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BOARD-INVITED REVIEW: Estrogen and progesterone signaling: Genomic and nongenomic actions in domestic ruminants

Departments of Animal Sciences and Biochemistry/Biophysics, Oregon State University, Corvallis 97331

	Abstract

Top Abstract INTRODUCTION CHARACTERISTICS OF NUCLEAR... CHANGES IN UTERINE AND... P4, ESTRADIOL, AND UTERINE... UTERINE BLOOD FLOW DURING... NONGENOMIC ACTIONS OF ESTROGEN NONGENOMIC ACTIONS OF PROGESTINS BIOLOGICAL SIGNIFICANCE LITERATURE CITED

 
Progesterone and estrogens play key roles in regulating various physiological phenomena related to normal growth, development, and reproduction of domestic animals. This review focuses on the mechanisms by which progesterone and estrogens regulate the reproductive processes in these animals. The majority of research on the actions of progesterone and estrogens on the reproductive systems of cattle, sheep, and pigs has been genomic in nature and represents attempts to better understand how these steroids regulate gene expression. Results of recent research suggest that progesterone and estrogens can alter target cell responses nongenomically via membrane receptors. The characteristics of membrane receptors for progesterone and estrogen in various cell types are described and the intracellular signal pathways defined. Estrogens acting via membrane receptors can suppress LH secretion by gonadotropes and stimulate rapid increases in uterine blood flow. Progesterone acting via a membrane receptor has been shown to inhibit binding of oxytocin to oxytocin receptors in isolated endometrial plasma membranes and stimulate capacitation of spermatozoa. Results of research suggest that progesterone and estrogens can act nongenomically to alter target cell responses in domestic animals. The biological implications of this mode of action in these animals are discussed.

Key Words: estradiol-17β • luteinizing hormone • membrane receptor • oxytocin • progesterone • uterus

	INTRODUCTION

Top Abstract INTRODUCTION CHARACTERISTICS OF NUCLEAR... CHANGES IN UTERINE AND... P4, ESTRADIOL, AND UTERINE... UTERINE BLOOD FLOW DURING... NONGENOMIC ACTIONS OF ESTROGEN NONGENOMIC ACTIONS OF PROGESTINS BIOLOGICAL SIGNIFICANCE LITERATURE CITED

 
Steroid hormones regulate a host of physiological processes in domestic animals, but chief among those that affect productivity are mammary gland development, reproductive successes, and hypothalamus-hypophyseal functions. In domestic animals, steroids have been used primarily to promote growth, synchronize estrus, and maintain pregnancy. Since the discovery of nuclear steroid receptors and their roles as transcription factors, it has generally been assumed that all steroids act directly to regulate gene expression, a "genomic" action. However, in recent years, experimental evidence has been presented indicating that steroids can provoke responses in target cells independent of gene transcription, a "nongenomic" action by the steroid. For purposes of classification, a nongenomic steroid effect is one detectable within seconds to minutes after exposure to the steroid. By contrast, genomic responses to steroids generally require hours before being detectable.

From the standpoint of steroids and domestic animals, most interest has focused on the roles of progesterone (P₄) and estrogen because of their effects on the reproductive tract and anterior pituitary gonadotropin secretion. Thus, for comparative purposes this review provides a brief overview of the genomic actions of estrogen and P₄ in domestic animals. However, major emphasis is placed on describing the current state of knowledge regarding the nongenomic action of estrogen and P₄ in domestic animals with reference to relevant research in other species.

	CHARACTERISTICS OF NUCLEAR ESTROGEN AND P4 RECEPTORS

Top Abstract INTRODUCTION CHARACTERISTICS OF NUCLEAR... CHANGES IN UTERINE AND... P4, ESTRADIOL, AND UTERINE... UTERINE BLOOD FLOW DURING... NONGENOMIC ACTIONS OF ESTROGEN NONGENOMIC ACTIONS OF PROGESTINS BIOLOGICAL SIGNIFICANCE LITERATURE CITED

 
Estrogen and P₄ receptors (PR) belong to a superfamily of nuclear receptors that include not only steroid receptors but vitamin D, thyroid hormone, and orphan receptors (Carson-Jurica et al., 1990; Tsai and O’Malley, 1994). The basic structure of these receptors consists of a C-terminal or ligand-binding domain (LBD), a highly conserved DNA-binding domain near the center of the receptor, and an N-terminal domain of variable length. Within these domains are at least 2 transcription activation subdomains or functions, referred to as AF (Tsai and O’Malley, 1994; Edwards, 2005). The AF-1 in the N-terminal domain and ligand-dependent AF-2 present within the LBD are essential for gene transcription. Receptors to which estrogen or P₄ are bound become activated by their disassociation from protein chaperone molecules, dimerization, and subsequent binding of the steroid-receptor complex to a steroid response element usually localized in the promoter region of the gene (Tsai and O’Malley, 1994; Kumar and Thompson, 2003). Transcription of genes by the steroid-receptor complex may be altered by recruitment of protein coactivators or corepressors to AF-1, AF-2, or both (McKenna et al., 1999; Smith and O’Malley, 2004). The resulting coactivators or corepressors in association with the ligand-bound receptor ultimately facilitate the assembly of the RNA polymerase II complex that promotes gene transcription.

Two forms of the nuclear estrogen receptor (ER) are found in mammalian species. These 2 receptor subtypes are products of different genes and are designated ER (ER) and ERβ. Both monomeric forms of each receptor subtype are approximately 60 kDa, and both subtypes bind estradiol-17β with high affinity but differ in DNA-binding affinity. Estrogen receptor and ERβ can be coexpressed in some target tissues but they also exhibit different tissue or cell expression patterns (Hall and McDonnell, 1999; Heldring et al., 2007).

The PR also exists as 2 isoforms, designated as PR-A and PR-B (Edwards, 2005). The 2 isoforms of PR arise from a single gene as a result of alternating promoter start sites that give rise to 2 different PR mRNA. Progesterone receptor-A and PR-B have identical homologies of the LBD, DNA-binding domain, and AF-1 of the N-terminal domain; however, PR-A differs from the full-length PR-B by truncation of the most N-terminal 164 AA. The 2 isoforms of PR differ in their functional activities. Both isoforms are coexpressed in most target tissues, but the ratio of the 2 forms of PR can vary depending on cell type or physiological state. An AF-3 domain within the most N-terminal 164 AA of PR-B promotes a stronger transcriptional activity by PR-B (Li and O’Malley, 2003). It has been reported that PR-A can act as a transrepressor of PR-B and perhaps other steroid receptors such as ER (Edwards, 2005). In PR-A and PR-B knockout mice, PR-A has been found to play a major role in mediating the actions of P₄ in the uterus and ovary, whereas PR-B is more important in mammary gland development (Mulac-Jericevic et al., 2000, 2003).

	CHANGES IN UTERINE AND HYPOPHYSEAL NUCLEAR ER AND PR DURING VARIOUS REPRODUCTIVE STATES

Top Abstract INTRODUCTION CHARACTERISTICS OF NUCLEAR... CHANGES IN UTERINE AND... P4, ESTRADIOL, AND UTERINE... UTERINE BLOOD FLOW DURING... NONGENOMIC ACTIONS OF ESTROGEN NONGENOMIC ACTIONS OF PROGESTINS BIOLOGICAL SIGNIFICANCE LITERATURE CITED

 
Early reports documented the presence of nuclear estrogen and PR in the oviduct, uterus, and pituitary of laboratory animals. These studies served as the impetus to examine the concentrations of nuclear estrogen and PR in these target tissues in domestic animals. Uterine (endometrial) concentrations of nuclear estrogen and PR, as measured by radioreceptor assays, were found to increase during proestrus, attain maximal levels at estrus and metestrus, and then gradually decrease during the midluteal phase of the bovine and ovine estrous cycle (Senior, 1975; Koligian and Stormshak, 1977a; Miller et al., 1977; Zelinski et al., 1982; Robinson et al., 2001). Similarly, in the ewe, steady-state concentrations of endometrial ER mRNA and PR mRNA, found predominantly in the luminal and glandular epithelium, were maximal at estrus to metestrus, markedly reduced during the midluteal phase of the cycle, and then increased late (d 11 to 15) in the luteal phase (Spencer and Bazer, 1995). In pregnant ewes, changes in endometrial ER and PR mRNA, as well as concentrations of respective receptors, were similar to those of the nonpregnant ewe at comparable stages after estrus, except concentrations of mRNA and protein in the luminal and glandular epithelium remained as at the midluteal phase and were very low or absent between d 13 to 25 of gestation (Spencer and Bazer, 1995).

By simply examining the relationship between uterine concentrations of ER and PR and systemic levels of estrogen and P₄, one might deduce that estrogen upregulates both ER and PR. Indeed, this aspect of estrogen action in the endometrium was confirmed by using ovariectomized ewes treated with estrogen or P₄ alone or in combination (Koligian and Stormshak, 1977b).

Comparatively few studies have been conducted to quantify estrogen and PR in the anterior pituitary or hypothalamus of domestic animals. Early research established the presence of androgen and ER in the hypothalamus and anterior pituitary of the ram and ewe (Pelletier and Caraty, 1981; Glass et al., 1984). Subsequent research revealed that estrogen and androgen receptors were present in various morphological nuclei throughout the ovine hypothalamus. However, those cells containing the greatest concentrations of ER in the hypothalamus of the ewe were located in the preoptic area, ventromedial nucleus, and arcuate nucleus (Herbison et al., 1993; Lehman et al., 1993). Similarly, these same sites in the hypothalamus of the ram were found to be richly endowed with androgen receptors (Herbison, 1995). Alexander et al. (1993) examined quantities of ER in the hypothalamus, medial amygdala, and anterior pituitary of high and low sexually performing rams. The total number of ER in the anterior pituitary was nearly 100-fold greater than the number present in hypothalamic regions. The proportion of total ER in the preoptic area of the hypothalamus that was bound by estradiol-17β was significantly greater in high-performing than in low-performing rams. In contrast, the proportion of ER that was occupied with estradiol-17β in the anterior pituitary of high-performing rams tended to be less than that in low-performing rams. These differences in ER did not appear to affect secretion of LH during sexual behavior testing because high and low sexually performing rams had similar LH profiles relative to systemic basal concentrations, number of secretory pulses, and pulse amplitude of the gonadotropin.

During the estrous cycle of the cow, quantities of estrogen and GnRH receptors in the anterior pituitary are markedly increased prior to the preovulatory surge of LH (Nett et al., 1987). Estrogen of follicular origin apparently causes upregulation of the ER and GnRH receptors (Turzillo and Nett, 1999). Significant increases in estradiol receptors in the hypothalamus and anterior pituitary of ewes on d 22 postpartum were found to immediately precede the onset of ovarian cyclicity (Wise et al., 1986). In postpartum cows, receptors for both GnRH and estradiol in the anterior pituitary are lowest immediately after parturition, then increase to maximal concentrations by 2 wk postpartum (Nett et al., 1988). Pituitary content of LH in the cow is low following parturition and increases significantly through d 30 postpartum. Exposure of the anterior pituitary to the high systemic concentrations of placental estrogen that are prevalent during the late stage of gestation may be responsible for downregulating estrogen and GnRH receptors and for synthesis of LH. Indeed, chronic treatment of ovariectomized ewes with estradiol for 3 wk caused a reduction in mRNA for the LH and β subunits in the anterior pituitary (Nilson et al., 1983). Collectively, these reports suggest that the estradiol exhibits a paradoxical effect on LH synthesis, with low physiological levels stimulating and chronic levels inhibiting the synthesis of this gonadotropin. On the other hand, estradiol, whether chronically administered to ovariectomized ewes or added to primary ovine pituitary cell cultures at physiological concentrations, significantly increased the synthesis and secretion of prolactin (Shupnik et al., 1979; Vician et al., 1979). Progesterone suppressed the estradiol-induced increase in preprolactin mRNA production and prolactin synthesis.

