Human Reproduction Update, Vol.15, No.1 pp.

119 138, 2009 Advanced Access publication on October 19, 2008 doi:10.1093/humupd/dmn044

Non-genomic progesterone actions in female reproduction

B. Gellersen 1,3, M.S. Fernandes 2, and J.J. Brosens 2,3
1 Endokrinologikum Hamburg, Falkenried 88, 20251 Hamburg, Germany 2Institute for Reproductive and Developmental Biology, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 0NN, UK 3 Correspondence addresses: Tel: 49-40-47196591; Fax: 49-40-47196599; E-mail: gellersen@endokrinologikum.com (B.G.); Tel: 44-20-75942164; Fax: 44-20-75942183; E-mail: j.brosens@imperial.ac.uk (J.J.B.)

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Introduction Materials and Methods Progesterone and female reproduction Genomic versus non-genomic steroid actions Rapid progesterone actions Putative receptors implicated in rapid progesterone actions Conclusions and perspective

background: The steroid hormone progesterone is indispensable for mammalian procreation by controlling key female reproductive events that range from ovulation to implantation, maintenance of pregnancy and breast development. In addition to activating the progesterone receptors (PRs)-B and -A, members of the superfamily of ligand-dependent transcription factors, progesterone also elicits a variety of rapid signalling events independently of transcriptional or genomic regulation. This review covers our current knowledge on the mechanisms and relevance of non-genomic progesterone signalling in female reproduction. methods: PubMed was searched up to August 2008 for papers on progesterone actions in ovary/breast/endometrium/myometrium/ brain, focusing primarily on non-genomic signalling mechanisms. results: Convergence and intertwining of rapid non-genomic events and the slower transcriptional actions critically determine the functional response to progesterone in the female reproductive system in a cell-type- and environment-specic manner. Several putative progesterone-binding moieties have been implicated in rapid signalling events, including the classical PR and its variants, progesterone receptor membrane component 1, and the novel family of membrane progestin receptors. Progesterone and its metabolites have also been implicated in the allosteric regulation of several unrelated receptors, such as g-aminobutyric acid type A, oxytocin and sigma1 receptors.

conclusions: Identication of the mechanisms and receptors that relay rapid progesterone signalling is an area of research fraught with difculties and controversy. More in-depth characterization of the putative receptors is required before the non-genomic progesterone pathway in normal and pathological reproductive function can be targeted for pharmacological intervention.
Key words: progesterone / reproduction / non-genomic / progesterone receptor / membrane progestin receptor

Introduction
The cloning and characterization of the progesterone receptors B and A (PR-B and -A), members of the superfamily of ligand-activated transcription factors, and their subsequent ablation in mice have yielded invaluable insights into the diverse and often indispensable actions of progesterone in female reproduction (Kastner et al., 1990; Vegeto

et al., 1993; Ismail et al., 2003; Li and OMalley, 2003; Mulac-Jericevic and Conneely, 2004). Pregnancy and lactation are dependent on a series of inammatory events associated with intense tissue remodelling, such as ovulation, implantation, decidualization, parturition and breast development, all of which critically rely on progesterone actions (Critchley et al., 2001; Norman et al., 2007; Conneely et al., 2007; Richards et al., 2008). In addition, progesterone not only serves as

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an intermediate in the biosynthesis of androgens and estrogens but also has important actions on sexual behaviour, gonadotrophin secretion and on many non-reproductive organs (Graham and Clarke, 1997; Jamnongjit and Hammes, 2006). Among progesterones actions in male reproduction is an involvement in the induction of the acrosome reaction in sperm (Schuffner et al., 2002; Oettel and Mukhopadhyay, 2004). In agreement with its ubiquitous role in female reproduction, impaired progesterone responses have now been implicated in a wide spectrum of human reproductive disorders, including abnormal menstrual bleeding, broids, endometriosis and adenomyosis, breast and endometrial cancer, miscarriage and preterm labour (Wu et al., 2006; Burney et al., 2007; Ehn et al., 2007; Ito et al., 2007; Salazar and Calzada, 2007; Yin et al., 2007; Boruban et al., 2008; SzekeresBartho et al., 2008). Not surprisingly, natural progesterone, progestins, antiprogestins and the emerging selective PR modulators are among the most widely used compounds in reproductive medicine, gynaecology and obstetrics (Chabbert-Buffet et al., 2005; Chwalisz et al., 2005). Not all effects of progesterone, however, can be explained by the classical model of steroid action and, like every other steroid hormone, progesterone exerts rapid effects on diverse signalling pathways, independently of transcriptional or genomic regulation (Falkenstein et al., 2000b; Schmidt et al., 2000; Losel and Wehling, 2003). Although compelling evidence has emerged indicating that some of these non-genomic actions are mediated by activation of the cytoplasmic fraction of the nuclear PR (nPR), more specically the B-isoform (Boonyaratanakornkit et al., 2001, 2007), rapid progesterone responses are also detected in cells and tissues devoid of nPR, such as T-lymphocytes, platelets, the rat corpus luteum and PR knockout (PRKO) mice (Park-Sarge et al., 1995; Ehring et al., 1998; Bar et al., 2000; Frye et al., 2006). It is widely believed that non-genomic progesterone actions are initiated at the cell surface by specic membrane-bound receptors yet unequivocal proof for this model is as yet lacking. A major obstacle is that several putative non-genomic PRs have been reported but none has been characterized in-depth. After summarizing the role of progesterone in regulating female reproductive function, we will elaborate on the current controversies that surround these putative membrane-bound PRs and their proposed modes of actions.

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Losel et al., 2003; Schumacher et al., 2007) is beyond the scope of this review. Here, we can only highlight some of the salient actions of progesterone in the female reproductive system, focusing predominantly on the human situation but drawing on the observations made in knockout mice that are devoid of one or both nPR isoforms.

Ovary
The ovary, and more specically the corpus luteum, is the major source of progesterone during the menstrual cycle, whereas the placenta takes over this role after 9 weeks gestation. The adrenal gland and central nervous system are additional sources of progesterone production (Graham and Clarke, 1997; Frye et al., 2006; Schumacher et al., 2007). Progesterone synthesis from cholesterol in the ovary is dependent on gonadotrophin stimulation (Graham and Clarke, 1997; Peluso, 2006). Once secreted, progesterone is carried in the blood by specic binding proteins, such as corticosteroid-binding globulin (Rosner, 1991). Interestingly, there are striking interpopulational differences in circulating progesterone levels. For instance, the meanpeak luteal progesterone levels in ovulatory cycles of Bolivian Aymara women are  70% lower when compared with those in women from Chicago (Vitzthum et al., 2004). Despite these lower progesterone levels, there is no evidence of a lower fecundity rate in this population. In the ovary, progesterone signalling has been implicated in follicular growth, ovulation and luteinization (Peluso, 2006). The luteinizing hormone (LH) surge up-regulates both nPR isoforms in human granulosa cells of pre-ovulatory follicles (Iwai et al., 1990), thereby setting in motion a cascade of inammatory events leading to ovulation (Richards et al., 2008). Two lines of evidence provided unequivocal proof of the essential role of progesterone signalling in ovulation. First, the antiprogestin RU486 (mifepristone) suppresses ovulation (Loutradis et al., 1991). Second, mice that lack both nPR isoforms fail to ovulate, even in response to ovulation induction and despite the presence of normally developed follicles that possess mature oocytes (Lydon et al., 1995). In contrast to PR-A knockout mice, ovarian function is not affected upon selective PR-B ablation, indicating that the A isoform is both necessary and sufcient to mediate the ovulatory response (Conneely et al., 2002). The mechanism whereby activated PR-A initiates ovulation is not fully understood but likely involves induction of metalloproteinases, such as ADAMTS-1 and cathepsin-L, essential for formation of an extracellular hyaluronan-rich matrix by the cumulus oocyte complex, a process called expansion (Robker et al., 2000; Richards, 2005). After ovulation, progesterone promotes its own secretion and inhibits cell proliferation and apoptosis in luteinized granulosa cells (Peluso and Pappalardo, 1998; Peluso et al., 2001, 2002, 2005).

Materials and Methods

PubMed was searched up to August 2008 for publications on PR/membrane progestin receptor (mPR)/PGRMC in combination with ovary/ breast/endometrium/myometrium/brain and non-genomic/rapid signalling, and for progesterone in combination with microtubule-associated protein 2 (MAP2) or g-aminobutyric acid (GABA) receptor/oxytocin receptor/sigma1 receptor (s1R).

