aimed to review the novel actions of progesterone, which otherwise is considered as a female reproductive hormone. The article focuses on its important physiological actions in males too and gives an overview of its novel perspectives in disorders of central and peripheral nervous system. Progesterone may have a potential benefit in treatment of traumatic brain injury, various neurological disorders and male related diseases like benign prostatic hypertrophy (BPH), prostate cancer and osteoporosis. Norethisterone (NETA), a progesterone derivative, decreases bone mineral loss in male castrated mice suggesting its role in osteoporosis. In the future, progesterone may find use as a male contraceptive too, but still needs confirmatory trials for safety, tolerability and acceptability. Megestrol acetate, a progesterone derivative is preferred in prostatic cancer. Further, it may find utility in nicotine addiction, traumatic brain injury (recently entered Phase III trial) and Alzheimer's disease, diabetic neuropathy and crush injuries. Studies also suggest role of progesterone in stroke, for which further clinical trials are needed. The non genomic actions of progesterone may be in part responsible for these novel actions. Although progesterone has shown promising role in various non-hormonal benefits, further clinical studies are needed to prove its usefulness in conditions like stroke, traumatic brain injury, neuropathy and crush injury. In male related illnesses like BPH and prostatic Ca, it may prove a boon in near future. New era of hormonal male contraception may be initiated by use of progesterone along with testosterone.

Cross-talk between rapid and long term effects of progesterone on vascular tissue

We tested the hypothesis whether; the non-genomic action of progesterone (Pg) on vascular tissue would be associated with hormonal long term effect on the modulation of cell growth. Using rat aortic strips, we showed that the stimulatory effect of Pg on nitric oxide synthesis involved both kinase and phosphatase pathways. The increase in the vasoactive production was prevented by the MAPK inhibitor (PD98059). In addition, preincubation with a phosphatase antagonist potentiated the hormonal effect. Pg increased PKC activity, but the inhibition of PKC did not alter the stimulatory action of the hormone on nitric oxide generation. In endothelial cell cultures (EC), 24 h treatment with Pg significantly diminished cell proliferation. This antiproliferative effect was suppressed by the PKC inhibitor chelerythrine (chel) and l-NAME (nitric oxide synthase inhibitor). We also observed that Pg stimulates EC migration. In summary, the present findings provide evidence of an integration of genomic and non-genomic effects in the mechanism of action displayed by Pg in vascular tissue. The fast effects elicited by the hormone implies signal transduction activation required for the regulation of vasoactive production, but also necessary for the modulation of endothelial cells growth.

Non-Genomic Actions of Progesterone in the Normal and Neoplastic Mammalian Ovary

This review summarizes recent findings on the non-genomic or membrane-initiated actions of progesterone that regulate the function of the normal and neoplastic mammalian ovary. This review focuses on three receptors: the classic progesterone receptor, the membrane progesterone receptors (MPRalpha, beta, and gamma) that were initially cloned from seatrout ovaries, and a progesterone binding protein referred to as progesterone receptor membrane component-1 (PGRMC1). Specifically, the structure of each of these receptors is compared and related to their capacity to activate various signal transduction pathways. Then the biological effects of P4 on the function of granulosa cells, luteal cells, ovarian surface epithelial cells, and ovarian cancers that are derived from the ovarian surface epithelial cells are discussed in relationship to the expression of each of these receptors. Whenever possible, studies involving human cells and tissues are presented, although data from other mammalian species are used to supplement the human studies to provide a more complete picture of this complex and rapidly developing area of membrane-initiated actions of progesterone.

Nongenomic effects of progesterone on the contraction of muscle cells from the guinea pig colon

Progesterone (PG) affects muscle cells by genomic mechanisms through nuclear receptors and by nongenomic mechanisms through unidentified pathways. This study aimed to determine the pathways mediating its nongenomic actions. Experiments were performed in dissociated muscle cells from guinea pig colons. Nongenomic actions were defined as those occurring within 10 min of PG exposure. PG blocked the contraction to CCK-8 and NKA (10(-7) M) but did not impair ACh (10(-7) M) and KCl (2.5 x 10(-2) M)-induced contraction. Both CCK-8 and NKA contract muscle cells by releasing calcium from intracellular stores, whereas ACh and KCl can utilize extracellular calcium. PG also blocked the contraction induced by inositol 1,4,5-trisphosphate, thapsigargin, and caffeine, agents that contract muscle cells by releasing calcium from storage sites. The nongenomic actions of PG were transient because they were absent 1 h after the first PG dose, remaining unresponsive after a second PG dose was administered. Furthermore, PG had no effect on the contraction induced by CCK-8 and thapsigargin in muscle cells from animals pretreated with daily intramuscular PG for 4 days. Cytosolic incorporation experiments of [(3)H]PG showed that pretreatment with unlabeled PG significantly reduced the radiolabeled PG incorporation in the cytosol. We conclude that the nongenomic actions of PG on colonic muscle cells transiently blocked calcium release from storage sites, and this response became rapidly desensitized. This effect does not appear to be specific to PG because other steroid hormones such as aldosterone and testosterone can also induce it.