	P4, ESTRADIOL, AND UTERINE OXYTOCIN RECEPTORS

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Binding of oxytocin (OT) to its receptor in the uterine endometrium of the nonpregnant ruminant stimulates pulsatile secretion of PGF₂, which causes regression of the corpus luteum (McCracken et al., 1999). Because the uterus is a target organ for P₄ and estrogen, considerable research has been conducted to elucidate the role of these ovarian steroids in regulating the uterine expression of the OT receptor (OTR). In the ewe and cow, concentrations of OTR localized in the uterine luminal and superficial glandular epithelium of the endometrium are maximal at estrus, become virtually absent or nondetectable during the midluteal phase of the estrous cycle, and on d 14 (ewe) or 15 (cow) reappear in these cells and continue to increase in number until onset of behavioral estrus (Sheldrick and Flint, 1985; Fuchs et al., 1990; Jenner et al., 1991; Wallace et al., 1991; Robinson et al., 2001). In the ewe, the increase in endometrial concentrations of OTR coincides with the rise in ovarian venous estradiol-17β and the reduction in systemic concentrations of P₄ as a consequence of luteolysis (Sheldrick and Flint, 1985). On the basis of these observations, it would seem logical to assume that estrogen upregulates and P₄ downregulates the uterine OTR. Such effects of estrogen and P₄ are consistent with reported changes in expression of receptors for these steroids in uterine tissues during the course of the estrous cycle. Luminal and superficial glandular epithelial cells of the ovine uterus are endowed with ER mRNA and protein on d 14 and 15, but there is an absence of PR mRNA and nuclear receptor from d 6 to 13 of the cycle (Spencer and Bazer, 1995). Thus, the appearance of OTR in the luminal epithelium in the absence of functional nuclear PR (nPR) may be due to the unimpeded action of estradiol being produced by the developing ovarian follicle(s).

Experimental evidence also indicates that exogenous estradiol can upregulate uterine concentrations of OTR. Treatment of ovariectomized ewes with P₄ (5 to 12 d) followed by 2 d of estradiol treatment caused an increase in the uterine endometrium OTR as compared with treatments with P₄ only (Vallet et al., 1990). Further, exposure of bovine endometrial explants to estradiol stimulated an increase in OTR compared with levels of receptor present in control explants (Leung and Wathes, 2000). Whereas the reporter region of the bovine OTR gene contains at least 3 estrogen response element (ERE) half-sites, there is no firm evidence that the ligand-bound ER binds directly to these sites to stimulate OTR expression (Telgmann et al., 2003). Similarly, the promoter region of the ovine OTR gene contains neither full ERE or ERE half-sites (Fleming et al., 2006). Deletion and mutation analysis of the promoter region of the ovine OTR revealed that stimulation of the gene by estradiol is dependent on a guanine-cytosine-rich SP1 binding site at –104 and –64 bp.

Overall, experimental evidence favors a conclusion that estrogen upregulates and P₄ downregulates uterine OTR. However, the intracellular dynamics of OTR expression in uterine target cells, as affected by exposure to estrogen and P₄ during the estrous cycle, may be more complex than presently envisioned.

Pregnancy recognition in the ruminant encompasses the production of interferon- (IFN-) by the developing conceptus, which inhibits development of the endometrial luteolytic mechanism and pulsatile release of PGF₂ (Bazer, 1992). It has been shown experimentally that IFN- acts to prevent increases in endometrial ER mRNA and protein, with a consequent reduction in OTR and a suppression in the uterine release of PGF₂(Spencer et al. 1995; Spencer and Bazer, 1996). It has been proposed that the antiluteolytic effects of IFN- are mediated by the direct inhibition of ER gene transcription, thereby preventing ligand-bound ER from stimulating OTR gene transcription (Fleming et al., 2006).

	UTERINE BLOOD FLOW DURING THE ESTROUS CYCLE

Top Abstract INTRODUCTION CHARACTERISTICS OF NUCLEAR... CHANGES IN UTERINE AND... P4, ESTRADIOL, AND UTERINE... UTERINE BLOOD FLOW DURING... NONGENOMIC ACTIONS OF ESTROGEN NONGENOMIC ACTIONS OF PROGESTINS BIOLOGICAL SIGNIFICANCE LITERATURE CITED

 
The roles of estrogens and P₄ in regulating uterine blood flow have been studied extensively for more than 30 yr. Uterine blood flow in the cow, ewe, and sow varies during the estrous cycle in synchrony with the changing ratios of estrogen to P₄ concentration in systemic blood (Greiss and Anderson, 1969; Ford and Christenson, 1979; Ford et al., 1979). Uterine blood flow is markedly greater immediately prior to and during the onset of estrus, followed by a reduced flow during the luteal phase of the cycle (Ford, 1982). Thus, P₄ promotes vasoconstriction, which may contribute to the reduced uterine blood flow. In unilaterally ovulating ewes and cows, removal of segments of the uterine artery ipsilateral to the ovary bearing the corpus luteum (CL) respond in vitro to periarterial nerve stimulation with greater contractility than arterial segments on the contralateral side (Ford et al., 1976). These data were interpreted to suggest that the uterine artery supplying the horn ipsilateral to the ovary bearing the CL were exposed to a greater local concentration of P₄ than the contralateral uterine artery. Indeed, it was subsequently demonstrated that in the cow (Pope et al., 1982) and ewe (Weems et al., 1989) uterine tissues on the side ipsilateral to the ovary with the CL contain a greater concentration of P₄ than tissues on the contralateral side. This difference in tissue concentration of P₄ between the ipsilateral and contralateral sides of the uterus is attributed to lymphatics draining the ovary (Lindner et al., 1964), which are in close association with the utero-ovarian vasculature (Morris and Sass, 1966).

The precise mechanism by which P₄ promotes vasoconstriction of uterine arteries is unknown. However, either directly or indirectly, P₄ causes an increase in the concentration of uterine arterial smooth muscle ₁-adrenergic receptors (Ford et al., 1984). Activation of these adrenergic receptors by norepinephrine emanating from periarterial sympathetic nerves results in vasoconstriction of the uterine vascular bed (Greiss and Pick, 1964).

Exposure of the uterine artery to estrogen results in a rapid increase in blood flow. Injection of estrogen directly into the uterine arteries of ovariectomized ewes increases blood flow within 30 min (Killam et al., 1973). Similarly, in ovariectomized cows an increase in uterine blood flow occurred within 20 to 40 min after injection of 1 µg of estradiol-17β into the uterine artery (Ford and Reynolds, 1983). The speed with which this uterine response to estrogen occurs suggests that the steroid is acting nongenomically in the vasculature.

Estrogen synthesized by porcine and bovine conceptuses is believed to act locally to promote transient increases in blood flow to the gravid uterine horn during early gestation. In sows, uterine venous blood and uterine flushings contained increased quantities of estrogen coincident with a transient increase in uterine blood flow on d 13 of gestation (Ford et al., 1982). Similarly, in cows a transient increase in blood flow to the gravid horn occurs on d 14 to 18, and during this time the venous blood draining this horn contains increased concentrations of estradiol-17β (Ford et al., 1981). The fact that the estrogen produced by the conceptuses of these species acts locally to cause an abrupt transient increase in uterine blood flow suggests that estrogen may be acting nongenomically, at least initially, to promote this uterine vasculature response, just as it does in the ovariectomized cow given an intraarterial injection of estradiol-17β, as described above.

	NONGENOMIC ACTIONS OF ESTROGEN

Top Abstract INTRODUCTION CHARACTERISTICS OF NUCLEAR... CHANGES IN UTERINE AND... P4, ESTRADIOL, AND UTERINE... UTERINE BLOOD FLOW DURING... NONGENOMIC ACTIONS OF ESTROGEN NONGENOMIC ACTIONS OF PROGESTINS BIOLOGICAL SIGNIFICANCE LITERATURE CITED

 
Nature of the Membrane-Associated Receptors
Evidence that estrogens could act to stimulate cell responses via an extranuclear mechanism was first advanced by the report of Pietras and Szego (1975) in the mid-1970s. However, because investigators of hormone action were at the time focused on characterizing nuclear steroid receptors and their function, the significance of this report was ignored. In a subsequent study, Pietras and Szego (1977) incubated estradiol-derivatized nylon monofilament fibers with cells of the endometrium, intestinal mucosa, and liver. Only endometrial and liver cells were found to adhere to the estrogen-derivatized fibers, but not nontarget cells from the intestine. The results of this study provided direct proof that specific binding sites for estradiol were resident in the plasma membrane of target cells. Interest in the plasma membrane ER was piqued by the report of Razandi et al. (1999). These investigators transfected cDNA for mouse ER and ERβ into Chinese hamster ovary (CHO) cells. Expression of the cDNA for each receptor resulted in production of a single transcript that gave rise to both membrane and nuclear receptors. The membrane receptor number was only 3% as great as the nuclear receptor density. Treatment of cells with estradiol-17β stimulated a phosphoinositide cascade as well as activation of adenylate cyclase, suggesting that the receptors were coupled to G_q and G_s proteins. More importantly, exposure of the transfected cells to estradiol provoked an increase in extracellular-regulated kinase (ERK) activity within 10 min, which was considered to be the consequence of the nongenomic action of the steroid (Figure 1). The estrogen antagonist, Imperial Chemical Industries (ICI) 182,780, inhibited estradiol-17β and estradiol-17β-BSA conjugate activation of the mitogen-activated protein kinase (MAPK) components. Subsequently, Song et al. (2002), using MCF-7 cells, provided evidence for a rapid direct physical association between ER and Sarc homology collagen (Shc) adaptor protein upon stimulation of cells with estradiol-17β. Using a mutagenesis approach, the investigators demonstrated that Src homology (SH) 2 and phosphotyrosine binding domains of Shc and the AF-1 region of ER are the interacting molecular sites of association. The presence of estradiol-17β rapidly induced Shc phosphorylation, presumably by a Src protein family member, with the resultant formation of an Shc-growth factor receptor-binding protein-2–son-of-sevenless (SOS, a guanine nucleotide exchange factor) complex with a consequent phosphorylation of ERK-1/-2 and activation of the transcription factor Elk-1. Estradiol-17β rapidly induced (within 20 min) formation of membrane ruffles and pseudopodia in modified MCF-7 cells. The estradiol-induced morphological changes were prevented by the antiestrogen ICI 182,780. In a subsequent study, Song et al. (2004) examined how estradiol-17β interacts with ER localized at the plasma membrane and the mechanisms by which Shc and IGF-I receptor (IGF-IR) mediate this process. They were able to demonstrate that estradiol-17β rapidly induced formation of a ternary protein complex consisting of Shc, ER, and IGF-IR. Knockdown of Shc with a specific small inhibitory RNA decreased the association of ER and IGF-IR by 87%, suggesting that Shc is a crucial molecule in the formation of this ternary complex. Results of their experiments demonstrated that Shc and IGF-IR serve as key localized membrane elements that facilitate ER-mediated rapid estrogen action.