Endometrium
The uterine mucosa is a major target for ovarian steroid hormones. The postovulatory rise in circulating progesterone levels inhibits the proliferative activity of the estrogen-primed human endometrium and induces profound tissue remodelling in preparation of embryo implantation and placenta formation (Dey et al., 2004; Brosens and Gellersen, 2006). The antiproliferative effect of progesterone is, at least in epithelial cells, mediated by inhibition of multiple genes that

Progesterone and female reproduction

A detailed discussion of progesterone actions on extra-reproductive tissues such as bone, cardiovascular and respiratory systems, thymus, kidney and adipose tissue (reviewed in: Graham and Clarke, 1997;

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encode for factors needed for DNA replication licensing, including the various minichromosome maintenance (MCM) proteins (Pan et al., 2006). In concert, progesterone initiates a differentiation programme, characterized initially by growth and coiling of the spiral arteries, secretory transformation of the glands, inux of distinct immune cells, especially specialized uterine natural killer (uNK) (CD56bright/ CD162) cells and subsequently by decidualization of the stromal compartment (Gellersen et al., 2007). uNK cells are a rich source of growth and angiogenic factors and are capable of modulating T cell function at the feto-maternal interface through expression of glycodelin A and galectin-1 (Koopman et al., 2003; Dosiou and Giudice, 2005). Interestingly, peripheral blood NK cells, but not uNK cells, express both PR-B and PR-A and are susceptible to progesterone-induced apoptosis (Arruvito et al., 2008). It is widely thought that progesterone modulates uNK cell function indirectly, via paracrine signals derived from PR-positive cells in the stromal compartment, although direct PR-independent pathways cannot be excluded (Anne Croy et al., 2006). Transformation of stromal broblasts into secretory epithelioidlike decidual cells during the late secretory phase of the cycle bestows some unique features upon the endometrium, essential for coordinated trophoblast invasion in the case of pregnancy. Decidualization heralds the end of the mid-secretory phase implantation window, dened as the limited period during which progesterone-driven changes in the luminal epithelium allow apposition, attachment and invasion of a developmentally competent blastocyst (Horcajadas et al., 2007; Wang and Dey, 2006). Decidualizing endometrial stromal cells form a cuff around the changing spiral arteries that are characterized by endothelial swelling, vacuolation, and disorganization of the smooth muscle media (Craven et al., 1998). Biochemically, decidual cells play a decisive role in ensuring tissue haemostasis by expressing the brinolysis inhibitor plasminogen activator type 1 as well as tissue factor, a membrane-anchored glycoprotein that serves as the receptor for coagulation factor VII/VIIa (Lockwood et al., 2008). Upon secretory transformation, decidualizing endometrial stromal cells also acquire the means to respond to trophoblast signals and to provide histiotrophic support to the early conceptus (Burton et al., 2002; Hess et al., 2007). Moreover, decidual cells are highly adapted to resist oxidative insults by expressing a variety of intra- and extra-cellular free radical scavengers and through disabling of stress-dependent pro-apoptotic signalling pathways (Kajihara et al., 2006; Gellersen et al., 2007). Expression of a decidual phenotype is, however, strictly dependent upon elevated progesterone levels. In the absence of pregnancy, falling progesterone levels not only reverse the decidual phenotype but also induce the expression of a gene network that encodes for chemokines, pro-inammatory cytokines, matrix metalloproteinases and apoptotic factors, leading to inux of inammatory cells, proteolytic breakdown of the extracellular matrix, cell death, focal bleeding and menstruation (Gellersen and Brosens, 2003; Jabbour et al., 2006; Gellersen et al., 2007). Selective nPR knockout studies demonstrated that progesterone antagonises estrogen-induced epithelial proliferation and drives stromal cell differentiation strictly dependent upon PR-A activation. Female mice that lack this receptor isoform are sterile due to defective ovulation, implantation and decidualization (MulacJericevic and Conneely, 2004).

Myometrium
High levels of progesterone secreted by the placenta throughout pregnancy are critical to maintain the human myometrium in a quiescent state. The ability of antiprogestins to induce parturition demonstrates the importance of nPR signalling in ensuring myometrial relaxation during pregnancy (Herrmann et al., 1982; Avrech et al., 1991). A single dose of mifepristone sensitizes the myometrium for the procontractile action of prostanoids. Consequently, a sequential regimen of mifepristone followed by misoprostol has become the standard method for medical termination of pregnancy (Sitruk-Ware, 2006). Conversely, progestational agents are increasingly used to prevent preterm birth, which may be effective at least in part by preventing premature cervical ripening (Xu et al., 2008). Like ovulation and menstruation, parturition is an inammatory process that encompasses the fetal membranes, decidua, myometrium and cervix (Norman et al., 2007). Progesterone is generally considered an anti-inammatory steroid (Szekeres-Bartho et al., 2001). It opposes prostaglandin production in the uterus of pregnancy, partially by inhibiting cyclooxygenase (COX-2) expression, an enzyme involved in prostaglandin biosynthesis, and by up-regulating 15-prostaglandin dehydrogenase, a prostaglandin catabolizing enzyme (Graham and Clarke, 1997; Greenland et al., 2000; Hardy et al., 2006). As opposed to most mammals, parturition in humans is not preceded by a precipitous fall in circulating maternal progesterone levels. The onset of labour is thought to be the result of decreased myometrial progesterone responsiveness, commonly referred to as functional progesterone withdrawal (Astle et al., 2003b; Brown et al., 2004; Mesiano, 2007). Increased expression of contractionassociated proteins, the so-called CAPs, then transforms the myometrium to a highly contractile state. CAPs include oxytocin and its receptor, COX-2, prostaglandin F2a receptor, gap junction proteins, such as connexin 43, and ion channels (Astle et al., 2003b; Mesiano and Welsh, 2007). Arguably, parturition can either be viewed as a sequence of events that activate stimulatory pathways or as a loss of mechanisms that pez Bernal, 2003). The physicomaintain uterine quiescence (Lo chemical basis of myometrial contractility is the interaction of actin and myosin in myometrial smooth muscle cells, which is controlled by the Ca2-calmodulin-dependent activity of myosin light chain kinase (MLCK). MLCK phosphorylates and activates myosin light chain (MLC), leading to contraction. This is opposed by the dephosphorylation of MLC by myosin phosphatase, causing relaxation. Important regulatory inputs into this cycle are the enhanced phosphorylation of MLC by the mitogen-activated protein kinases (MAPKs) ERK1/2, and the enforced dephosphorylation by stimulation of myosin phosphatase through cyclic nucleotides. This underlies the relaxant effect of high cAMP levels throughout pregnancy (Abdel-Latif, 2001; Smith, 2007). Progesterone contributes to the maintenance of elevated cAMP concentrations by inhibiting phosphodiesterase PDE4 activity, thus limiting cAMP turnover (Konas et al., 1990). MLC phosphorylation is also enhanced by the bioactive lysophospholipids sphingosine 1-phosphate (Sph-1-P) and lysophosphatidic acid (Moore et al., 2000; Essler et al., 2002). The transient phosphorylation of sphingosine to Sph-1-P is catalysed by sphingosine kinase (SphK) (Ye, 2008). Progesterone induces SphK1 expression in the rat uterus during pregnancy and in isolated rat myometrial cells,

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which in turn elevates MLC phosphorylation and cyclin D1 protein levels (Jeng et al., 2007). Although progesterone-mediated induction of the cell-cycle regulator cyclin D1, a phenomenon also seen in mammary cells (see Section Breast), might partially account for myometrial proliferation in pregnancy, a stimulation of MCL activation by progesterone during pregnancy would be detrimental and must be opposed. Release from counteracting signals might then operate with the onset of parturition. Although progesterone-mediated MLC phosphorylation via induction of SphK1 expression requires several hours (Jeng et al., 2007), more rapid progesterone actions independent on de novo protein synthesis have been described. In muscle strips isolated from lower uterine segment of term pregnant women, progesterone increases the frequency and tonus of contractions in a Ca2-dependent manner (Fu et al., 1997). However, it has to be noted that there are conicting reports on the precise effect of progesterone on contractile frequency, amplitude, duration and area of activity in term myometrial strips (Mesiano and Welsh, 2007), which may in part be due to the fact that human myometrium is a heterogeneous tissue, consisting of outer myometrium and a functionally distinct inner myometrial layer, termed the uterine junctional zone (Daels, 1974; Brosens et al., 1995; Fusi et al., 2006). Parturition is normal in PR-B knockout mice. PR-A decient mice are, however, infertile, which precludes analysis of the function of this receptor isoform in parturition (Mulac-Jericevic et al., 2000; Conneely et al., 2003). In humans, PR-B seems to be important for maintaining the myometrium in a quiescent state while up-regulation of PR-A towards term may contribute to induction of the contractile phenotype (Merlino et al., 2007). Several other mechanisms to explain the onset of labour at term include down-regulation of steroid receptor coactivators (Condon et al., 2003), induction of truncated nPR isoforms (see Section nPR variants) and altered expression of mPRs (see Section Membrane progestin receptors). However, although the concept of functional progesterone withdrawal is attractive, none of the proposed mechanisms are as yet supported by irrefutable scientic or clinical evidence.