Involvement of phosphoinositide-3-kinase and phospholipase C transduction systems in the non-genomic action of progesterone in vascular tissue

We investigate the participation of tyrosine kinase, phosphatidylinositol-3-kinase, phospholipase C systems in the intracellular transduction pathways involved in the non-genomic stimulation of vasodilators compounds synthesis induced by progesterone (Pg). Using aortic strips isolated from female fertile Wistar rats, we showed that physiological concentrations of progesterone markedly increase prostacyclin synthesis in a very short time interval (45 s to 10 min) as well as nitric oxide release (5–30 min). The stimulatory action of progesterone on nitric oxide synthase (NOS) activity was maintained even in the presence of an antagonist of progesterone receptor, compound RU486. In contrast, in the presence of tyrosine kinase inhibitor (1 μM genistein) or phosphatidylinositol-3-kinase inhibitor (1 μM LY294002), the enhancement of nitric oxide elicited by 10–100 nM progesterone was completely suppressed. The steroid stimulates phopholipase C activity, inducing significant increase in diacylglycerol generation (5–15-min treatment). The presence of an inhibitor of protein kinase C (PKC) impaired the anti-aggregatory action of the hormone. Due to the fact that phospholipase C activation implies calcium mobilization, we investigate the role of changes in calcium fluxes on progesterone nitric oxide generation. We found that calcium influx from extracellular medium and calcium mobilization from internal pools was required. The present results suggest that, tyrosine kinase and phosphatidylinositol-3-kinase cascades are involved in progesterone nitric oxide synthase stimulation and that diacilglicerol/protein kinase C system may be relevant for physiological regulation of platelet aggregation process.

Nongenomic action of progesterone in rat aorta: Role of nitric oxide and prostaglandins

The mechanism of action of progesterone (Pg) on rat vascular tissue was investigated. We obtained evidence that 10-nM Pg inhibited platelet aggregation at 1–5 min. Previously, we reported that nitric oxide (NO) mediated this antiaggregatory effect. Rat aortic strips (RAS) NO synthase (NOS) activity in response to “in vitro” treatment with other sex steroids hormones was measured. The stimulatory action of Pg on NO production was specific for ovarian hormones and depends on sex. The effect was nongenomic since cycloheximide did not suppress the increment in NO induced by Pg. Finally, we demonstrated that Pg (5 min) increased prostacyclin release (42–182% above control) in a dose-dependent manner (1–100 nM). Indeed, indomethacin (10 μM) completely suppressed the increment in citrulline levels induced by the hormone. These results suggest that Pg exerts a direct nongenomic action on rat aortic metabolism, which involves NOS and cyclooxygenase (COX) activation and a cross-talk between NO- and prostacyclin (PGI 2)-dependent pathways.

Inhibitory non-genomic effects of progesterone on Na+ absorption in epithelial cells from Xenopus kidney (A6).

The effect of the steroid hormone progesterone on transepithelial sodium transport was measured in confluent monolayers of the A6-cell line from Xenopus kidney. Apical permeabilization with Amphotericin B enabled us to measure the Na+/K+-pump current, and current-fluctuation analysis was used to analyze changes in apical channel density and gating characteristics. Basolateral progesterone (22.2 microM) had a rapid inhibitory effect on the Na+/K+-pump current, and a corresponding decrease in Na+ channel density. The effect occurred within some minutes and took about 50 min to reach a new steady state, in which 45% of the macroscopic current (ISC) was inhibited. Progesterone also inhibits the hypo-osmotic stimulation of Na+ channels which occurs in untreated monolayers. Compared with the known effects of adrenal steroids, our results show a rapid inhibitory action of a steroid hormone on Na+ absorption. The time profile of the progesterone effect suggests, at least in the first minutes, a non-genomic action of progesterone.