View larger version (14K): [in this window] [in a new window]   Figure 1. Nongenomic estrogen signaling pathways involving estrogen receptor (ER) and G-protein receptor (GPR30). Biochemical components of the pathways are as follows: E = estradiol-17β; P = phosphate; AC = adenylate cyclase; Gβ = heterotrimeric G-protein; cAMP = cyclic adenosine monophosphate; PKA = protein kinase A; MMP = a metalloproteinase; HB-EGF = heparin-bound epidermal growth factor; EGFR = epidermal growth factor receptor; ER = endoplasmic reticulum; Grb2 = growth factor receptor binding protein-2; Shc = Sarc homology collagen adaptor protein; SOS = son-of-sevenless, a guanine nucleotide exchange factor; Ras = a GTPase activated by GTP; Raf = a kinase also referred to as MAP kinase, kinase; MEK = a kinase also referred to as MAP kinase, kinase; ERK-1/-2 = extracellular regulated kinases also referred to as MAP kinase; Elk-1, c-fos, c-jun = transcription factors; TK = Tyr kinase; PI3K = phosphatidylinositol-3 kinase; PI4,5P2 = phosphatidylinositol-4,5-bisphosphate; PI3,4,5P3 = phosphatidyl inositol-3, 4,5-trisphosphate; Akt/PKB = protein kinase B; PLC = phospholipase C; and IP3 = inositol-1,4,5-trisphosphate. Pathway interactions designated with a question mark are presently not well defined.

Mobilization of Ca²⁺ may contribute to the observed nongenomic actions of estrogens. An early report by Morley et al. (1992) contained data indicating that exposure of pig granulosa cells to 10^–6 to 10^–10 M estradiol-17β caused an immediate 4- to 8-fold increase in intracellular Ca²⁺. This cellular response is specific of estrogens because various progestins and androgens were ineffective. The rapid estrogen-induced increase in intracellular Ca²⁺ was not reduced by incubating the cells in Ca²⁺-free medium or by pretreating with Ca²⁺ channel blockers. It appears then that the release of Ca²⁺ is from intracellular stores triggered by inositol trisphosphate generated by an ER-induced hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate.

There are some who believe the nongenomic action of estrogen, at least in some tissues, is mediated by a G protein-coupled receptor referred to as GPR30 (Figure 1). This is a bizarre protein whose role as an ER and whose location within the cell are unsettled and controversial. Human SKBR3 breast cancer cells fail to express ER and ERβ but do contain the GPR30 protein. Filardo et al. (2000) showed that exposure of these cells to estradiol-17β (1 nM) for 5 min resulted in phosphorylation of ERK-1/-2. Surprisingly, the estrogen antagonist, ICI 182,780 (1 µM), was as effective as estradiol-17β in causing phosphorylation of ERK-1/-2. Further, in MDA-MB-231 cells, which express some ERβ but little GPR30 protein, there is no activation of ERK in response to either estradiol or the estrogen antagonist, suggesting that the classical ER are not mediators of estrogen action in this cell type. Proof of this was provided by results of an experiment in which a GPR30 cDNA was transfected into MDA-MB-231 cells, causing overexpression of the GPR30 protein. Exposure of these transfected cells to estradiol or ICI 182,780 caused phosphorylation of ERK-1/-2, whereas estradiol-17 and P₄ were ineffective. Filardo et al. (2000) provided evidence suggesting that estradiol signaling occurred via a Gβ subunit-dependent, pertussis toxin-sensitive pathway that ultimately results in transactivation of epidermal growth factor (EGF) receptor (EGFR) via the autocrine release of heparin-bound EGF. Binding of heparin-bound EGF to EGFR promotes receptor dimerization and autophosphorylation of Tyr residues within the cytoplasmic domain of the receptor. Specific recognition of these phosphotyrosines by the adapter proteins Grb-2, Shc, or both, and the guanine nucleotide exchange factor SOS forms the Grb-2-Shc-SOS adaptor protein complex, which links activated EGFR to MAPK via the monomeric GTPase, Ras. The Ras recruits Raf, which promotes cascade phosphorylation and activation of Mek-1 and its substrates ERK-1/-2. The response of human breast carcinoma cells to estradiol-17β in terms of phosphorylation of ERK-1/-2 is transient, rapidly returning to basal levels 30 to 60 min after initial exposure to estradiol. Filardo et al. (2002) hypothesized that estrogens also act via GPR30 to stimulate adenylate cyclase to inhibit the EGFR to MAPK pathway. Exposure of membranes from human SKBR3 breast cancer cells and MDA-MB-231 cells (that were forced to overexpress GPR30 protein) to estradiol and ICI 182,780 responded to treatment with an increase in adenylate cyclase activity as measured by production of cyclic adenosine monophosphate (cAMP). By contrast, estradiol and ICI 182,780 failed to cause stimulation of cAMP production by membranes isolated from MDA-MB-231 cells (which contain ERβ but neglible quantities of GPR30) or promote blockade of EGF-induced activation of ERK-1/-2. Evidence is presented suggesting that estrogen-induced suppression of ERK-1/-2 activity in cancer cells expressing GPR30 may be due to protein kinase A phosphorylation of Ser residues in Raf-1. Although the reports by Filardo et al. (2000, 2002) suggested that GPR30 was a resident of the plasma membrane, the more recent research of Revankar et al. (2005) indicated otherwise. To determine the cellular localization of the receptor, these latter investigators expressed GPR30 as a fusion protein with green fluorescent protein in COS-7 (monkey kidney fibroblast) cells. The receptor was expressed with the β₂-adrenergic receptor, a G-protein-coupled receptor. Confocal fluorescent microscopy revealed that the β₂-adrenergic receptor was localized to the plasma membrane, whereas GPR30-green fluorescent protein was localized in the endoplasmic reticulum and the Golgi apparatus. The same intracellular location for GPR30 was found for MCF-7, SKBR3, and MDA-MB-231 cells. Exposure of COS-7 cells bearing GPR30 to estradiol-17β causes a mobilization of intracellular Ca²⁺. Because GPR30 had been shown by Filardo et al. (2000, 2002) to activate the MAPK pathway via EGFR and mobilization of intracellular Ca²⁺ is mediated by phospholipase C (PLC)-dependent inositol-1,4,5-trisphosphate production, an experiment was conducted to determine whether either of these pathways was involved in estrogen-stimulated Ca²⁺ mobilization by ER and GPR30. The GPR30-mediated Ca²⁺ mobilization was completely blocked by an inhibitor of EGFR, but not by an inhibitor of PLC, whereas the ER response was completely blocked by the PLC inhibitor, but not measurably so by the EGRF inhibitor. These data suggest that ER- and GPR30-provoked Ca²⁺ mobilization are mediated by distinctly different pathways.

Because of equivocal data regarding the cellular location of GPR30, Filardo et al. (2007) conducted a series of experiments to prove that GPR30 is present in the plasma membrane. For these experiments, HEK-29 cells transfected with a hemagglutin-epitope tag incorporated at the amino terminus of human GPR30 and the SKBR3 breast cancer cells were utilized. Using a variety of techniques, including confocal microscopy, western blotting of subcellular fractions, estrogen binding, and estradiol activation of G-protein in cell compartments, these investigators provided strong evidence that GPR30 is found in the plasma membrane. However, it should be noted that these cancer cells were, in some experiments, exposed to exceedingly high concentrations of estradiol.

Rapid Estrogen-Induced Responses by the Pituitary
Administration of estradiol-17β to ovariectomized ewes resulted in an almost immediate reduction in systemic concentration of LH, followed in approximately 12 h by an ovulatory-type surge of the gonadotropin (Nett et al., 1984). The almost instantaneous response to estrogen suggested an inhibitory effect on the hypothalamus or hypophysis and precluded it being the result of the traditional effect of the steroid on gene expression. Recognizing that this response must be due to an extranuclear cellular site of action, Arreguin-Arevalo and Nett (2005) began to explore the mechanism by which estradiol-17β was able to evoke such a rapid response by the pituitary. Using a primary culture of ovine pituitary cells, these investigators showed that preincubation of cells for 15 min with estradiol-17β or estradiol-17β conjugated to BSA or a peptide (PEP) prevented GnRH-induced secretion of LH (Figure 2). This was a specific response to estradiol-17β because pretreatment of cells with P₄, testosterone, hydrocortisone, or estradiol-17 failed to affect secretion of LH by themselves, and did not suppress GnRH-induced secretion of LH. Further, when pituitary cells were coin-cubated with these steroids plus estradiol-17β, they did not block the ability of estradiol-17β to inhibit GnRH-induced release of LH. Treatment of cells with tamoxifen, hydroxytamoxifen, or ICI 182,780 blocked inhibition by estradiol-17β (E₂) and the estradiol-17β conjugates (E₂-BSA, E₂-PEP). When cells were treated with selective ER agonists, the ER-agonist (propylprazoletriol), but not the ERβ agonist (diarylpropionitrile), decreased the GnRH-induced secretion of LH. The fact that E₂-BSA and E₂-PEP mimicked the action of estradiol-17β suggests that this effect was mediated by an ER associated with the plasma membrane.

View larger version (12K): [in this window] [in a new window]   Figure 2. Primary cultures of ovine pituitary cells (2 x 105 cells per well) were preincubated for 15 or 60 min in the presence of estradiol-17β (E2), E2-BSA, or E2-peptide (PEP) and then exposed to 2 nM GnRH for 15 min. Mean ± SEM of LH released represent combined responses to all 3 forms of E2 because no significant interactions between incubation time and dosage or between treatment and dosage were found. a–cMeans with different letters differ (P < 0.01). Reproduced from Arreguin-Arevalo and Nett (2005).

View larger version (11K): [in this window] [in a new window]   Figure 3. Representative response of an ovariectomized ewe given i.v. infusion of estradiol-17β (E2) for 4 h. The scale on the left represents serum concentrations of LH in blood samples collected every 15 min from 4 h before to 5 h after E2. The scale on the right depicts LH concentrations attained during the LH surge. Note the rapid decrease in LH secretion upon initiation of treatment. Reproduced from Arreguin-Arevalo and Nett (2006).

View larger version (20K): [in this window] [in a new window]   Figure 4. Representative response of an ovariectomized ewe given i.v. infusion of estradiol-17β (E2)-BSA for 4 h. The scale on the left represents serum concentrations of LH in blood samples collected every 15 min from 4 h before to 5 h after E2-BSA treatment. The scale on the right depicts LH concentrations during the predicted time of the LH surge. Note the rapid decrease in LH secretion upon initiation of E2-BSA treatment and the absence of the LH surge. Reproduced from Arreguin-Arevalo and Nett (2006).