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development, characterized by decreased pregnancy-associated side branching of the ductal epithelium, absence of terminal end buds and complete inhibition of lobulo-alveolar differentiation in response to exogenous estrogen and progesterone treatment (Lydon et al., 1995). Importantly, progesterone still elicits side-branching and lobular alveolar development in PR-A knockout mice, whereas this is no longer the case in PR-B decient animals. Thus, in contrast to the lower female reproductive tract, expression of PR-B is both sufcient and indispensable for progesterone actions on proliferation and differentiation of the mammary gland (Mulac-Jericevic et al., 2000, 2003; Mulac-Jericevic and Conneely, 2004). A recent clinical study demonstrated that mifepristone strongly inhibits breast epithelial cell proliferation in premenopausal women (Engman et al., 2008). Conversely, several epidemiological studies reported an increased risk of breast cancer if estrogen replacement therapy was combined with progestins like medroxyprogesterone acetate (MPA) (Seeger and Mueck, 2008). The Womens Health Initiative, the only prospective placebo-controlled interventional study to date, calculated an odds ratio of 1.24 (CI: 1.01 1.54) for a mean duration of treatment of 5.6 years. Other studies reported no increased risk when estrogen therapy was combined with micronized progesterone or dydrogesterone (de Lignieres et al., 2002; Fournier et al., 2008). Progestins have been shown to stimulate proliferation, inhibit apoptosis and enhance invasiveness of breast cancer cells (Kato et al., 2005; Saitoh et al., 2005; Moore et al., 2006; Salatino et al., 2006). Interestingly, mifepristone treatment of mice that lack the murine homologues of BRCA1 and p53, two tumour suppressors frequently mutated in breast cancer, has been shown to prevent mammary tumourigenesis (Poole et al., 2006). Together, these observations suggest that antiprogestins like mifepristone may well be useful in managing patients at increased risk of breast cancer.

Brain
Progesterone acts at the level of the hypothalamic pituitary axis to regulate the secretion of gonadotrophin-releasing hormone and LH, respectively, thereby establishing a feedback mechanism that regulates steroidogenesis in the ovary (Goodman and Karsch, 1980; Karsch, 1987; OByrne et al., 1991). Moreover, progesterone plays an integral role in the neuroendocrine regulation of feminine sexual behaviour. In ovariectomized rats and guinea pigs, injection of estradiol followed 48 h later with an injection of progesterone induces the sexually receptive posture, termed copulatory behaviour or lordosis (Blaustein, 2008). Conversely, PRKO mice do not exhibit lordosis (Lydon et al., 1995). Although nPR is necessary for lordosis, ovarian progesterone is not. For example, dopamine agonists can substitute for progesterone in facilitating sexual behaviour in estradiol-primed rats and mice by a mechanism that requires nPRs but not progesterone (Mani et al., 1994a, b; Mani et al., 1996). More recently, several other signalling pathways, involving the opioid receptor and the second messengers cAMP and cGMP, have been shown to facilitate sexual behaviour in a PR-dependent mechanism (Acosta-Martinez et al., 2006; Mani et al., 2006). In contrast, progesterone, or more accurately progestins, is widely considered to have inhibitory effects on sexual desire in women, although the evidence is scant (Dennerstein et al., 1980). Instead, estrogen and androgen replacement therapy has been

Breast
During the menstrual cycle, the mammary gland goes through sequential waves of proliferation and apoptosis. Unlike the endometrium, proliferation of human mammary epithelium is considerably greater in the luteal phase, peaking around Day 24 of the cycle. Moreover, the proliferative index in breast correlates with circulating progesterone levels (Navarrete et al., 2005). However, only a minor subpopulation of luminal epithelial cells express nPR and estrogen receptors (ER). These cells are often non-dividing but usually lie adjacent to proliferating cells, suggesting that progesterone drives the proliferation of PR-negative cells by promoting the expression of growth-stimulating factors, such as WNTs, insulin-like growth factor-II (IGF-II) or stromaderived hepatocyte growth factor (Ismail et al., 2003; Rosen, 2003). Progesterone, in concert with estrogen and growth factors such as epidermal growth factor and IGF-I, drives the formation of lobular alveolar structures during pregnancy, essential for subsequent lactation (Graham and Clarke, 1997). Again, the role of nPR in mediating progesterone actions in the mammary gland is supported by studies in PRKO mice. Null mutation for both nPR isoforms results in impaired mammary gland

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expression. In addition to these genomic actions, all steroid hormones exert rapid effects, taking place in seconds or minutes, on various signal transduction pathways and second messenger systems and without the involvement of transcriptional modulation. These rapid responses are referred to as non-classic, non-genomic or extranuclear steroid effects (Falkenstein et al., 2000b; Losel and Wehling, 2003; Norman et al., 2004). Several criteria have been proposed that may help in distinguishing non-genomic from genomic steroid actions. In general, non-genomic effects are (i) too rapid to be compatible with transcriptional activation and protein synthesis; (ii) not abolished upon addition of inhibitors of transcription or translation; (iii) sometimes observed in isolated cell membranes or in cells devoid of nuclei, such as erythrocytes and platelets; (iv) inducible by cell-impermeable steroid protein conjugates and (v) generally not blocked by antagonists of nuclear steroid receptors (Revelli et al., 1998; Losel et al., 2003). In the reproductive tract, non-genomic and genomic actions inevitably converge to produce a tissue- and cell-specic progesterone response. Transcription factors, including nuclear steroid receptors and their cofactors, are highly modied by a multitude of posttranslational modications, such as phosphorylation, ubiquitination, sumoylation, methylation and acetylation (Yang, 2005; Faus and Haendler, 2006; Jones et al., 2006; Daniel et al., 2007; Heine and Parvin, 2007). These covalent modications are often interdependent and the presence of a particular combination or code can lead to functionally distinct activities of a given nuclear factor. The posttranslational code on a variety of downstream effector molecules can change rapidly in response to upstream signalling events. Thus, although non-genomic steroid actions may be rapid and transient, the downstream consequences can be profound and sustained.

extensively marketed to treat psychosexual and mood disorders, especially those associated with the menopause (Studd, 2007). Progesterone is thought to have important but poorly characterized effects on brain development in the fetus and neonate (Wagner, 2008). Interestingly, there is clear sexual dimorphism in the temporalspatial expression of nPR in various regions of the fetal and neonatal brain, at least in rodents (Wagner et al., 2001; Quadros et al., 2002). For instance, in the perinatal period, nPR immunoreactivity is high in the medial preoptic nucleus in male but not female rodents, suggesting that developmental windows exist during which progesterone signalling critically regulates neuroendocrine functions (Wagner, 2008). Progesterone combined with estrogen has also been used in the treatment of premature infants during the rst weeks of life. The rationale behind these clinical trials is that these infants are deprived of the hormonal uterine environment towards term. Interestingly, premature infants receiving hormonal treatment achieved normal psychomotor development earlier than untreated premature infants (Trotter et al., 2001). In addition, progesterone also has important neuroprotective and promyelinating effects in the adult brain (Schumacher et al., 2007). The lower risk of stroke in premenopausal women compared with men of the same age has also been attributed to the neuroprotective effects of progesterone and estrogens (Sacco et al., 1997; Cai et al., 2008). Following the menopause, the incidence of stroke in women increases rapidly (Hodis and Mack, 2007). Interestingly, MPA, a progestin widely used in hormone replacement therapy, does not confer neuroprotection, unlike natural progesterone (Nilsen and Brinton, 2003). Two recent clinical trials indicated that progesterone treatment may also improve neurologic outcome after traumatic brain injury in adults (Wright et al., 2007; Xiao et al., 2008). Although still unresolved, progesterone may exert its effect after trauma by down-regulating the associated inammatory cascade, limiting cellular necrosis and apoptosis, or by protecting the blood-brain barrier (Singh, 2006; Vandromme et al., 2008).