Nongenomic Action of Progesterone: Activation of XenopusOocyte Phospholipase C through a Plasma Membrane-Associated Tyrosine Kinase

Nongenomic Action of Progesterone: Activation of XenopusOocyte Phospholipase C through a Plasma Membrane-Associated Tyrosine Kinase

Nongenomic Action of Progesterone: Activation of XenopusOocyte Phospholipase C through a Plasma Membrane-Associated Tyrosine Kinase*

Using a plasma membrane-cortex preparation (wherein the nucleus and >90% of the total cell protein are removed), progesterone stimulated tyrosine kinase activity that stimulated phospholipase C. Although it has been known for over 20 yr that progesterone acts at the plasma membrane of Xenopus oocytes to induce oocyte maturation, this is the first report that progesterone stimulates this tyrosine kinase activity that is associated with the oocyte plasma membrane and cortex. A tyrosine kinase inhibitor (tyrphostin B46) inhibited steroid stimulation of tyrosine kinase and phospholipase C (PLC) activities, but did not block lipase C stimulation by G protein activators. A fusion protein that contains tandem N- and C-terminal SH2 domains of PLCgamma also blocked progesterone stimulation of PLC (a fusion protein with the SH2 domain from Shc was ineffective). Lowering the Ca2+ concentration in the medium inhibited progesterone, but not guanosine 5'-O-(3-thiotriphosphate), stimulation of PLC, and the effects of progesterone and a G protein agonist were additive. However, neither progesterone nor insulin increased phosphotyrosine on PLCgamma. To evaluate another tyrosine kinase path involving phosphatidylinositol 3-kinase, we added wortmannin to our membrane preparation, but wortmannin did not inhibit progesterone's ability to activate PLC.

Stimulation of specific binding of [ 3H]-progesterone to bovine luteal cell-surface membranes: specificity of digitonin

Non-genomic actions of progesterone have been described in the ovary, and luteal membranes of several species have been shown to possess specific binding sites for [ 3H]-progesterone. However, binding of radiolabelled progesterone to luteal membranes was demonstrable only in the presence of digitonin. Digitonin is a non-ionic detergent which is thought to act by forming one-to-one complexes with certain sterols. It is also a cardiotonic agent, inhibiting (Na +–K +) ATPase activity by interaction with the extracellular (ouabain/K +) binding site. We therefore investigated which properties of digitonin were responsible for its stimulatory actions on progesterone binding to bovine luteal membranes. A range of compounds with detergent, cardiotonic and/or cholesterol-complexing activities were tested for their effects on [ 3H]-progesterone binding to bovine luteal membrane fractions, and on haemolysis of rat erythrocytes. Stimulation of progesterone binding to luteal membranes was highly specific for digitonin, and a number of ionic and non-ionic detergents, cardenolides, saponins and cholesterol-complexing reagents tested failed either to stimulate [ 3H]-progesterone binding to bovine luteal membranes in the absence of digitonin, or to inhibit binding specifically in the presence of digitonin. When digitonin was first reacted with excess cholesterol or pregnenolone to form the respective digitonides, stimulatory activity was greatly reduced, suggesting that the ability of digitonin to interact with (an) endogenous steroid(s) may be important in its action. High performance liquid chromatography (HPLC)-mass spectrometry of commercially available digitonin preparations indicated the presence of numerous minor impurities in most commercial digitonin preparations. Three major UV-absorbing peaks were isolated and characterised by mass spectrometry: all stimulated progesterone binding to bovine luteal membrane receptors in a dose-dependent manner, though to differing extents. Our data suggest that the unique action of digitonin on luteal membrane progesterone receptors is not related to its detergent or cardiotonic properties, but appears to be related to its ability to complex with membrane sterols.

Bicarbonate-dependent lipid ordering and protein aggregation are part of the nongenomic action of progesterone on capacitated spermatozoa.

The nongenomic action of progesterone (P) on capacitated sperm in mediating acrosomal exocytosis operates through transmembrane signal transduction involving increased intracellular calcium ions and modulation of protein kinases and phospholipid metabolism through a second messenger pathway. Conflicting views exist regarding the nature of the nongenomic receptor of P. It is thought to be a cell-surface receptor having the properties of a calcium channel, chloride channel, bicarbonate/chloride exchanger, and gamma aminobutyric acid type A (GABA(A)). In this study, we tried to understand the role of bicarbonate and/or P in inducing membrane perturbations in capacitated and acrosome-reacting spermatozoa. We also attempted to characterize the membrane responses in P-stimulated and bicuculline-poisoned sperm. The presence of a high level of bicarbonate in the medium favored high rotational mobility of lipids and proteins in capacitated sperm. However, the capacitated sperm exhibited low lipid ordering and unaltered protein ordering in response to the high bicarbonate concentration. P and bicuculline appeared to bind to the same or similar binding sites, probably a GABA(A) receptor, eliciting similar motional perturbations in sperm membranes. It appears that both P and bicuculline can induce receptor aggregation and lipid ordering in sperm membranes, which alter the capability of sperm to bind zona pellucida, and that the P action on sperm takes effect through a bicarbonate-dependent signal transduction mechanism.