Rapid Estrogen Signaling in the Vascular System
As mentioned in an earlier section of this review, ovarian steroids have been shown to exert a local effect on the uterine arteries of domestic ruminants. In particular, the presence of estrogen was found to markedly increase arterial dilation and blood flow. Simoncini et al. (2000) reported that estrogen activated the phosphatidylinositol-3-kinase (PI3K)-Akt/protein kinase B pathway (Figure 1), with consequent activation of endothelial nitric oxide synthase (eNOS). Using human vascular endothelial cells, these investigators demonstrated that ER binds in a ligand-dependent manner to the p85 regulatory subunit of PI3K. Stimulation with estrogen increases ER-associated PI3K activity. This kinase catalyzes the phosphorylation of phosphatidylinositol-4,5-bisphosphate to form phosphatidylinositol-3,4,5-trisphosphate. The phosphatidylinositol-3,4,5-trisphosphate associates with 3-phosphoinositide-dependent protein kinase-1, a protein kinase that phosphorylates Akt/protein kinase B and leads to enhanced activity of eNOS with a resultant increase in NO, which causes relaxation of vascular smooth muscle. Estrogen receptor β did not interact with p85 or recruit PI3K activity after estrogen stimulation. To determine whether estradiol-17β-stimulated eNOS activation was mediated by Akt, the investigators transiently transfected bovine aortic endothelial cells with adeno-viruses containing constitutively active and dominant negative Akt mutants. Transfection of these endothelial cells with the constitutively active Akt resulted in a substantial increase in eNOS activity, whereas overexpression of the dominant negative Akt completely abolished estradiol-17β-stimulated eNOS activity. The short duration over which endothelial cells responded to estradiol-17β suggested that recruitment and activation of PI3K was by ligand-bound ER but independent of gene transcription. In contrast to these observations, Chen et al. (2004) reported that estrogen-induced activation of eNOS in ovine uterine arterial endothelial cells (UAEC) occurred by a different pathway. According to these investigators, exposure of UAEC to estradiol increased eNOS activity and NO production within 2 min as a consequence of the estradiol binding to ER in the plasma membrane. Exposure of UAEC to estradiol resulted in rapid phosphorylation of ERK-1/-2, which was maximal within 5 min after treatment of cells with the estrogen. Treatment of cells with the ER antagonist ICI 182,780 or PD98059, an inhibitor of the MAPK pathway, abolished estradiol-17β-induced phosphorylation of ERK-1/-2 and eNOS activity. However, exposure of UAEC to the PI3K inhibitor LY294004 failed to prevent estradiol-induced production of NO by these cells. The investigators concluded that rapid activation of eNOS to produce NO in ovine UAEC by estrogen is mediated, at least in part, through the MAPK pathway with activation of ERK, and not via activation of the PI3K pathway. The production of NO would result in rapid uterine vasodilation by estrogen, a phenomenon shown to occur in vivo.

	NONGENOMIC ACTIONS OF PROGESTINS

Top Abstract INTRODUCTION CHARACTERISTICS OF NUCLEAR... CHANGES IN UTERINE AND... P4, ESTRADIOL, AND UTERINE... UTERINE BLOOD FLOW DURING... NONGENOMIC ACTIONS OF ESTROGEN NONGENOMIC ACTIONS OF PROGESTINS BIOLOGICAL SIGNIFICANCE LITERATURE CITED

 
Nature of the Membrane-Associated Receptors
Progesterone can activate several different nongenomic pathways dependent on the tissue or cell type and receptor type present in the system. A nongenomic action by the classical nPR has been implicated by several studies (Skinner et al., 1998; Bagowski et al., 2001; Vallejo et al., 2005; Boonyaratanakornkit et al., 2007; Evaul et al., 2007; Lange et al., 2007). There is evidence that P₄ binding to a plasma membrane-localized or plasma membrane-associated nPR induces proliferation of murine endometrial stromal cells, via activation of MAPK, as a consequence of cross-talk with the classical ERβ (Vallejo et al., 2005). At low levels of expression, nPR and ERβ form a complex, which activates both the MAPK pathway and the Akt pathway, and thereby stimulates the cell to progress through the mitotic cycle. Because both PR and ERβ are at low concentrations during the process of decidualization in the murine uterus, this may be an important mechanism in the rodent by which P₄ promotes the maintenance of pregnancy.

An SH3-interacting domain motif located in the N-terminus between AA residues 421 to 428 (near the AF-1 domain) was discovered by Boonyaratanakornkit et al. (2001) in both nPR-B and nPR-A. The SH3-interacting domains are polyproline sequence motifs characteristic of a class II consensus PEP for Src kinase-like SH3 domains. Polyproline sequences form a left-handed helix that recognizes the hydrophobic pocket of SH3 domain-containing proteins. In coimmunoprecipitation assays, the SH3 domain of nPR interacted with a wide variety of Src Tyr kinase family members, including Src Tyr kinase (Edwards et al., 2003). The SH3 domain of Src recognizes the polyproline sequence, and this interaction participates in the activation of the Tyrkinase activity of Src. The Src Tyr kinase activates the MAPK cascade, which ultimately results in the phosphorylation or activation of several transcription factors, including c-fos and c-jun. Activation of the MAPK cascade rapidly induces transcription of cyclin proteins, which then cause the cell to enter into mitosis. Boonyaratanakornkit et al. (2007), using breast cancer epithelial cells, determined that nPR-B was the isoform that preferentially activated Src and MAPK, and was important for inducing expression of the cyclin D1 gene. Mutated nPR-B (mutated in the proline-X-X-proline motif) was not able to induce cell-cycle progression or expression of cyclin D1. Although these responses seem to be genomic, because they occur within a few hours via activation of kinase cascades, they are considered to be nongenomic functions of the steroid receptor because the receptor itself does not interact with the DNA. These experiments were confirmed by the research of Faivre et al. (2005), who observed an induction of phosphorylated ERK-1/-2 by treatment with progestin R5020 within 5 to 10 min, and activated MAPK was required for progestin to stimulate cyclin D1 and cdk 2 expression in MCF-7 and T47D breast cancer cells, both of which have high expression of nPR. Subsequent research by this group provided evidence for a biphasic response to P₄ treatment, with both P₄-dependent upregulation of the EGF/Wnt-1 pathway in addition to the MAPK pathway needed to induce P₄-mediated proliferation of progestin-responsive breast cancer cells (Faivre and Lange, 2007).

The PI3K pathway may be induced by an isoform of nPR to mediate oocyte maturation and germinal vesicle breakdown in frogs (Xenopus laevis). The Xenopus PR is related to the mammalian PR-B isoform and is termed XPR-1. The XPR-1 has been observed localized to the plasma membrane of oocytes, and treatment with P₄ can activate PI3K within 30 min (Bagowski et al., 2001). However, there is evidence that P₄-mediated induction of the G-protein subunits β actually induce Xenopus oocyte maturation via Gβ stimulation of adenylate cyclase (Guzmán et al., 2005). A recent study implicated classical steroid hormone receptors for both P₄ and testosterone in signaling through Gβ in Xenopus by pharmacological methods, that is, by using the specific ligands R5020 and R881 (Evaul et al., 2007). However, another study by Josefsberg et al. (2007) demonstrated that a recently discovered membrane PR β (mPRβ) ortholog, XmPRβ, mediates oocyte maturation in Xenopus. These researchers used microinjection of a neutralizing antibody to XmPRβ into the oocyte and could block the P₄-induced maturation. There was also an induction of MAPK within 10 min after transfected (XmPRβ) CHO cells were exposed to 30 nM P₄. The effect of XmPRβ on Gβ signaling was not investigated. Thus, the exact protein that mediates rapid P₄-initiated events in Xenopus oocytes remains to be determined. It may be that all of the above signaling cascades are necessary, or this may represent a redundancy in this important system.

A novel membrane-associated PR has been identified that is functionally and structurally distinct from genomic nPR: a G-protein-coupled receptor first characterized in spotted seatrout ovaries (Thomas et al., 2002). In the spotted seatrout, a progestin (17,20β,21-trihydroxy-4-pregnen-3-one) rapidly mediates the induction of oocyte maturation (Thomas et al., 2002). This action occurs at the cell surface, is not blocked by inhibiting transcription, and is induced rapidly within a few minutes of progestin administration, suggesting these actions are not mediated by an nPR. Zhu et al. (2003b) described this protein: a 352-AA protein with a molecular weight of 40 kDa with 7 transmembrane domains characteristic of a G-protein-coupled receptor. Homologous genes for this mPR were found to exist in humans, mice, and swine via a BLAST search (Zhu et al., 2003a). Similar proteins have been identified by other researchers in human myometrium (Fernandes et al., 2005) and in ovine hypothalamus, pituitary, uterus, ovary, and corpus luteum (Ashley et al., 2006). There are some conflicting data that suggest these proteins cannot mediate plasma membrane events (Ashley et al., 2006; Krietsch et al., 2006).

Novel mPR isoforms have been shown to decrease cAMP levels in Atlantic croaker (Micropogonias undulates) oocytes, and germinal vesicle breakdown (GVBD; a marker of oocyte maturation) was blocked by inhibition of the PI3K/Akt pathway (Pace and Thomas, 2005). Although MAPK activation was observed in croaker oocytes, blockage of MAPK activation did not inhibit GVBD, suggesting that signaling via PI3K/Akt is the primary mechanism by which GVBD occurs in response to P₄ in croaker oocytes.

Signaling through the ovine mPR in transfected CHO cells demonstrated rapid (within 1 min) increases in intracellular calcium (Ashley et al., 2006). When cellular localization was studied, the ovine mPR localized to the endoplasmic reticulum. Because the increase in intracellular calcium was via liberation of cellular calcium stores, the mPR could function in this manner to stimulate rapid steroid responses. It is notable, however, that some researchers claim the mPR is localized to the plasma membrane and mediates plasma membrane-associated responses (Thomas, 2003; Zhu et al., 2003b; Pace and Thomas, 2005). Other studies in transfected cells in addition to that by Ashley et al. (2006) have visualized the mPR localized to either the endoplasmic reticulum (human mPR, β, and ; Krietsch et al., 2006) or to an intracellular tubular network (human mPR isoform only; Fernandes et al., 2005). There may be species differences in the cellular localization (and signaling pathways) of these proteins. Interestingly, Krietsch et al. (2006) duplicated the materials and methods of Zhu et al. (2003b) and were unable to stimulate either cAMP production or MAPK activity with transfected human mPR and both pufferfish and spotted seatrout mPR sequences (seatrout mPR sequence was provided by Zhu et al. 2003b). Because the seatrout mPR sequence was amplified by PCR before inserting into the vector, some small mutations may have been induced that affected the protein sequence of the transfected seatrout mPR.

Membrane P₄-binding proteins have been identified in spermatozoa, bovine follicular and luteal cell membranes, and murine granulosa cells by using the c-terminal PR antibody c-262 (Peluso et al., 2001; Bramley et al., 2002; Luconi et al., 2002). There is some debate regarding whether the P₄-binding protein in spermatozoa could be nPRC (Luconi et al., 2002).

Peluso et al. (2001, 2003) later characterized the P₄-binding protein in the membranes of murine granulosa cells. Progesterone prevents these cells from entering into an apoptotic pathway. Because these cells in the rat ovary do not express nPR before the preovulatory surge of gonadotropin, it is unlikely the binding protein is nPR. Peluso et al. (2004) cloned this P₄-binding protein and found it to consist of 407 AA, similar to a homolog expressed in the mouse lung. After a domain analysis, this protein was termed plasminogen activator inhibitor mRNA binding protein-1 (PAIRBP-1) for its homology to other proteins (Peluso et al., 2006). The analysis provided no evidence of a transmembrane domain, but did identify several hyaluronic acid binding sites. Further investigation of PAIRBP-1 by Peluso et al. (2006) led to the identification of a membrane-bound partner, PR membrane component-1 (PGRMC-1), that is required for PAIRBP-1 to exert the effects mediated by P₄ on these cells. The PGRMC-1 is a relatively small protein (28 kDa) and possesses a short N-terminal extracellular domain, a single transmembrane domain, and a cytoplasmic domain that contains several potential SH2 and SH3 domains. The PGRMC-1 protein is homologous to the IL-6 receptor, a member of a growth factor receptor superfamily that includes the cytokines, growth hormone, and prolactin. Progesterone receptor membrane component-1 has been observed as a dimer (56 kDa), but can form aggregates up to 200 kDa. When spontaneously immortalized granulosa cells are treated with an antibody that blocks the N-terminal extracellular domain, treatment with P₄ will not result in a decrease in the total number of apoptotic spontaneously immortalized granulosa cells (Peluso et al., 2006).