Rapid progesterone actions

Over 65 years ago, Hans Selye (1942) reported that intraperitoneal injection of progesterone in rats induces a prompt anaesthetic effect, an observation that arguably provided the rst evidence of a rapid non-genomic steroid effect. Since then, a plethora of rapid progesterone actions has been described in very diverse tissue or cell systems (Graham and Clarke, 1997; Calogero et al., 2000; Falkenstein et al., 2000b; Losel et al., 2003; Jamnongjit and Hammes, 2005; Mani, 2006; Correia et al., 2007; Fu and Simoncini, 2007; Lange et al., 2007; Schumacher et al., 2007) (Table I). Historically, two model systems have been exhaustively used to analyse non-genomic progesterone actions; the acrosome reaction in human spermatozoa and the induction of oocyte maturation in Xenopus laevis. In human sperm, progesterone stimulates rapid inux of extracellular Ca2 and efux of Cl2, both essential for induction of the acrosome reaction (Meizel et al., 1997). Although classical nPRs have been described in human sperm (Gadkar et al., 2002; Losel et al., 2005), progesterone-mediated acrosome reaction cannot be blocked by mifepristone (Baldi et al., 1991), an observation that complements other evidence pointing towards the existence of distinct but uncharacterized PRs on the plasma membrane of human sperm (Blackmore et al., 1991). This is further underpinned by the observation that male PRKO mice are fertile (Lydon et al., 1995). The Ca2dependent increase in cAMP and subsequent phosphorylation of

Genomic versus non-genomic steroid actions

Nuclear receptors such as PR-B and PR-A are DNA-binding proteins that upon ligand binding recognize specic cis-acting hormone response elements typically located in the promoter region of target genes. Modulation of gene transcription, however, requires recruitment of cofactors to the DNA-bound receptor. These cofactors are generally classied into coactivators or corepressors, depending on their ability to induce or inhibit transcriptional output, respectively (Parker et al., 2006; Rosenfeld et al., 2006; OMalley et al., 2008). Many cofactors do not bind DNA directly but possess enzymatic activities capable of modulating the chromatin or alternatively serve as bridging molecules that facilitate the assembly of the RNA polymerase II initiation complex (Rosenfeld et al., 2006). Approximately 300 cofactors have been described to date and many have surprisingly diverse additional functions, such as RNA chain elongation, splicing and termination (Lonard et al., 2007; Yu et al., 2007). Thus, the cardinal feature of nuclear receptors is their ability to interact with DNA in response to hormone binding or other signals and then to recruit multiprotein complexes that control gene

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Table I Rapid effects of progesterone in target tissues

Physiological action Acrosome reaction/ capacitation Oocyte maturation Immunoregulatory function Platelet aggregation Anti-apoptotic effects Muscle contraction Vasoreactivity Steroidogenesis and LH action Lordosis Transepithelial resistance Actin cytoskeleton remodelling/cell movement Neuroprotection Retinal neuronal activity Cell/tissue/organism Human spermatozoa Amphibian and sh oocytes Human T-lymphocytes Human platelets Rat granulosa cells Human intestinal smooth muscle cells Rat vascular smooth muscle cells Rodent Leydig cells Female mice Human fetal membranes Human umbilical vein endothelial cells, human breast cancer cells Mouse cerebral cortex, rat hippocampal neurons Mouse rod bipolar cells Not assessed G-protein activation, PI3 kinase and RhoA/ROCK-2 cascade activation PI3 kinase activation, ERK1/2 activation, Ca2 inux inhibition PI3 kinase activation Signalling pathway Ca2 inux, Cl2 efux, cAMP increase G-protein activation and cAMP decrease, ERK1/2 activation, PI3 kinase activation G-protein activation, K channel inhibition Ca2 inux MAPK kinase (MEK) inhibition, Ca2 homeostasis, Protein kinase G activation Ca2 currents reduction Ca2 inux regulation Na inux

Reference Luconi et al. (2004), Blackmore et al. (1991) and Kirkman-Brown et al. (2000) Zhu et al. (2003b), Thomas et al. (2002), Maller (2001) and Bagowski et al. (2001) Dosiou et al. (2008) and Ehring et al. (1998) Bar et al. (2000) and Blackmore (1999, 2008) Peluso et al. (2001) and Peluso and Pappalardo (2004) Bielefeldt et al. (1996) Barbagallo et al. (2001) Rossato et al. (1999) and El-Hefnawy and Huhtaniemi (1998) Frye et al. (2006) Verikouki et al. (2008) Fu et al. (2008a, b) Kaur et al. (2007), Nilsen and Brinton (2003) and Cai et al. (2008) Koulen et al. (2008)

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spermatozoa proteins are thought to be important intermediate events in the initiation of the acrosome reaction in response to progesterone (Harrison et al., 2000). Importantly, the extent of progesterone-induced Ca2 response correlates with the fertilization rate in oligozoospermic men (Baldi et al., 1995), further underscoring the importance of rapid steroid signalling in human reproduction. In X. laevis oocytes, progesterone elicits numerous responses including an increase in intracellular Ca2, a sudden rise in intracellular pH and a decrease in membrane conductance (Revelli et al., 1998). Progesterone signals the resumption of meiotic division in oocytes arrested in the G2 phase, which involves inhibition of adenylate cyclase, leading to decreased intracellular cAMP levels (FinidoriLepicard et al., 1981). Notably, oocyte maturation can also be induced efciently with glucocorticoids, androgens and mineralocorticoids and to a lesser extent mifepristone (Bayaa et al., 2000; Edwards, 2005). Irrefutable evidence has accumulated showing that steroidmediated oocyte maturation is mediated exclusively by a non-genomic mechanism. Transcription is almost completely suppressed during oocyte maturation, and transcription inhibitors do not affect the rate or potency of oocyte maturation. Further, oocyte maturation occurs even in enucleated Xenopus oocytes (Losel et al., 2003; Edwards, 2005). A surprising observation is the diversity of second messenger systems and signalling pathways that reportedly relay non-genomic progesterone actions in amphibian and mammalian cells (Table I) pointing towards the existence of multiple hormone binding moieties involved in progesterone signalling. Notably, systematic large-scale microarray studies in human breast cancer cell lines and primary decidualizing endometrial stromal cell cultures demonstrated that nPR

governs the expression of a surprisingly large number of genes that encode for ligands, membrane-bound receptors, calcium-binding proteins and signalling molecules (Richer et al., 2002; Cloke et al., 2008). Functionally, knockdown of nPR expression in endometrial cells was sufcient to abolish activation of WNT/b-catenin, transforming growth factor b/SMAD and signal transducer and activator of transcription pathways upon decidualization (Cloke et al., 2008). Thus, while progesterone is capable of triggering diverse cytoplasmic signalling events, activation of nPRs in turn programmes the cellular responses to cytokines, growth factors and other signal molecules.

Putative receptors implicated in rapid progesterone actions

There are several reasons as to why the search and characterization of bona de non-genomic PRs has turned out to be fraught with difculties. First, steroid hormones can elicit rapid but receptor-independent effects by affecting physicochemical membrane properties, albeit only at micromolar concentrations (Falkenstein et al., 2000a). For example, progesterone, in contrast to estradiol or testosterone, interacts with membrane vesicles, decreases membrane uidity, induces aggregation of these vesicles and renders them permeable to hydrophilic molecules (Shivaji and Jagannadham, 1992). Second, many mammalian cells express nPR or other nuclear receptors for which progesterone may function as an agonist or antagonist. A case in point is the pregnane X receptor, a nuclear receptor activated by progesterone metabolites, which has been implicated in regulating the vascular tone in pregnancy (Hagedorn et al., 2007). Third, there are important

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methodological pitfalls when studying rapid cytoplasmic events. For instance, progesterone when conjugated to bovine serum albumin is theoretically membrane-impermeable and any biological effect is therefore readily attributed to activation of a membrane-bound receptor. However, these conjugate preparations are often contaminated with unbound steroid at levels sufciently high to trigger intracellular events (Stevis et al., 1999; Hammes, 2003). Moreover, simple cell culture manipulations, such as changing medium, elicit rapid cellular responses that, if not appropriately controlled for, can be misinterpreted as hormone-specic. Several receptor-dependent mechanisms have been proposed to account for the diversity of steroid hormone actions in general and for progesterone actions in particular. These include activation of a subpopulation of the classical nPR that resides in signalling complexes in the cytoplasm or at the plasma membrane (see Section Nuclear progesterone receptor), effects mediated by truncated variants of the classical nPR (Section nPR variants), allosteric regulation of unrelated receptors by progesterone or its metabolites (Sections GABAA and oxytocin receptors and MAP2 and sigma1 receptor) and activation of transmembrane (TM) receptors that are structurally unrelated to nuclear hormone receptors (Sections Membrane progestin receptors and Progesterone receptor membrane component 1).