Progesterone stimulates p42 extracellular signal-regulated kinase (p42erk) in human spermatozoa

Mitogen-activated protein kinases (MAPK), also known as extracellular signal-regulated kinases (ERKs) are cytoplasmic and nuclear serine/threonine kinases involved in signal transduction of several extracellular effectors. Recently, we have demonstrated that ERKs are present in spermatozoa and are involved in the regulation of the process of capacitation. We report here the effect of progesterone, a well-known inducer of the acrosome reaction in mammalian spermatozoa, on the immunolocalization, phosphorylation and activity of ERKs in capacitated human spermatozoa. We demonstrated that short-term incubation of spermatozoa with progesterone induces phosphorylation and activation of ERKs, resulting in redistribution of the proteins from the post-acrosomal region to the equatorial segment within the sperm head. To investigate the role of ERKs on the biological effects of progesterone, we used the MAPK cascade inhibitor PD098059, which strongly inhibited progesterone-induced activation of ERK-2. This compound did not inhibit progesterone-induced acrosome reaction, although it prevented redistribution of the enzyme to the equatorial region of the sperm head. These results suggest that the two processes, although temporally related, are independent. In conclusion, we provide new insight into the signal transduction pathways involved in the non-genomic action of progesterone in spermatozoa and suggest a possible involvement of ERKs in the process of fertilization.

Genomic and Non-genomic Actions of Progesterone in the Control of Female Hamster Sexual Behavior

Progesterone (P) in both the ventromedial hypothalamus (VMH) and the ventral tegmental area (VTA) is necessary to facilitate sexual receptivity in estrogen-primed hamsters. The mechanism of P may be different in the VMH and VTA, as there are many intracellular progestin receptors (PR) in the VMH but few in the VTA. Progesterone conjugated to bovine serum albumin (P-3-BSA) does not bind well to intracellular PR or permeate the surface of neuronal membranes. However, VTA application of P-3-BSA rapidly increases sexual receptivity if P has been applied earlier to the VMH. P-3-BSA is ineffective when applied to the VMH. The membrane-limited effect of P may be related to the ability of some progestins to modulate the GABAA-benzodiazepine receptor complex (GBRC). We have found that infusions of a GABAA agonist, muscimol, into the VTA enhance and a GABAA antagonist, bicuculline, inhibit receptivity. Because P itself is not highly effective at the GBRC, and since the most potent modulators of the GBRC, the 5α-reduced progestins, do not bind well to PRs, progestin metabolites were applied to the VTA. Only the potent GBRC modulators facilitated sexual receptivity when applied to the VTA concurrent with P to the VMH. The reverse treatment, with a progestin metabolite implanted into the VMH, was ineffective. VTA infusions of an inhibitor of 5α-reductase also attenuated behavioral estrus in hamsters. These data are consistent with P facilitation of sexual receptivity being genomically mediated in the VMH, while the non-genomic actions of P in the VTA may be a result of metabolism and subsequent interaction with the GBRC.

Progesterone and the neural mechanisms of hamster sexual behavior

Stimulation of both the ventral medial hypothalamus (VMH) and the ventral tegmental area (VTA) by progesterone is necessary to facilitate sexual behavior in female hamsters. Recently obtained evidence indicates that progesterone exerts its behaviorally relevant actions in the VTA by acting on cell membranes. When progesterone conjugated to bovine serum albumin, which cannot permeate the cell membrane, is applied to the VTA concurrent with free progesterone to the VMH, estrogen-primed hamsters become sexually receptive. Since the reverse treatment is ineffective, this suggests that progesterone's nongenomic effects in the VTA may require concurrent genomic activation by progesterone in the VMH. The nongenomic action of progesterone on sexual receptivity may involve the GABA A receptor complex, as progestins are known to modulate this receptor complex. VTA infusions of GABA A agonists enhance, and antagonists inhibit, progesterone's effectiveness on receptivity. Finally, the behavioral effectiveness of progesterone metabolites in the VTA, concurrent with progesterone in the VMH, is consistent with their relative biochemical efficacy at the GABA A complex. These data suggest that progesterone may exert its behavioral effects in the VTA through GABA A. However, it is not yet clear whether progesterone normally acts directly on GABA A in the VTA. Progesterone may also act at some other membrane binding site and GABA A may represent an indirect mechanism for progesterone.

Progesterone-facilitated sexual receptivity: A review of arguments supporting a nongenomic mechanism

Progesterone facilitates sexual receptivity in estradiol-primed female rats. While many experiments suggest a genomic mechanism for the behavioral action of progesterone, some results appear better explained by nongenomic mechanisms. Furthermore, the presence of membrane binding sites for progesterone and rapid modifications of neuronal excitability induced by this steroid suggest that a nongenomic action of progesterone could be membrane related. However, in spite of the discovery of such membrane-related actions of progesterone, their relation to progesterone-facilitated sexual behavior remains unclear.