Rapid Progestin Inhibition of the Hypothalamus
The first evidence of a nongenomic action of P₄ in the brain was reported in the 1940s by Hans Selye (1942), who observed an anesthetic effect almost immediately after administration of a bolus of P₄ to rats. Researchers in 1959 later observed depressed electroencephalogram activity in female rabbits following the postcoitis rise in P₄ (Sawyer and Kawakami, 1959).

Later research demonstrated in the hypothalamus that allopregnanolone (3,5-tetrahydroprogesterone) enhances the inhibitory effect of gamma-aminobutyric acid (GABA) on GABA type A (GABA_A) receptor-expressing neurons, possibly through P₄ secreted from neighboring glial cells (Rønnekleiv and Kelly, 2005).

Evidence suggests that P₄ is rapidly metabolized in the brain, and the metabolites (i.e., allopregnanolone) actually mediate the rapid effects of P₄ in the brain. However, there is also evidence for a direct effect of P₄ in the hypothalamus. Hamsters primed for 2 h with P₄, upon receiving a bolus injection of P₄ (or P₄ conjugated to BSA) directly into the midbrain ventral tegmental area rapidly exhibit lordosis behavior (within 10 min; DeBold and Frye, 1994). Progesterone administered concurrently with estradiol-17β in the hypothalamus/ preoptic area of the ewe can rapidly inhibit the estradiol-induced preovulatory surge of GnRH and LH (Richter et al., 2005). These actions are presumed to be mediated by the nPR, because of the observations of Skinner et al. (1998) that, upon pretreatment with RU 486 (an nPR antagonist), P₄ can no longer inhibit estradiol-induced GnRH secretion. In ovariectomized estradiol-primed ewes, removal of a P₄ implant will rapidly increase the frequency of pulsatile GnRH release (within 175 min), and these effects are seen only in estradiol-primed females. Further investigation by Richter et al. (2005) revealed that the inhibition by P₄ was most likely at the level of estradiol signaling, and not at the level of GnRH release, because treatment with P₄ after the onset of the estradiol-induced surge did not inhibit GnRH release.

Expression of OT neurons in the supraoptic nucleus and the paraventicular nucleus of the hypothalamus are under control of GABA via GABA_A receptors in these neurons (Brussaard et al., 2000). Treatment of OT neurons with GABA decreases the rate of firing, seen as an increase in the amount of time required to resensitize Cl^– channels. During late pregnancy in female rats, metabolites of P₄ have been shown to inhibit firing of OT neurons by preventing protein kinase C-induced modulation of GABA_A receptors. During parturition, GABA_A receptors are less sensitive to P₄ metabolites, and counteraction of protein kinase C no longer occurs. This is accompanied by an increase in firing activity of OT neurons at the time of parturition (Brussaard et al., 2000).

In some reports, the short time between exposure to P₄ and the detected hypothalamic response suggest that the effects of P₄ may be mediated by membrane-associated steroid binding proteins. Results of research involving the use of PR antagonists (see Skinner et al., 1998) revealed that the membrane-associated binding protein may in fact be the nPR. The true identity of the hypothalamic membrane progestin binding protein(s) remains to be determined.

Rapid Progestin Inhibition of Uterine OTR Activation
In studies of the bovine endometrium, a relatively high molar concentration of P₄ (10^–5 M) was able to inhibit the OT-stimulated release of PGF₂ from dissociated endometrial epithelial cells in the presence of actinomycin D (which blocks RNA synthesis from DNA; Bogacki et al., 2002). In these cells, P₄ blocked both the binding of OT to its receptor and OT-stimulated Ca²⁺ increase. Various steroids were then investigated (progestins and testosterone at 10^–5 M; Duras et al., 2005) for their ability to inhibit OT signaling. All steroids investigated inhibited OT-induced PGF₂ secretion and intracellular Ca²⁺ response. The observed cellular responses were attributed to a nongenomic action of steroids. But the authors did acknowledge the possibility that such responses might actually represent artificial, nonspecific effects of the steroids because of a long (4-h) incubation period and a comparatively high concentration of steroids utilized.

This premise was tested by Burger et al. (1999), who investigated the effects of progestins at a range of doses between 10 to 200 µM on human OTR transfected into various cell lines. These researchers reported that their data demonstrated that the effects of P₄ on OT binding and OTR function were nonspecific steroid effects, that is, a steroid overloading of the plasma membrane, which then interferes with the ability of the OTR to interact with signaling proteins. Wenz and Barrantes (2003), using artificial membrane bilayers, tested the effects of various steroids, including P₄, on the integrity of lipid domains. Their data indicated that the lower the hydrophobicity of the steroid (hydrophobicity determined by the group bound to carbon 17), the more lipid domain-disrupting activity the steroid displayed at higher molar concentrations. Progesterone and all naturally occurring metabolites of P₄ that possess low hydrophobicity groups attached to carbon 17 demonstrated domain-disrupting activity in the artificial lipid domains, but only at high molar concentrations (10µ M P₄).

In ovine endometrial plasma membranes, a lower, more physiological concentration of P₄ (16 nM) can specifically inhibit the binding of OT to the OTR (Figure 5; Dunlap and Stormshak, 2004). The fact that RU 486 (antagonist) can block the inhibiting effect of P₄ on binding of OT suggests the possibility that membrane-localized nPR may somehow be associated with the OTR. Inhibition of OT binding by P₄ may reflect an induced allosteric alteration in the structural conformation of the OTR monomer or dimer, which is directly or indirectly relieved or prevented by an antagonist to the nPR such as RU 486. Similarly, inhibition of OT binding by P₄ and progestins at physiological levels (below 1 µ M) of P₄ has been reported from studies of the murine and the human OTR (Grazzini et al., 1998). Specific and saturable membrane binding of progestins has been characterized in the ovine endometrium, and the binding sites for OT and progestin appear to be closely associated in this tissue (Dunlap and Stormshak, 2004). In the ewe, the inhibition of OT binding to membrane preparations by low levels of P₄ (2 to 16 nM) is dose dependent (Figure 6; Bishop and Stormshak, 2006). As stated above, there is evidence suggesting that P₄ inhibits OT binding either by competing with OT for its receptor site or by binding to a closely associated protein that alters OTR conformation. Bishop and Stormshak (2006) demonstrated that preexposure of ovine endometrial explants to P₄ (8 nM) rapidly inhibited OT-induced phosphoinositide hydrolysis (within 30 min; Figure 7) and OT-induced PGF₂ production (within 2 h). Thus, there is evidence in the ruminant endometrium for a physiologically relevant nongenomic action of P₄ to interfere with the ability of OT to signal through the OTR. The data generated from the bovine model (Bogacki et al., 2002; Duras et al., 2005) may represent an artificial, in vitro effect of steroid as suggested by Burger et al. (1999). However, the effects of lower concentrations of P₄ in the ovine endometrium (Dunlap and Stormshak, 2004; Bishop and Stormshak, 2006) may represent a more specific effect because progestins display lipid-domain effects at high molar concentrations only (Wenz and Barrantes, 2003). The ovine endometrium also displays specific and saturable binding to P₄ by the radioligand binding assay (Dunlap and Stormshak, 2004). The ovine mPR has been observed in the uterus of the ewe (Ashley et al., 2006), but whether this protein is involved in the inhibition of the OTR remains to be investigated. The fact that this protein appears to be localized to the endoplasmic reticulum makes a direct protein-protein interaction between the mPR and the OTR unlikely, but the mPR may mediate other downstream signaling events that interfere with OTR function.

View larger version (12K): [in this window] [in a new window]   Figure 5. [3H]-oxytocin ([3H]-OT) specifically bound to isolated endometrial plasma membranes after 1 h of in vitro exposure of membranes to vehicle (control), 5 ng/mL of progesterone (P4), or P4 + 5 ng/mL of P4 receptor antagonist RU 486. a,bMeans ± SEM with different letters differ (P < 0.05). Reproduced from Dunlap and Stormshak (2004).

View larger version (13K): [in this window] [in a new window]   Figure 6. Dose-response depicting effects of increasing concentrations of progesterone on specific binding of oxytocin to ovine endometrial cell membranes. The 95% confidence interval is represented by the dashed lines. Reproduced from Bishop and Stormshak (2006). Copyright 2006, The Endocrine Society.

View larger version (11K): [in this window] [in a new window]   Figure 7. Incorporation of [3H]-myoinositol into inositol-1,4,5-trisphosphate (IP3) in ovine endometrial explants after incubation in the presence of vehicle, 2.5 ng/mL progesterone (P4), 100 nM oxytocin (OT), or P4 + OT. a–cMeans ± SEM with different letters differ (P < 0.05). Reproduced from Bishop and Stormshak (2006). Copyright 2006, The Endocrine Society.

Treatment of diestrus-II rat uteri with P₄ (40 µg/mL) and metabolites of P₄ (3β-hydroxy-5β-pregnan-20-one, 5β-pregnan-3,20-dione, and 3-hydroxy-5-pregnan-20-one) will inhibit uterine contractions within 2 min (Putnam et al.,1991). The effects of P₄ only are blocked by treatment with RU 486, but a GABA_A receptor antagonist (picrotoxin) will block the inhibitory effects of the metabolites. Picrotoxin does not affect the ability of P₄ to inhibit contractions. This suggests that during gestation in the rat, P₄ metabolites may act through a GABA_A receptor system to inhibit uterine contractions (Putnam et al., 1991).

It has been reported that P₄ may bind to an mPR in bovine T lymphocytes to prevent proliferation of stimulated cells via inhibition of the subunit of the IL-2 receptor (Cannon and Pate, 2004). More recently, Ndiaye et al. (2007) demonstrated the presence of mPR, β, and and PCRMC-1 but not nPR, in T lymphocytes.

Spermatozoa respond to stimulation by P₄ and they lack a transcriptionally active nucleus. These cells lack both nPR-A and nPR-B, but P₄ treatment can stimulate calcium influx, Tyr phosphorylation of proteins, efflux of chloride, and increases in cAMP (Luconi et al., 2002). In the travels of spermatozoa to the oocyte, they do come into contact with increased concentrations of P₄ within the cumulus oophorus (1 to 10 µg/mL of P₄ has been measured in cells surrounding human oocytes; Luconi et al., 2002). Progesterone stimulates capacitation of human spermatozoa and induces the acrosome reaction in caprine spermatozoa (Somanath et al., 2000). Possible candidates for a PR have been identified by using the c-terminal PR antibody c-262, as mentioned before (Luconi et al., 2002), and the 56- to 57-kDa protein may be similar to the one identified by Peluso et al. (2001) as being present in the ovary. However, characterization of this protein has yet to be performed. There is some discussion about whether this putative PR could be another isoform of the genomic PR, the PR-C form (Luconi et al., 2002).

	BIOLOGICAL SIGNIFICANCE

Top Abstract INTRODUCTION CHARACTERISTICS OF NUCLEAR... CHANGES IN UTERINE AND... P4, ESTRADIOL, AND UTERINE... UTERINE BLOOD FLOW DURING... NONGENOMIC ACTIONS OF ESTROGEN NONGENOMIC ACTIONS OF PROGESTINS BIOLOGICAL SIGNIFICANCE LITERATURE CITED

 
Responses to the genomic action of steroids in vivo are sometimes so profound they can be observed visually or, at the very least, be readily quantified by using appropriate laboratory techniques. On the other hand, responses to the nongenomic effects of steroids in animals that can actually be measured have been few, and consequently skepticism exists regarding the biological significance of this mode of steroid action. Perhaps the best example of an in vivo nongenomic effect of a steroid is the ability of exogenous estradiol-17β to cause a rapid reduction in the secretion of LH in ovariectomized ewes, as discussed previously. Similarly, estradiol-17β has been shown to promote rapid increases in uterine blood flow at estrus in the cow and ewe. This rapid response is attributed to the ability of estradiol to stimulate eNOS in endothelial cells to produce the vasodilator NO. In vivo responses to the nongenomic effects of P₄ are less pronounced than those for estrogen. Nevertheless, experimental evidence suggests that P₄ can interfere with the binding of OT to its receptor in the uterus of the cow and ewe.