Nuclear progesterone receptor

As alluded to, nPR has been implicated in rapid cytoplasmic progesterone signal transduction events. Like other nuclear receptors, PR-B and -A are modular proteins consisting of a centrally located, highly conserved DNA-binding domain (DBD), a carboxy-terminal ligand-binding domain (LBD) and a variable amino-terminal domain (Fig. 1). Both isoforms arise from differential promoter usage in a single gene, and PR-A differs from PR-B only in that it is 164 amino acids shorter at the amino-terminus (Kastner et al., 1990). Embedded within this modular structure are subdomains critical for transcriptional activation (activating functions) or inhibition (inhibitory domain), respectively, and receptor dimerization (Giangrande et al., 2000; Leonhardt et al., 2003; Heneghan et al., 2006). In addition, the presence of nuclear localization and export sequences in nPR ensures continual shuttling of the receptor between the nuclear and cytoplasmic cell compartments (Tyagi et al., 1998; Hager et al., 2000; Li et al., 2005). In many cells, however, this dynamic equilibrium favours nuclear compartmentalization of the receptor, especially of the A-isoform, which is further enforced upon addition of ligand (Clemm et al., 2000; Arnett-Manseld et al., 2004). nPR carries a short proline-rich motif (amino acids 421428) that, upon progesterone binding, mediates interaction between the cytoplasmic fraction of the receptor and the Src-homology 3 (SH3) domain of Src tyrosine kinases at the plasma membrane. This interaction triggers rapid activation of the Ras/Raf-1/MAPK pathway, which is entirely abolished upon mutation of the polyproline motif in PR (Boonyaratanakornkit et al., 2001, 2007). In contrast to PR-B, PR-A does not mediate progesterone activation of the Src/MAPK signalling pathways in human breast cancer cells, presumably because of the predominantly nuclear localization of this isoform (Boonyaratanakornkit et al., 2007). Intriguingly, the ability of nPR to directly interact with SH3 domains is not shared with other steroid hormone receptors.

Figure 1 Modular structures of nPRs, PGRMC1 and SERBP1.

(A) Functional domains of PR-B and PR-A, including the DBD and LBD, are indicated. AF-3, -1 and -2 are activating functions, ID is an inhibitory domain operative in PR-A only (Giangrande et al., 2000; Leonhardt et al., 2003). The proline-rich region interacting with the SH3 domain of Src tyrosine kinase is underlined (Boonyaratanakornkit et al., 2001), and phosphorylation sites are indicated by asterisks (Weigel and Moore, 2007). The N-terminally truncated isoform PR-C, proposed to be translated from residue 595, is shown in parenthesis as its natural occurrence is debatable (Wei et al., 1990; Samalecos and Gellersen, 2008). (B) PGRMC1 comprises a single Nterminal TM domain (TM) and a cytochrome (cyt) b5 domain. Interaction sites for SH2 and SH3 domains, kinase binding sites and phosphorylation sites for tyrosine (Tyr) and serine/threonine (S/T) kinases are indicated by asterisks. A C-terminal putative endoplasmic reticulum retention motif (KXX) is underlined (Cahill, 2007). The proposed interaction partner of PGRMC1, SERBP1, has a central hyaluronan binding domain (HABP4), and motifs typical of a number of RNA binding proteins: N-terminal R- and RG-rich sequences, and a C-terminal G-rich box found in mammalian glycine-rich RNA-binding proteins (GRPs) (Maruyama et al., 1999; Huang et al., 2000; Heaton et al., 2001). A cluster of SH3 interaction domains, binding sites for kinases and lipids and putative S/T phosphorylation sites are indicated (http://scansite.mit.edu/motifscan_seq.phtml).

Whether or not PR-mediated Src activation also requires the presence of the ER remains a matter of debate. In breast cancer cells and rat endometrial stromal cells, a direct interaction of PR-B with unliganded ER has been shown to promote proliferation in response to progesterone (Vallejo et al., 2005; Ballare et al., 2006). The physiological role of PR-B mediated MAPK activation in regulating reproductive function awaits further elucidation. Cyclin D1 appears

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to be an important downstream target of this signalling pathway, at least in breast cancer cell lines. This cardinal cell cycle regulator is overexpressed in  45% of breast tumour samples (Lange, 2004, 2007; Faivre et al., 2005). The Src/MAPK pathway has also been implicated in the ability of progesterone to confer protection against acute ischaemic neuronal damage (Cai et al., 2008). In addition, the proposed role for nPR in mediating cytoplasmic progesterone effects in mammalian cells is akin to the role of its amphibian homologue, termed X-PR, in Xenopus oocyte maturation. However, although the LBD and DBD of X-PR and human PR are highly similar, there is very little homology in the amino-terminal domains of these receptors. Moreover, X-PR localizes predominantly to the cytoplasm in contrast to the mammalian receptors (Martinez et al., 2007). Progesteronemediated X-PR activation is also coupled to the MAPK pathway in Xenopus oocytes as well as to the phosphatidylinositol 3-kinase signalling pathway (Bagowski et al., 2001). The rapid reduction of potassium inux in Xenopus oocytes in response to progesterone involves attenuated activity of plasma membrane Gbg, rather than Ga subunit of heterotrimeric G proteins, and has also been attributed to X-PR (Evaul et al., 2007). Although several studies have implicated X-PR in progesterone-dependent oocyte maturation, others have questioned this (Maller, 2001).

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PR antibodies revealed excellent specicity of antibodies directed against the N-terminal portion of the receptor (used to detect PR-B or PR-A), whereas antibodies raised against C-terminal epitopes (necessary to visualize PR-C or PR-M) produce prominent nonspecic signals and artefacts on Western blot analysis, which can easily be mistaken for the elusive PR variants (Samalecos and Gellersen, 2008).

GABA type A and oxytocin receptors

The GABA type A (GABAA) receptor is a member of the cysteine-cys-loop family of membrane ligand-gated ion channels and mediates most of the synaptic inhibition in the mammalian brain by altering Cl2 conductance and neuron excitability (Belelli and Lambert, 2005; Schumacher et al., 2007). GABAA receptors are targeted by clinically important drugs, and many pregnane steroids, some of which are synthesized de novo in the brain, can potently and specically modulate GABAA receptor function in a non-genomic manner and consequently produce anxiolytic, analgesic, anticonvulsant, sedative, hypnotic and anaesthetic effects (Belelli and Lambert, 2005). Progesterone metabolites like allopregnanolone (3a-hydroxy5a-pregnan-20-one) are positive allosteric modulators of GABAA receptors and therefore act as inhibitory neurosteroids. In contrast, pregnenolone sulphate and dehydroepiandrosterone sulphate are negative modulators of the GABAA receptor and positive modulators of the N-methyl-D-aspartate receptor, therefore acting as excitatory steroids (Monnet and Maurice, 2006). Allopregnanolone has antiseizure effects in the rat brain, mediated by the GABAA receptor (Frye and Scalise, 2000). In addition, allopregnanolone stimulates the proliferation of rodent and human neural progenitor cells via GABAA receptor-activated voltage-gated L-type Ca2 channels (Wang et al., 2005), and accelerates myelination in rat cerebellar cultures via nPR and GABAA receptors (Ghoumari et al., 2003). A recent study demonstrated the presence of two discrete steroid binding sites on GABAA receptors. Allopregnanolone is thought to potentiate GABA responses by binding to a cavity formed by the a subunit TM domains and to activate GABAA receptors directly by binding to interfacial residues between a and b subunits (Hosie et al., 2006). Oxytocin induces myometrial contractions that can be counteracted by progesterone. This has been attributed to direct binding of progesterone to the oxytocin receptor (OXTR), thereby allosterically hindering oxytocin activation of the receptor. Although this may be true in rodents, it is almost certainly not the case for the human OXTR (Grazzini et al., 1998). The notion that not progesterone but its 5b-dihydroprogesterone metabolite binds the human OXTR is also controversial (Thornton et al., 1999; Astle et al., 2003a). Arguably, 5b-reduced forms of progesterone may interact with myometrial GABAA receptors to inhibit smooth muscle contractions (Putnam et al., 1991; Mesiano, 2007). Progesterone has also been reported to antagonise oxytocin binding to its receptor in ovine endometrial plasma membranes, an effect that was reversible by mifepristone (Dunlap and Stormshak, 2004).