Responses to the nongenomic action of steroids may also be so subtle as to be nondetectable. As an example, there is evidence that steroids may act nongenomically to sensitize intracellular systems for the purpose of enhancing their subsequent genomic transcriptional efficiency. This possibility is supported by the data of Vasudevan et al. (2001), who subjected SK-NE-BE(2)C neuroblastoma cells transfected with human (h) ER to a 2-pulse exposure of estrogen. Cells were first exposed for 20 min to 10^–9 M estradiol-17β-BSA, followed after 4 h by exposure to a second pulse of 10^–9 M estradiol-17β. This treatment sequence significantly increased transcriptional expression of hER as compared with controls or cells having been exposed to only estradiol-17β (E₂). A reverse-pulse sequence in which cells were first exposed to E₂ and then E₂-BSA did not potentiate transcription, demonstrating that E₂-initiated membrane events are essential for later transcriptional effects. Of course, it is possible that the apparent beneficial nongenomic effect of estradiol-17β is attributed to free E₂ having been leached off the E₂-BSA, and thus entering the cell to directly initiate the transcription processes 4 h prior to exposure of cells to a larger concentration of E₂. The result of this latter scenario could be an enhanced transcription of the hER.

In conclusion, the literature is replete with a preponderance of reports describing the nongenomic actions of estrogens and progestins in transfected or cancer cells. However, although these studies have been instrumental in promoting interest in the field, there is a need for further research to document the importance of the nongenomic action of steroids to the biology of the living animal.

	Footnotes

 
² Present address: Oregon National Primate Research Center, Division of Reproductive Sciences, Oregon Health & Science University, Beaverton, OR 97006.

¹ Corresponding author: Fred.Stormshak{at}oregonstate.edu

Received for publication August 2, 2007. Accepted for publication October 24, 2007.

	LITERATURE CITED

Top Abstract INTRODUCTION CHARACTERISTICS OF NUCLEAR... CHANGES IN UTERINE AND... P4, ESTRADIOL, AND UTERINE... UTERINE BLOOD FLOW DURING... NONGENOMIC ACTIONS OF ESTROGEN NONGENOMIC ACTIONS OF PROGESTINS BIOLOGICAL SIGNIFICANCE LITERATURE CITED

 


Alexander, B. M., A. Perkins, E. A. VanKirk, G. E. Moss, and J. A. Fitzgerald. 1993. Hypothalamic and hypophyseal receptors for estradiol in high and low sexually performing rams. Horm. Behav. 27:296–307.[CrossRef][Medline]

Arreguin-Arevalo, J. A., and T. M. Nett. 2005. A nongenomic action of 17β-estradiol as the mechanism underlying the acute suppression of secretion of luteinizing hormone. Biol. Reprod. 73:115–122.[Abstract/Free Full Text]

Arreguin-Arevalo, J. A., and T. M. Nett. 2006. A nongenomic action of estradiol as the mechanism underlying the acute suppression of secretion of luteinizing hormone in ovariectomized ewes. Biol. Reprod. 74:202–208.[Abstract/Free Full Text]

Ashley, R. L., C. M. Clay, T. A. Farmerie, G. D. Niswender, and T. M. Nett. 2006. Cloning and characterization of an ovine intracellular seven transmembrane receptor for progesterone that mediates calcium mobilization. Endocrinology 147:4151–4159.[Abstract/Free Full Text]

Bagowski, C. P., J. W. Myers, and J. E. Ferrell Jr. 2001. The classical progesterone receptor associates with p42 MAPK and is involved in phosphatidylinositol 3-kinase signaling in Xenopus oocytes. J. Biol. Chem. 276:37708–37714.[Abstract/Free Full Text]

Bazer, F. W. 1992. Mediators of maternal recognition of pregnancy in mammals. Proc. Soc. Exp. Biol. Med. 199:373–384.[CrossRef][Medline]

Bishop, C. V., and F. Stormshak. 2006. Nongenomic action of progesterone inhibits oxytocin-induced phosphoinositide hydrolysis and prostaglandin F₂ secretion in the ovine endometrium. Endocrinology 147:937–942.[Abstract/Free Full Text]

Bogacki, M., W. J. Silvia, R. Rekawiecki, and J. Kotwica. 2002. Direct inhibitory effect of progesterone on oxytocin-induced secretion of prostaglandin F₂ from bovine endometrial tissue. Biol. Reprod. 67:184–188.[Abstract/Free Full Text]

Boonyaratanakornkit, V., E. McGowan, L. Sherman, M. A. Mancini, B. J. Cheskis, and D. P. Edwards. 2007. The role of extranuclear signaling actions of progesterone receptor in mediating progesterone regulation of gene expression and the cell cycle. Mol. Endocrinol. 21:359–375.[Abstract/Free Full Text]

Boonyaratanakornkit, V., M. P. Scott, V. Ribon, L. Sherman, S. M. Anderson, J. L. Maller, W. T. Miller, and D. P. Edwards. 2001. Progesterone receptor contains a proline-rich motif that directly interacts with SH3 domains and activates c-Src family tyrosine kinases. Mol. Cell 8:269–280.[CrossRef][Medline]

Bramley, T. A., G. S. Menzies, M. T. Rae, and G. Scobie. 2002. Nongenomic steroid receptors in the bovine ovary. Domest. Anim. Endocrinol. 23:3–12.[CrossRef][Medline]

Brussaard, A. B., J. Wossink, J. C. Lodder, and K. S. Kits. 2000. Progesterone-metabolite prevents protein kinase C-dependent modulation of gamma-aminobutyric acid type A receptors in oxytocin neurons. Proc. Natl. Acad. Sci. USA 97:3625–3630.[Abstract/Free Full Text]

Burger, K., F. Fahrenholz, and G. Gimpl. 1999. Nongenomic effects of progesterone on the signaling function of G protein-coupled receptors. FEBS Lett. 464:25–29.[CrossRef][Medline]

Cannon, M. J., and J. L. Pate. 2004. Presence of membrane-bound progesterone receptor mRNA in bovine peripheral blood mononuclear cells. Page 206 in Int. Vet. Immunol. Symp.

Carson-Jurica, M. A., W. T. Schrader, and B. O’Malley. 1990. Steroid receptor family: Structure and functions. Endocr. Rev. 11:201–220.[Abstract/Free Full Text]

Chen, D.-B., I. M. Bird, J. Zheng, and R. R. Magness. 2004. Membrane estrogen receptor-dependent extracellular signal-regulated kinase pathway mediates acute activation of endothelial nitric oxide synthase by estrogen in uterine artery endothelial cells. Endocrinology 145:113–125.[Abstract/Free Full Text]

DeBold, J. F., and C. A. Frye. 1994. Genomic and nongenomic actions of progesterone in the control of female hamster sexual behavior. Horm. Behav. 28:445–453.[CrossRef][Medline]

Dunlap, K. A., and F. Stormshak. 2004. Nongenomic inhibition of oxytocin binding by progesterone in the ovine uterus. Biol. Reprod. 70:65–69.[Abstract/Free Full Text]

Duras, M., J. Mlynarchzuk, and J. Kotwica. 2005. Nongenomic effect of steroids on oxytocin-stimulated intracellular mobilization of calcium and on prostaglandin F₂ and E₂ secretion from bovine endometrial cells. Prostaglandins Other Lipid Mediat. 76:105–116.[Medline]

Edwards, D. P. 2005. Regulation of signal transduction pathways by estrogen and progesterone. Annu. Rev. Physiol. 67:335–376.[CrossRef][Medline]

Edwards, D. P., S. E. Wardell, and V. Boonyaratanakornkit. 2003. Progesterone receptor interacting coregulatory proteins and cross talk with cell signaling pathways. J. Steroid Biochem. Mol. Biol. 83:173–186.

Evaul, K., M. Jamnongjit, B. Bhagavath, and S. R. Hammes. 2007. Testosterone and progesterone rapidly attenuate plasma membrane Gβ-mediated signaling in Xenopus laevis oocytes by signaling through classical steroid receptors. Mol. Endocrinol. 21:186–196.[Abstract/Free Full Text]

Faivre, E. J., and C. A. Lange. 2007. Progesterone receptors upregulate Wnt-1 to induce epidermal growth factor receptor transactivation and c-Src-dependent sustained activation of Erk1/2 mitogen-activated protein kinase in breast cancer cells. Mol. Cell. Biol. 27:466–480.[Abstract/Free Full Text]

Faivre, E., A. Skildum, L. Pierson-Mullany, and C. A. Lange. 2005. Integration of progesterone receptor mediated rapid signaling and nuclear actions in breast cancer cell models: Role of mitogen-activated protein kinases and cell cycle regulators. Steroids 70:418–426.[CrossRef][Medline]

Fernandes, M. S., V. Pierron, D. Michalovich, S. Astle, S. Thornton, H. Peltoketo, E. W. Lam, B. Gellersen, I. Huhtaniemi, J. Allen, and J. J. Brosens. 2005. Regulated expression of putative membrane progestin receptor homologues in human endometrium and gestational tissues. J. Endocrinol. 187:89–101.[Abstract/Free Full Text]

Filardo, E. J., J. A. Quinn, K. I. Bland, and A. R. Frackelton. 2000. Estrogen-induced activation of Erk-1 and Erk-2 requires the G-protein-coupled receptor homolog, GPR30, and occurs via transactivation of the epidermal growth factor receptor through release of HB-EGF. Mol. Endocrinol. 14:1649–1660.[Abstract/Free Full Text]

Filardo, E. J., J. A. Quinn, A. R. Frackelton Jr., and K. I. Bland. 2002. Estrogen action via the G protein-coupled receptor, GRP30: Stimulation of adenyl cyclase and cAMP-mediated attenuation of the epidermal growth factor-to-MAPK signaling axis. Mol. Endocrinol. 16:70–84.[Abstract/Free Full Text]

Filardo, E., J. Quinn, Y. Pang, C. Graeber, S. Shaw, J. Dong, and P. Thomas. 2007. Activation of the novel estrogen receptor G protein-coupled receptor 30 (GPR30) at the plasma membrane. Endocrinology 148:3236–3245.[CrossRef][Medline]

Fleming, J. G. W., T. E. Spencer, S. H. Safe, and F. W. Bazer. 2006. Estrogen regulates transcription of the ovine oxytocin receptor gene through GC-rich SP1 promoter elements. Endocrinology 147:899–911.[CrossRef][Medline]

Ford, S. P. 1982. Control of uterine and ovarian blood flow throughout the estrous cycle and pregnancy of ewes, sows, and cows. J. Anim. Sci. 55(Suppl. 2):32–42.[Medline]

Ford, S. P., J. R. Chenault, R. K. Christenson, S. E. Echternkamp, and J. J. Ford. 1981. Effects of preimplantation bovine and porcine conceptuses on blood flow and steroid content of the uterus. Pages 436–438 in Cellular and Molecular Aspects of Implantation. S. R. Glasser and D. W. Bullock, ed. Plenum Press, New York, NY.

Ford, S. P., J. R. Chenault, and S. E. Echternkamp. 1979. Uterine blood flow of cows during the oestrous cycle and early pregnancy: Effect of the conceptus on the uterine blood supply. J. Reprod. Fertil. 56:53–62.[Abstract/Free Full Text]

Ford, S. P., and R. K. Christenson. 1979. Blood flow to uteri of sows during the estrous cycle and early pregnancy: Local effect of the conceptus on the uterine blood supply. Biol. Reprod. 21:617–624.[Abstract]

Ford, S. P., R. K. Christenson, and J. J. Ford. 1982. Uterine blood flow and uterine arterial, venous and luminal concentration of oestrogens on days 11, 13 and 15 after oestrus in pregnant and nonpregnant sows. J. Reprod. Fertil. 64:185–190.[Abstract/Free Full Text]

Ford, S. P., and L. P. Reynolds. 1983. Role of adrenergic receptors in mediating estradiol-17β-stimulated increases in uterine blood flow of cows. J. Anim. Sci. 57:665–672.[Abstract/Free Full Text]

Ford, S. P., L. P. Reynolds, D. B. Farley, R. K. Bhatnagar, and D. E. Van Orden. 1984. Interaction of ovarian steroids and periarterial alpha 1-adrenergic receptors in altering uterine blood flow during the estrous cycle of gilts. Am. J. Obstet. Gynecol. 150:480–484.[Medline]

Ford, S. P., L. J. Weber, and F. Stormshak. 1976. In vitro response of ovine and bovine uterine arteries to prostaglandin F₂ and periarterial sympathetic nerve stimulation. Biol. Reprod. 15:58–65.[Abstract]

Fuchs, A. R., O. Behrens, H. Helmer, C.-H. Liu, C. M. Barros, and M. J. Fields. 1990. Oxytocin and vasopressin receptors in bovine endometrium and myometrium during the estrous cycle and early pregnancy. Endocrinology 127:629–636.[Abstract/Free Full Text]

Glass, J. D., R. P. Amann, and T. M. Nett. 1984. Effects of season and sex on the distribution of cytosolic estrogen receptors within the brain and the anterior pituitary gland of sheep. Biol. Reprod. 30:894–902.[Abstract]

Grazzini, E., G. Guillon, B. Mouillac, and H. H. Zingg. 1998. Inhibition of oxytocin receptor function by direct binding of progesterone. Nature 392:509–512.[CrossRef][Medline]

Greiss, F. C., Jr., and S. G. Anderson. 1969. Uterine vascular changes during the ovarian cycle. Am. J. Obstet. Gynecol. 103:629–640.[Medline]

Greiss, F. C., Jr., and J. R. Pick Jr. 1964. The uterine vascular bed adrenergic receptors. Obstet. Gynecol. 23:209–213.[Medline]

Guzmán, L., X. Romo, R. Grandy, X. Soto, M. Montecino, M. Hinrichs, and J. Olate. 2005. A Gβ stimulated adenylyl cyclase is involved in Xenopus laevis oocyte maturation. J. Cell. Physiol. 202:223–229.[CrossRef][Medline]

Hall, J. M., and D. P. McDonnell. 1999. The estrogen receptor β-isoform (ERβ) of the human estrogen receptor modulates ER transcriptional activity and is a key regulator of the cellular response to estrogens and antiestrogens. Endocrinology 140:5566–5578.[Abstract/Free Full Text]

Heldring, N., A. Pike, S. Anderson, J. Matthews, G. Cheng, J. Hartman, M. Tujague, A. Ström, E. Treuter, M. Warner, and J.-A. Gustafsson. 2007. Estrogen receptors: How do they signal and what are their targets?Physiol. Rev. 87:905–931.[Abstract/Free Full Text]

Herbison, A. E. 1995. Neurochemical identity of neurones expressing oestrogen and androgen receptors in sheep hypothalamus. J. Reprod. Fertil. Suppl. 49:271–283.[Medline]

Herbison, A. E., J. E. Robinson, and D. C. Skinner. 1993. Distribution of oestrogen receptor-immunoreactive cells in the preoptic area of the ewe: Co-localization with glutamic acid decarboxylase but not luteinizing hormone-releasing hormone. Neuroendocrinology 57:751–759.[Medline]

Jenner, L. J., T. J. Parkinson, and G. E. Lamming. 1991. Uterine oxytocin receptor in cyclic and pregnant cows. J. Reprod. Fertil. 105:165–175.[CrossRef]

Josefsberg, B.-Y. L., A. L. Lewellyn, P. Thomas, and J. L. Maller. 2007. The role of Xenopus membrane progesterone receptor β in mediating the effect of progesterone on oocyte maturation. Mol. Endocrinol. 21:664–673.[Abstract/Free Full Text]

Killam, A. P., R. Rosenfeld, F. C. Battaglia, E. L. Makowski, and G. Meschia. 1973. Effect of estrogens on the uterine blood flow of oophorectomized ewes. Am. J. Obstet. Gynecol. 115:1045–1052.[Medline]

Koligian, K. B., and F. Stormshak. 1977a. Nuclear and cytoplasmic estrogen receptors in ovine endometrium during the estrous cycle. Endocrinology 101:524–533.[Abstract/Free Full Text]

Koligian, K. B., and F. Stormshak. 1977b. Progesterone inhibition of estrogen receptor replenishment in ovine endometrium. Biol. Reprod. 17:412–416.[Abstract]

Krietsch, T., M. S. Fernandes, J. Kero, R. Losel, M. Heyens, E. W. Lam, I. Huhtaniemi, J. J. Brosens, and B. Gellersen. 2006. Human homologs of the putative G protein-coupled membrane progestin receptors (mPR, β, and ) localize to the endoplasmic reticulum and are not activated by progesterone. Mol. Endocrinol. 20:3146–3164.[Abstract/Free Full Text]

Kumar, R., and E. Thompson. 2003. Transactivation functions of the N-terminal domains of nuclear hormone receptors: Protein folding and coactivator interactions. Mol. Endocrinol. 17:1–10.[Abstract/Free Full Text]

Lange, C. A., D. Gioeli, S. R. Hammes, and P. C. Marker. 2007. Integration of rapid signaling events with steroid hormone receptor action in breast and prostate cancer. Annu. Rev. Physiol. 69:171–199.[CrossRef][Medline]

Lehman, M. N., F. J. P. Ebling, S. M. Moenter, and F. J. Karsch. 1993. Distribution of estrogen receptor-immunoreactive cells in the sheep brain. Endocrinology 133:876–886.[Abstract/Free Full Text]

Leung, S. T., and D. C. Wathes. 2000. Oestradiol regulation of oxytocin receptor expression in cyclic bovine endometrium. J. Reprod. Fertil. 19:287–292.

Li, X., and B. W. O’Malley. 2003. Unfolding the action of progesterone receptors. J. Biol. Chem. 278:39261–39264.[Free Full Text]

Lindner, H. R., M. B. Sass, and B. Morris. 1964. Steroids in the ovarian lymph and blood of conscious ewes. J. Endocrinol. 30:361–376.[Medline]

Luconi, M., L. Bonaccorsi, L. Bini, S. Liberatori, V. Pallini, G. Forti, and E. Baldi. 2002. Characterization of membrane nongenomic receptors for progesterone in human spermatozoa. Steroids 67:505–509.[CrossRef][Medline]

McCracken, J. A., E. E. Custer, and J. C. Lamsa. 1999. Luteolysis: A neuroendocrine-mediated event. Physiol. Rev. 79:263–323.[Abstract/Free Full Text]

McKenna, N. J., R. B. Lanz, and B. W. O’Malley. 1999. Nuclear receptor coregulators: Cellular and molecular biology. Endocr. Rev. 20:321–344.[Abstract/Free Full Text]

Menzies, G. S., K. Howland, M. T. Rae, and T. A. Bramley. 1999. Stimulation of specific binding of [³H]-progesterone to bovine luteal cell-surface membranes: Specificity of digitonin. Mol. Cell. Endocrinol. 153:57–69.[CrossRef][Medline]

Miller, B. G., L. Murphy, and G. M. Stone. 1977. Hormone receptor levels and hormone, RNA and protein metabolism in the genital tract during the oestrous cycle of the ewe. J. Endocrinol. 73:91–98.[Abstract/Free Full Text]

Morley, P., J. F. Whitfield, B. C. Vanderhyden, B. K. Tsang, and J. L. Schwartz. 1992. A new nongenomic estrogen action: The rapid release of intracellular calcium. Endocrinology 131:1305–1312.[Abstract/Free Full Text]

Morris, B., and M. B. Sass. 1966. The formation of lymph in the ovary. Proc. Soc. Exp. Biol. Med. 164:577–591.

Mulac-Jericevic, B., J. P. Lydon, F. J. DeMayo, and O. M. Conneely. 2003. Defective mammary gland morphogenesis in mice lacking the progesterone receptor B isoform. Proc. Natl. Acad. Sci. USA 100:9744–9749.[Abstract/Free Full Text]

Mulac-Jericevic, B., R. A. Mullinax, F. J. DeMayo, J. P. Lydon, and O. M. Conneely. 2000. Subgroup of reproductive functions of progesterone mediated by progesterone receptor-B isoform. Science 289:1751–1754.[Abstract/Free Full Text]

Ndiaye, K., D. Poole, and J. Pate. 2007. Expression of mRNA for membrane progesterone receptors by bovine T lymphocytes. Biol. Reprod. (Special Issue):123.

Nett, T. M., D. Cermak, T. Braden, J. Manns, and G. D. Niswender. 1987. Receptors for GnRH and estradiol and pituitary content of gonadotropins in beef cows. I. Changes during the estrous cycle. Dom. Anim. Endocrinol. 4:123–132.[CrossRef][Medline]

Nett, T. M., D. Cermak, T. Braden, J. Manns, and G. D. Niswender. 1988. Pituitary receptors for GnRH and estradiol, and pituitary content of gonadotropins in beef cows. II. Changes during the postpartum period. Dom. Anim. Endocrinol. 5:81–89.[CrossRef][Medline]

Nett, T. M., M. E. Crowder, and M. E. Wise. 1984. Role of estradiol in inducing an ovulatory-like surge of luteinizing hormone in sheep. Biol. Reprod. 30:1208–1215.[Abstract]

Nilson, J. H., M. T. Nejedlik, J. B. Virgin, M. E. Crowder, and T. M. Nett. 1983. Expression of subunit and luteinizing hormone β genes in the ovine anterior pituitary. J. Biol. Chem. 258:12087–12090.[Abstract/Free Full Text]

Pace, M. C., and P. Thomas. 2005. Steroid-induced oocyte maturation in Atlantic croaker (Micropogonias undulatus) is dependent on activation of the phosphatidylinositol 3-kinase/Akt signal transduction pathway. Biol. Reprod. 73:988–996.[Abstract/Free Full Text]

Pedram, A., M. Razandi, R. C. A. Sainson, J. K. Kim, C. C. Huges, and E. R. Levin. 2007. A conserved mechanism for steroid receptor translocation to the plasma membrane. J. Biol. Chem. 282:22278–22288.[Abstract/Free Full Text]

Pelletier, J., and A. Caraty. 1981. Characterization of cytosolic 5-DHT and 17β-estradiol receptors in the ram hypothalamus. J. Steroid Biochem. 14:603–611.[CrossRef][Medline]