nPR variants
Following the cloning of the full-length human nPR cDNA in 1987 (Misrahi et al., 1987), the analysis of PR transcripts in breast cancer cells by Northern blot hybridization revealed extensive heterogeneity in the 50 region of the transcripts, which led to the prediction of a third isoform, termed PR-C (Wei et al., 1990). Using an antibody against the carboxy-terminus of PR, PR-C was originally described as a 60 kDa amino-terminally truncated isoform, presumably arising by translation initiation at methionine residue 595 (Wei and Miner, 1994) (Fig. 1). Lacking the rst zinc nger of the DBD, PR-C must be devoid of DNAbinding activity but retain the ligand binding properties of full-length receptors. Thus, one mechanism of action of the C isoform would be to antagonize PR-A or -B activation by sequestering progesterone. An important role for PR-C in human parturition has recently been put forward when a dramatic up-regulation of this isoform was observed upon Western blot analysis of term myometrial biopsies (Condon et al., 2006). The identication of another isoform, PR-M, seemed of particular relevance for membrane-associated progesterone signalling events. PR-M arises by splicing of a novel leader exon M to the downstream exons 4 8 of PR mRNA. Exon M carries an in-frame start codon and adds 16 hydrophobic amino acids, representing a putative signal peptide, to the C-terminal portion of PR including the LBD (Saner et al., 2003). The structure of both PR-C and PR-M predicts extra-nuclear localization. Combined with the fact that they lack the polyproline motif involved in Src activation, these isoforms might therefore also antagonize the cytoplasmic signalling activities of PR-B. Our own extensive analyses, however, indicate that neither PR-C nor PR-M is expressed at appreciable levels. This is due to the fact that the proposed start codons for these isoforms are within a sequence context that does not favour translation initiation in vivo (Samalecos and Gellersen, 2008). Furthermore, a systematic evaluation of commercially available

MAP2 and sigma1 receptor

Microtubules are major structural components of the cytoskeleton and are essential in the growth and maintenance of axons and dendrites during neuronal differentiation. Rat brain cytosol and

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seven TM domains and are classical G protein-coupled receptors (GPCRs) (Fig. 2). However, Tang et al. proposed that PAQRs have an inside-out topology, i.e. with the amino-termini of the receptors residing in the cytoplasm and the carboxy-termini on the cell surface. They favoured this model as it would place the most highly evolutionary conserved residues intracellularly, although experimental data in support of this conjecture is as yet lacking (Tang et al., 2005). Initial functional characterization provided further support for the notion that the mPRs are promising novel pharmaceutical targets for the management of reproductive disorders. Not only was it reported that the mPRs are expressed on the plasma membrane of cells but also that they modulate the activity of various signal transduction cascades upon progesterone binding, including stimulation of ERK1/2 or p38 MAPKs, inhibition of cAMP production via coupling to an inhibitory Ga protein, and stimulation of intracellular Ca2 mobilization (Zhu et al., 2003a, b; Ashley et al., 2006; Hanna et al., 2006; Karteris et al., 2006; Thomas et al., 2007). Our analysis started with the proling of mPRa, b and g transcripts in cycling human endometrium and in gestational tissues before and after the onset of parturition (Fernandes et al., 2005). We found that the onset of labour was associated with a signicant reduction in myometrial mPRa and b mRNA levels. Yet a different study reported an increase in myometrial mPR expression at term. Moreover, the authors proposed that this induction could account for the onset of human parturition as activation of the mPRs reduces cAMP

microtubules contain a pregnenolone-binding protein termed MAP2 (microtubule-associated protein 2) (Yamamoto et al., 1983) and compelling evidence indicates that pregnenolone stimulates MAP2dependent microtubule assembly. Interestingly, progesterone also binds MAP2 with an afnity comparable with that of pregnenolone. However, progesterone does not stimulate microtubule polymerization but instead antagonizes the effect of pregnenolone (Murakami et al., 2000; Fontaine-Lenoir et al., 2006). Progesterone has also been shown to inhibit the activity of the neuronal nicotinic acetylcholine receptor (nAChR) as well as the s1R (Valera et al., 1992; Lena and Changeux, 1993; Monnet and Maurice, 2006). Although allosteric inhibition of nAChR by progesterone requires micromolar concentrations, the human s1R contains a steroid-binding component and reportedly binds progesterone with an afnity (Ki) as high as 30 nanomolar (Collier et al., 2007). The s1R is activated by a variety of chemically unrelated drugs, such as haloperidol, pentazocine and ditolylguanidine, collectively known as sigma ligands (Quirion et al., 1992). This 25 kDa receptor, characterized by a single putative TM domain, resides in the endoplasmic reticulum but, upon activation, translocates to the cell membrane where it modulates intracellular Ca2 levels and various neurotransmitters systems (Hayashi T et al., 2000; Cai et al., 2008). Interestingly, s1R also binds cholesterol and has been implicated in the formation of lipid rafts, membrane platforms that are important for intracellular signalling (Palmer et al., 2007). Although endogenous activating ligands of the s1R are yet to be found, this enigmatic receptor is overexpressed in a variety of cancers and high-density s1-binding sites have also been described in different reproductive organs, e.g. ovaries, testis and pituitary, as well as on peripheral lymphocytes (Su et al., 1988; SimonyLafontaine et al., 2000; Wang et al., 2004; Palmer et al., 2007).

Membrane progestin receptors

The ability of cell-impermeable progesterone conjugates to elicit rapid cellular responses has fuelled the search for cell surface progesterone binding sites for many years. However, direct identication of candidate moieties by cloning and sequencing has only been achieved recently. The two most promising candidate molecules are progesterone receptor membrane component 1 (PGRMC1) and the family of mPRs, which we will discuss in detail. The discovery of the mPRs in 2003 was considered a major breakthrough in the eld of rapid progesterone signalling. The mPRs belong to a larger family of proteins highly conserved from eubacteria through to higher mammals, termed progestin and adipoQ receptors (PAQRs). The PAQR family also includes the adiponectin receptors (adipoRs) 1 and 2 (Fernandes et al., 2005; Tang et al., 2005). Three mPRs (mPRa, b and g) were initially cloned from sh ovaries and subsequently identied in a variety of species, including human (Zhu et al., 2003a, b; Tokumoto et al., 2006). Several observations sparked enormous interest in the role of these novel receptors in female reproduction, including their involvement in mediating progesterone-dependent oocyte maturation in sh and amphibians, the predominant and regulated expression of mPRa in mammalian reproductive tissues and evidence implicating myometrial mPRa and b in the onset of human parturition (Zhu et al., 2003a, b; Karteris et al., 2006; Josefsberg Ben-Yehoshua et al., 2007). Furthermore, based on sequence analyses, the original reports predicted that these receptors comprise

Figure 2 Possible membrane topologies of mPRs, members of the PAQR family.

Upon their initial cloning, mPRa, b and g were proposed to be plasma membrane GPCRs with the typical 7TM domain structure, the N-terminus facing towards the extracellular space (top panel) (Zhu et al., 2003a, b). This assumption has been challenged by placing an extended group of mPRs (a, b, g, d and 1) in the Class II subfamily of PAQRs with predicted 8TM topology (Smith et al., 2008) (bottom panel). Positions of residues most highly conserved throughout the PAQR family are indicated by shaded ovals; a reverse membrane insertion of the mPRs would place these residues facing the cytoplasm (Fernandes et al., 2005; Tang et al., 2005; Smith et al., 2008), a prediction supported by experimental evidence (Smith et al., 2008). Whether the mPRs localize to the plasma membrane or the endoplasmic reticulum membrane is still unresolved (Fernandes et al., 2008). A putative C-terminal endoplasmic retention motif (KXX) common to mPRa, b, g and d is indicated.

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synthesis and activates MAPK signalling in human myocytes, leading to the acquisition of a contractile phenotype (Karteris et al., 2006). We also investigated the functional characteristics ascribed to mPRs using various human expression systems. The results of these exhausting investigations, however, failed to corroborate that mPRs are expressed on the cell surface or mediate progesterone-dependent signalling events, such as inhibition of cAMP production, activation of ERK1/2 or p38 MAPKs, or Ca2 mobilization. Moreover, in our hands, the mPRs did not couple to G proteins and, most importantly, failed to bind progesterone or progestins (Krietsch et al., 2006). We demonstrated that mPRs primarily reside in the endoplasmic reticulum, a view supported by others (Ashley et al., 2006; Krietsch et al., 2006), and concluded that they must be considered intracellular orphan receptors. In support of our notion, a recent report demonstrated mPRa and g in the murine kidney to reside in the endoplasmic reticulum, due to a C-terminal endoplasmic retention motif (KXX) operational at least in mPRa (Fig. 2). Furthermore, the endogenous receptors did not facilitate ERK phosphorylation or Ca2 release in response to progesterone in isolated proximal tubules (Lemale et al., 2008). For a detailed discussion of the contentious issues, the reader is referred to a specialized review (Fernandes et al., 2008). The controversy surrounding the mPRs has recently taken another unexpected turn, courtesy of expression studies in yeast (Saccharomyces cerevisiae). Yeast are eukaryotic cells devoid of endogenous progesterone responses, even when exposed to high doses. Using a PAQR reporter system, heterologous expression of human mPRa, b and g in yeast cells was sufcient to elicit a progesterone response with an EC50 in the low nanomolar range (Smith et al., 2008). Agonist proling revealed that the afnity of mPRa and g for different ligands was distinct from that of nPR. For example, 17a-hydroxyprogesterone (17a-OHP), which is a poor agonist of nPR but binds with high afnity to the surface of human sperm (McDonnell and Goldman, 1994; Blackmore et al., 1996), induced a response in transfected yeast cells with an EC50 of around 10 nM. Interestingly, 17a-OHP caproate is the progestin of choice for the prevention of preterm delivery (Meis et al., 2003), although its precise mechanism of action remains to be determined. The nPR antagonist mifepristone also exhibited agonistic effects on human mPRs when expressed in yeast, albeit only in the micromolar range (Smith et al., 2008). Clearly, the expression studies in yeast are in support of the original claims by Zhu and co-workers that mPRs are bona de PRs (Zhu et al., 2003a, b; Thomas et al., 2007). However, a more in-depth phylogenetic analysis of the PAQR proteins has cast considerable doubt on two important characteristics ascribed to mPRs: their seven TM domain structure and the coupling to G proteins upon progesterone binding. The PAQR family consists of three groups: mPR-related, adipoR-related and hemolysin III-related family members (Fernandes et al., 2005). Two additional PAQRs (PAQR6 and 9) cluster within the mPR-related group and also function as PRs in the heterologous yeast expression system (Smith et al., 2008). Thus, the mPR subfamily, also referred to as Class II receptors, now comprises ve members, termed mPRa, b, g, d and 1 (Smith et al., 2008). The adipoR-related PAQRs form the Class I receptors, and hemolysin-related proteins are placed in Class III (Smith et al., 2008) (Table II). Importantly, although computational topology predictions conrm that Class I proteins consist of seven TM-spanning domains, Class II proteins are predicted to have eight TM domains (Fig. 2). Moreover, dual topology reporter

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Table II Classication of human PAQRs

Class I Domain structure 7 TM PAQR1 (adipoR1) PAQR2 (adipoR2) PAQR3 PAQR4 PAQR5 (mPRg) PAQR6 (mPRd) PAQR9 (mPR1)
Within Class II, PAQR7 and 8 form one clade, PAQR5, 6 and 9 another. Alternative names are given in parentheses. TM, transmembrane domain. Modied after (Smith et al., 2008).

Class II 8 TM PAQR7 (mPRa) PAQR8 (mPRb)

Class III 7 TM PAQR10 PAQR11

........................................................................................

assays in yeast revealed that the C-termini of mPRs do not pass through the lumen of the endoplasmic reticulum co-translationally and thus, along with the N-termini, are predicted to face the cytosol irrespective of these receptors being resident in the endoplasmic reticulum or the plasma membrane (van Geest and Lolkema, 2000; Smith et al., 2008) (Fig. 2). Three lines of evidence argue against mPRs being GPCRs: (i) the conserved eight TM domain topology of all Class II members, (ii) the ability of mPRs to respond to progesterone when expressed in yeast even upon deletion of the C-termini of the receptors and (iii) their ability to relay progesterone responses in yeast strains that lack G proteins (Smith et al., 2008). Although the observations in yeast are interesting, numerous issues remain unresolved. For example, what is the exact membrane topology of mPRs and do they reside predominantly in the endoplasmic reticulum or at the cell surface? Why is it that the human mPRs elicit a progesterone response when expressed in a heterologous system, whereas it is so difcult to reproduce this in homologous cell systems? If not coupled to G proteins, what are the signalling components involved in progesterone-dependent mPR activation? Are mPRs in the yeast expression system activated by direct binding of ligand or indirectly, via binding to a multimeric protein complex? Furthermore, what is the physiological relevance of having at least ve closely related mPR isoforms with very similar ligand preferences? It is likely that the mPRs will continue to be the subject of a heated debate (Fernandes et al., 2008; Thomas, 2008) and that resolution of the current controversies might have to await the generation of knockout mice. Interestingly, selective ablation of mPRa produced no overt reproductive defects in either male or female mice (T. Wintermantel and I. Huhtaniemi, personal communication), indicating possible redundancy among mPR family members. This observation raises the daunting possibility that combinatorial deletions of several mPRs are required to reveal their true physiological relevance.

Progesterone receptor membrane component 1

Purication of progesterone binding sites from porcine liver microsomal fractions led to the cloning of PGRMC1 (Meyer et al., 1996). PGRMC1, and the closely related PGRMC2, is structurally different from both mPRs and nPR (Raza et al., 2001) (Fig. 1). PGRMC1

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possibly containing as yet unidentied components, transduces progesterone signalling in this cell type. Like the mPRs with the exception of mPR1, PGRMC1 and PGRMC2 also possess a lysine residue positioned three residues from the carboxy-terminus (PGRMC1, ESARKND; PGRMC2, KDHNKQD) (Figs 1 and 2). The position of this lysine is essential for the retention of some TM proteins in the endoplasmic reticulum (Jackson et al., 1990). Assembly with heteromeric interacting proteins, such as SERBP1, may lead to masking of the endoplasmic reticulum retention motifs in PGRMC1/2, thereby promoting translocation to the plasma membrane (Ren et al., 2003; Nasu-Nishimura et al., 2006). In the human placenta, PGRMC1 and SERBP1 show overlapping patterns of expression and are most abundant in smooth muscle cells of the placental vasculature (Zhang et al., 2008). In the rat ovary, the expression pattern of PGRMC1, PGRMC2 and SERBP1 suggests a role in primordial follicle formation (Nilsson et al., 2006). A possible involvement of PGRMC1 and SERBP1 in ovarian neoplasia is an active eld of investigation (Peluso, 2007). Cytoplasmic and nuclear PGRMC1 expression varies between human ovarian cancer cell lines, raising the possibility that its subcellular localization may be dependent on cell cycle progression (Losel et al., 2008). Furthermore, SERBP1 mRNA is overexpressed in human epithelial ovarian cancer and its expression level is associated with tumour progression and metastasis (Koensgen et al., 2007). In addition to a proposed role in progesterone signalling, PGRMC1 has been implicated in an amazing spectrum of biological functions, including steroidogenesis, cellular homeostasis, survival and stress responses (Cahill, 2007). Compared with PGRMC1, much less is known about the biological functions of PGRMC2. Its ability to bind progesterone has not yet been assessed. Loss of high copy number of PGRMC2, measured by comparative genomic hybridization, has been associated with nodal metastasis of endocervical adenocarcinomas of the uterus (Hirai et al., 2004). Although several reports implicate PGRMC1 in progesterone signalling, the evidence for its role in sterol metabolism is perhaps more compelling. For instance, in transfected COS-7 cells, human PGRMC1 interacts directly with insulin-induced gene 1 (INSIG-1) and sterol regulatory element-binding proteins (SREBP) cleavageactivating protein (SCAP), proteins involved in sterol metabolism (Suchanek et al., 2005). The synthesis of cholesterol and other membrane lipids in mammalian cells is regulated by the controlled transport of SREBPs from the endoplasmic reticulum to the Golgi complex. SREBPs are membrane-bound transcription factors that activate genes encoding enzymes required for lipid synthesis. After their synthesis in the endoplasmic reticulum membranes, SREBPs bind to SCAP. In the presence of cholesterol, INSIG-1 forms a complex with SCAP and facilitates retention of the SCAP/SREBP complex in the endoplasmic reticulum. In sterol-depleted cells, SCAP escorts SREBPs from the endoplasmic reticulum to the Golgi apparatus for proteolytic processing, thereby allowing SREBPs translocation to the nucleus to stimulate transcription of genes involved in cholesterol synthesis (Yang et al., 2002). Thus, the fact that PGRMC1 binds both INSIG-1 and SCAP is supportive of an endoplasmic reticulum localization and suggests that PGRMC1 could also have a sterol-sensing function. The most compelling evidence in support of this notion stems from a recent study demonstrating that PGRMC1 and its yeast homologue Dap1 bind to and regulate endoplasmic reticulum