Peluso, J. J., T. Bremner, G. Fernandez, A. Pappalardo, and B. A. White. 2003. Expression pattern and role of a 60-kilodalton progesterone binding protein in regulating granulosa cell apoptosis: Involvement of the mitogen-activated protein kinase cascade. Biol. Reprod. 68:122–128.[Abstract/Free Full Text]

Peluso, J. J., G. Fernandez, A. Pappalardo, and B. A. White. 2001. Characterization of a putative membrane receptor for progesterone in rat granulosa cells. Biol. Reprod. 65:94–101.[Abstract/Free Full Text]

Peluso, J. J., A. Pappalardo, G. Fernandez, and C. A. Wu. 2004. Involvement of an unnamed protein, RDA288, in the mechanism through which progesterone mediates its antiapoptotic action in spontaneously immortalized granulosa cells. Endocrinology 145:3014–3022.[Abstract/Free Full Text]

Peluso, J. J., A. Pappalardo, R. Losel, and M. Wehling. 2006. Progesterone membrane receptor component 1 expression in the immature rat ovary and its role in mediating progesterone’s antiapoptotic action. Endocrinology 147:3133–3140.[Abstract/Free Full Text]

Pietras, R. J., and C. M. Szego. 1975. Endometrial cell calcium and oestrogen action. Nature 253:357–359.[CrossRef][Medline]

Pietras, R. J., and C. M. Szego. 1977. Specific binding sites for oestrogen at the outer surfaces of isolated endometrial cells. Nature 265:69–72.[CrossRef][Medline]

Pope, W. F., R. R. Maurer, and F. Stormshak. 1982. Distribution of progesterone in the uterus, broad ligament, and uterine arteries of beef cows. Anat. Rec. 203:245–250.[CrossRef][Medline]

Putnam, C. D., D. W. Brann, R. C. Kolbeck, and V. B. Mahesh. 1991. Inhibition of uterine contractility by progesterone and progesterone metabolites: Mediation by progesterone and gamma aminobutyric acid_A receptor systems. Biol. Reprod. 45:266–272.[Abstract]

Rae, M. T., G. S. Menzies, and T. A. Bramley. 1998a. Bovine ovarian nongenomic progesterone binding sites: Presence in follicular and luteal cell membranes. J. Endocrinol. 159:413–427.[Abstract]

Rae, M. T., G. S. Menzies, A. S. McNeilly, K. Woad, R. Webb, and T. A. Bramley. 1998b. Specific nongenomic, membrane-localized binding sites for progesterone in the bovine corpus luteum. Biol. Reprod. 58:1394–1406.[Abstract/Free Full Text]

Razandi, M., A. Pedram, G. L. Greene, and E. R. Levin. 1999. Cell membrane and nuclear estrogen receptors (ERs) originate from a single transcript: Studies of ER and ERβ expressed in Chinese hamster ovary cells. Mol. Endocrinol. 13:309–319.

Revankar, C. M., D. F. Cimino, L. A. Sklar, J. B. Arterburn, and E. R. Prossnitz. 2005. A transmembrane intracellular estrogen receptor mediates rapid cell signaling. Science 307:1625–1630.[Abstract/Free Full Text]

Richter, T. A., J. E. Robinson, J. M. Lozano, and N. P. Evans. 2005. Progesterone can block the preovulatory gonadotropin-releasing hormone/luteinising hormone surge in the ewe by a direct inhibitory action on oestradiol responsive cells within the hypothalamus. J. Neuroendocrinol. 17:161–169.[CrossRef][Medline]

Robinson, R. S., G. E. Mann, G. E. Lamming, and D. C. Wathes. 2001. Expression of oxytocin, oestrogen and progesterone receptors in uterine biopsy samples throughout the oestrous cycle and early pregnancy in cows. Reproduction 122:965–979.[Abstract]

Rønnekleiv, O. K., and M. J. Kelly. 2005. Diversity of ovarian steroid signaling in the hypothalamus. Front. Neuroendocrinol. 26:65–84.[CrossRef][Medline]

Rueda, B. R., I. R. Hendry, W. J. Hendry III, F. Stormshak, O. D. Slayden, and J. S. Davis. 2000. Decreased progesterone levels and progesterone receptor antagonists promote apoptotic cell death in bovine luteal cells. Biol. Reprod. 62:269–276.[Abstract/Free Full Text]

Sawyer, C. H., and M. Kawakami. 1959. Characteristics of behavioral and electroencephalographic after-reactions to copulation and vaginal stimulation in the female rabbit. Endocrinology 65:622–630.[Medline]

Selye, H. 1942. Correlation between the chemical structure and the pharmacological actions of steroids. Endocrinology 30:437–453.[Abstract/Free Full Text]

Senior, B. E. 1975. Cytoplasmic oestradiol-binding sites and their relationship to oestradiol content in the endometrium of cattle. J. Reprod. Fertil. 44:501–511.[Abstract/Free Full Text]

Sheldrick, E. L., and A. P. F. Flint. 1985. Endocrine control of uterine oxytocin receptors in the ewe. J. Endocrinol. 106:249–258.[Abstract/Free Full Text]

Shupnik, M. A., L. A. Baxter, L. R. French, and J. Gorski. 1979. In vivo effects of estrogen on ovine pituitaries: Prolactin and growth hormone biosynthesis and messenger ribonucleic acid translation. Endocrinology 104:729–735.[Abstract/Free Full Text]

Simoncini, T., A. Hafezi-Moghadam, D. P. Brazil, K. Lay, W. W. Chin, and J. K. Liao. 2000. Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase. Nature 407:538–541.[CrossRef][Medline]

Skinner, D. C., N. P. Evans, B. Delaleu, R. L. Goodman, P. Bouchard, and A. Caraty. 1998. The negative feedback actions of progesterone on gonadotropin releasing hormone secretion are transduced by the classical progesterone receptor. Proc. Natl. Acad. Sci. USA 95:10978–10983.[Abstract/Free Full Text]

Smith, C. L., and B. W. O’Malley. 2004. Coregular function: A key to understanding tissue specificity of selective receptor modulators. Endocr. Rev. 25:45–71.[Abstract/Free Full Text]

Somanath, P. R., K. Suraj, and K. K. Gandhi. 2000. Caprine sperm acrosome reaction: Promotion by progesterone and homologous zona pellucida. Small Rumin. Res. 37:279–286.[CrossRef][Medline]

Song, R. X., C. J. Barnes, Z. Zhang, Y. Bao, R. Kumar, and R. J. Santen. 2004. The role of Shc and insulin-like growth factor 1 receptor in mediating the translocation of estrogen receptor to the plasma membrane. Proc. Natl. Acad. Sci. USA 101:2075–2081.

Song, R. X.-D., R. A. McPherson, L. Adam, Y. Bao, M. Shupnik, R. Kumar, and R. J. Santen. 2002. Linkage of rapid estrogen action to MAPK activation by ER-Shc association and Shc pathway activation. Mol. Endocrinol. 16:116–127.[Abstract/Free Full Text]

Spencer, T. E., and F. W. Bazer. 1995. Temporal and spatial alterations in uterine estrogen receptor and progesterone receptor gene expression during the estrous cycle and early pregnancy in the ewe. Biol. Reprod. 53:1527–1543.[Abstract]

Spencer, T. E., and F. W. Bazer. 1996. Ovine interferon tau suppresses transcription of the estrogen receptor and oxytocin receptor genes in the ovine endometrium. Endocrinology 137:1144–1147.[Abstract]

Spencer, T. E., W. C. Becker, P. George, M. A. Mirando, T. F. Ogle, and F. W. Bazer. 1995. Ovine interferon-tau regulates expression of endometrial receptors for estrogen and oxytocin but not progesterone. Biol. Reprod. 53:732–745.[Abstract]

Telgmann, R., R. A. D. Bathgate, S. Jaeger, G. Tillmann, and R. Ivell. 2003. Transcriptional regulation of the bovine oxytocin receptor gene. Biol. Reprod. 68:1015–1026.[Abstract/Free Full Text]

Thomas, P. 2003. Rapid, nongenomic steroid actions initiated at the cell surface: Lesions from studies with fish. Fish Physiol. Biochem. 28:3–12.[CrossRef]

Thomas, P., Y. Zhu, and M. Pace. 2002. Progestin membrane receptors involved in the meiotic maturation of teleost oocytes: A review with some new findings. Steroids 67:511–517.[CrossRef][Medline]

Tsai, M.-J., and B. O’Malley. 1994. Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu. Rev. Biochem. 63:451–486.[CrossRef][Medline]

Turzillo, A. M., and T. M. Nett. 1999. Regulation of GnRH receptor gene expression in sheep and cattle. J. Reprod. Fertil. Suppl. 54:75–86.[Medline]

Vallejo, G., C. Ballare, J. L. Baranao, M. Beato, and P. Saragueta. 2005. Progestin activation of nongenomic pathways via cross talk of progesterone receptor with estrogen receptor-β induces proliferation of endometrial stromal cells. Mol. Endocrinol. 19:3023–3037.[Abstract/Free Full Text]

Vallet, J. L., G. E. Lamming, and M. Batten. 1990. Control of endometrial oxytocin receptor and uterine response to oxytocin by progesterone and oestradiol in the ewe. J. Reprod. Fertil. 90:625–634.[Abstract/Free Full Text]

Vasudevan, N., L. M. Kow, and D. W. Pfaff. 2001. Early membrane estrogenic effects required for full expression of slower, genomic actions in nerve cell line. Proc. Natl. Acad. Sci. USA 98:12267–12271.[Abstract/Free Full Text]

Vician, L., M. A. Shupnik, and J. Gorski. 1979. Effects of estrogen on primary ovine pituitary cell cultures: Stimulation of prolactin secretion, synthesis, and preprolactin messenger ribonucleic acid activity. Endocrinology 104:736–743.[Abstract/Free Full Text]

Wallace, J. M., R. Helliwell, and P. J. Morgan. 1991. Autoradiographical localization of oxytocin binding sites on ovine oviduct and uterus throughout the oestrous cycle. Reprod. Fertil. Dev. 3:127–135.[CrossRef][Medline]

Weems, C. W., Y. S. Weems, C. N. Lee, and D. L. Vincent. 1989. Progesterone in uterine and arterial tissue and in jugular and uterine venous plasma of sheep. Biol. Reprod. 41:1–6.[Abstract]

Wenz, J. J., and F. J. Barrantes. 2003. Steroid structural requirements for stabilizing or disrupting lipid domains. Biochemistry 42:14267–14276.[CrossRef][Medline]

Wise, M. E., J. D. Glass, and T. M. Nett. 1986. Changes in the concentration of hypothalamic and hypophyseal receptors for estradiol in pregnant and postpartum ewes. J. Anim. Sci. 62:1021–1028.[Abstract/Free Full Text]

Zelinski, M. B., P. Noel, D. W. Weber, and F. Stormshak. 1982. Characterization of cytoplasmic progesterone receptors in the bovine endometrium during proestrus and diestrus. J. Anim. Sci. 55:376–383.[Abstract/Free Full Text]

Zhu, Y., J. Bond, and P. Thomas. 2003a. Identification, classification, and partial characterization of genes in humans and other vertebrates homologous to a fish membrane progesterone receptor. Proc. Natl. Acad. Sci. USA 100:2237–2242.[Abstract/Free Full Text]

Zhu, Y., C. D. Rice, Y. Pang, M. Pace, and P. Thomas. 2003b. Cloning, expression, and characterization of a membrane progestin receptor and evidence it is an intermediary in meiotic maturation of fish oocytes. Proc. Natl. Acad. Sci. USA 100:2231–2236.[Abstract/Free Full Text]

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