contains a single membrane-spanning domain and, in porcine hepatocytes, resides primarily in the endoplasmic reticulum and Golgi apparatus (Falkenstein et al., 1998). Furthermore, PGRMC1 was found in the inner acrosomal membrane of porcine spermatozoa (Losel et al., 2004), and a phosphorylated form of PGRMC1 was localized to the nucleus of HeLa cells (Beausoleil et al., 2004). Motif scanning of PGRMC1 revealed the presence of three binding interfaces for Src homology domains, including two SH2- and one SH3-target sequences (Peluso et al., 2006; Cahill, 2007) (Fig. 1). Thus, in analogy to PR-B, PGRMC1 could be involved in relaying progesterone actions via MAPK activation. PGRMC1 and PGRMC2 expression itself appears to be under the control of ovarian steroids. Female PRKO mice exhibit an elevated level of PGRMC1 in the brain compared with wild-type littermates (Krebs et al., 2000). Microarray studies demonstrated that human PGRMC1 transcripts are among the most dramatically down-regulated mRNAs upon transition of proliferative to secretory endometrium (Kao et al., 2002; Talbi et al., 2006). In the mouse uterus, estrous cycle-dependent changes in expression levels are more pronounced for PRGMC2 than PGRMC1 (Zhang et al., 2008). The role of PGRMC1 in reproductive function has most extensively been studied in the ovary. In spontaneously immortalized rat granulosa cells, at least a fraction of PGRMC1 localizes to the extracellular surface of the plasma membrane. Cell surface translocation of PGRMC1 in this cell type is mediated by interaction with serpine1 mRNA binding protein 1 (SERBP1, also known as CGI-55, Rda288 or PAIRBP1) (Peluso et al., 2006). SERBP1 is a multifunctional protein that reportedly binds to the mRNA of plasminogen activator inhibitor 1 (serpine1) to regulate its stability (Heaton et al., 2001) and interacts with chromatin remodelling factor CHD-3 (Lemos et al., 2003). Interestingly, SERBP1 was isolated from rat granulosa cells using a specic antibody against the hormone-binding domain of nPR, initially leading to the assumption that it has progesterone binding capacity (Peluso et al., 2001). The current view is that it is the complex of PGRMC1 with SERBP1 that mediates the antiapoptotic effect of progesterone in granulosa cells via activation of protein kinase G and regulation of intracellular Ca2 levels, with PGRMC1 being the actual progesterone binding unit within the complex (Peluso et al., 2002, 2004, 2005, 2006, 2007a; Peluso and Pappalardo, 2004; Peluso, 2006). Neutralizing antibodies to either PGRMC1 or SERBP1 prevent the anti-apoptotic action of progesterone in granulosa/luteal cells (Engmann et al., 2006). Intriguingly, SERBP1 co-immunoprecipitated with an antibody to the a1 chain of GABAA receptor (Peluso and Pappalardo, 1998). Furthermore, SERBP1 has hyaluronan binding domains (Peluso et al., 2004) (Fig. 1). Hyaluronan (or hyaluronic acid) is a glycosaminoglycan component of the extracellular matrix, interacts with cell surface receptors and couples to various signalling pathways including activation of Src, focal adhesion kinase, MAPKs and protein kinase C (Turley et al., 2002). Of note, hyaluronan is able to mimic the anti-apoptotic action of progesterone and to compete with progesterone for binding sites on spontaneously immortalized rat granulosa cells (Peluso et al., 2004). On the other hand, incubation of granulosa cell cultures with SERBP1 antibody attenuates the anti-apoptotic effect of progesterone without interfering with progesterone binding to the cells (Peluso et al., 2005). These observations are difcult to reconcile and strengthen the concept that a multimeric complex,

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cytochrome P450 enzymes in humans and yeast. Small interfering RNA-mediated knockdown of PGRMC1 expression in HEK293 cells resulted in an accumulation of toxic sterol intermediates, further underscoring the role of PGRMC1 in sterol homeostasis (DeboseBoyd, 2007; Hughes et al., 2007). Overall, it remains to be claried if PGRMC1 is a bona de PR. There is convincing evidence that PGRMC1 at least participates in formation of progesterone binding sites as (i) partially puried membrane fractions containing PGRMC1 bind progesterone (Peluso et al., 2007b), (ii) overexpression of PGRMC1 increases progesterone binding to isolated membranes of CHO and intact granulosa cells (Falkenstein et al., 1999; Peluso et al., 2006) and (iii) knockdown of PGRMC1 in granulosa cells diminishes progesterone binding (Peluso et al., 2007b). While compelling, this evidence for high-afnity binding of progesterone to PGRMC1 is not yet entirely conclusive and formal proof of direct binding is still lacking (Cahill, 2007; Losel et al., 2008).

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needed to substantiate and dene their roles in progesterone signalling. Many of these molecules, including s1R, PGRMC1 and mPRs, appear also involved in regulating basic cellular metabolism and homeostasis. While this undoubtedly adds an important level of complexity, it also raises the possibility that much more selective and effective pharmacological agents can be developed for the treatment of disorders of the reproductive tract and beyond.

Authors Role
All authors contributed to the writing of the paper.

Funding
This work was supported by the Deutsche Forschungsgemeinschaft ncia e a Tecnologia (DFG Ge 748/10-2; to B.G.), Fundac a o para a Cie (FCT), Portugal (Ph.D. Grant SFRH/BD/19418/2004 to M.S.F.) and Institute of Obstetrics and Gynaecology funding (to J.J.B.).

Conclusions and perspective

In recent decades, the focus in progesterone biology has largely been centred on the role of nPRs and rightly so. Knockout studies in mice demonstrated incontrovertibly that the nPRs are master regulators of female reproduction. However, the original model of nuclear receptor function, i.e. acting as ligand-dependent transcription factors that regulate gene expression simply by binding to distinct DNA response elements in the promoter region of target genes, cannot account for the very diverse tissue- and cell-specic responses to progesterone or other steroid hormones. It is now apparent that many additional levels of regulation are at play, which collectively control the expression, turnover and activity of nPR isoforms and their associated cofactors in a cell-specic manner. The ability of progesterone, like any other steroid hormone, to rapidly change the activity of dened signal transduction pathways and second messenger systems also constitutes an important level of regulation. While these signalling events may be rapid and transient, they are more than sufcient to trigger a profound cellular response, even in cells devoid of nuclei. In most cells, activation of these cytoplasmic pathways will inevitably also converge on nuclear factors, including the nPRs themselves and their cofactors, by altering the post-translational modication codes of their downstream target proteins. Importantly, nPR in turn regulates the expression of many genes that encode for signalling intermediates, as demonstrated in both breast and uterus. Thus, the non-genomic and genomic mechanisms of action are not only connected but tightly interwoven to produce a cell-specic progesterone response. There has been considerable interest in non-genomic progesterone actions in certain tissues, such as the brain and sperm. However, by and large, the physiological relevance of this particular mode of steroid action in female reproductive organs, such as ovary, uterus or placenta, has largely been unexplored. Consequently, the extent to which non-genomic progesterone events contribute to hormonedependent reproductive disorders also remains elusive. Clearly, this constitutes a massive black box in our understanding of progesterone action, which we can only start to unravel upon full characterization and subsequent manipulation of the receptors that bind progesterone and relay these cytoplasmic events. As outlined, several candidate receptors have emerged in recent years but much more work is

Conict of interest: The authors have nothing to disclose.

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Submitted on August 11, 2008; resubmitted on September 8, 2008; accepted on September 11, 2008