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Regular Article |
c Departments of Physiology and
d Obstetrics and Gynecology, University of Connecticut Health Center, Farmington, Connecticut 06030
ABSTRACT
Progesterone (P4) inhibits granulosa cell apoptosis in a steroid-specific, dose-dependent manner, but these cells do not express the classic nuclear P4 receptor. It has been proposed that P4 mediates its action through a 60-kDa protein that functions as a membrane receptor. The present studies were designed to determine the P4 binding characteristics of this protein. Western blot analysis using an antibody that recognizes the P4 binding site of the nuclear P4 receptor (C-262) confirmed that the 60-kDa protein was localized to the plasma membrane of both granulosa cells and spontaneously immortalized granulosa cells (SIGCs). To determine whether this protein binds P4, proteins were immunoprecipitated with the C-262 antibody, electrophoresed, transferred to nitrocellulose, and probed with a horseradish peroxidase-labeled P4 in the presence or absence of nonlabeled P4. This study demonstrated that the 60-kDa protein specifically binds P4. Scatchard plot analysis revealed that 3H-P4 binds to a single site (i.e., single protein), which is relatively abundant (200 pmol/mg) with a Kd of 360 nM. 3H-P4 binding was not reduced by dexamethasone, mifepristone (RU 486), or onapristone (ZK98299). Further studies with SIGCs showed that P4 inhibited apoptosis and mitogen-activated protein kinase kinase (MEK) activity, and maintained calcium homeostasis. These studies taken together support the concept that the 60-kDa P4 binding protein functions as a low-affinity, high-capacity membrane receptor for P4.
apoptosis, calcium, granulosa cells, progesterone receptor, signal transduction
INTRODUCTION
Ovarian follicles become atretic as a result of the granulosa cells undergoing apoptosis [1, 2]. Because the control of granulosa cell apoptosis is critical to the normal functioning of the ovary, it is regulated by many redundant and interacting pathways. The factors that influence granulosa cell viability either activate or inhibit these ill-defined pathways. Proapoptotic pathways can be activated by Fas ligand, tumor necrosis factor 
, interleukin-6, and GnRH-like peptides [3]. In contrast, a numerous and diverse group of growth factors and hormones inhibits granulosa cell apoptosis [4]. This group includes insulin-like growth factor-1, growth hormone, epidermal growth factor, hepatocyte growth factor, basic fibroblast growth factor, estrogen, and progesterone (P4) [4, 5].
Although P4 inhibits granulosa cell apoptosis, its mechanism of action is not clear. Previous studies have shown that the classic nuclear P4 receptor is only expressed between 4 and 6 h after the preovulatory LH surge [6]. That ligand activation of the classic nuclear P4 receptor mediates such LH-regulated events as prostaglandin and plasminogen-activator synthesis, and type-I pituitary adenylate cyclase activating polypeptide receptor gene expression; and ovulation has been confirmed using specific P4 receptor antagonists [7, 8]. Further, P4 receptor antagonists increase the susceptibility of LH-exposed preovulatory granulosa cells to undergo apoptosis [9, 10].
Although the role of P4 in regulating preovulatory events is clear, P4 has also been shown to inhibit apoptosis of granulosa cells isolated from immature rats [11]. These studies have shown that the action of P4 is dose-dependent and steroid-specific [11]. Whereas the antiapoptotic action of P4 is well documented, it is not likely that P4 mediates its action through the classic nuclear P4 receptor. This point is illustrated by the observation that granulosa cells of immature rats do not express the A, B, or C isoforms of the nuclear P4 receptor [6, 12–14]. This is consistent with the fact that a 100-fold molar excess of mifepristone (RU 486) is required to attenuate the action of P4 [15] when a equal molar treatment with RU 486 is sufficient to block actions of P4 that are transduced through the nuclear P4 receptor [16–18].
These observations raise a question about the nature of the receptor involved in transducing the actions of P4 in rat granulosa cells. There appear to be at least two possibilities. First, the ligand binding studies conducted in the early 1980s demonstrated that granulosa cells express a cytosolic protein that binds the synthetic P4, R5020 [19–22]. 3H-R5020 binding was not only displaced by P4, but also RU 486 and dexamethasone [20, 21]. Thus, this cytosolic protein could function as a P4 receptor. Alternatively, a 60-kDa protein has been detected within the granulosa cell membrane using an antibody directed against the P4 binding domain of the nuclear P4 receptor [23]. This protein is also expressed by spontaneously immortalized granulosa cells (SIGCs) and has been proposed to function as a membrane receptor for P4 [23]. The present studies were designed to further characterize this 60-kDa membrane protein. Specifically, ligand blot and ligand binding studies were conducted to determine P4 binding characteristics. Additional studies assessed the ability of P4 to regulate cell viability, mitogen-activated protein kinase kinase (MEK) activity, and intracellular free calcium ([Ca2+]i).
MATERIALS AND METHODS
Animals and Reagents
Immature female Wistar rats (22 days of age) were obtained from Charles River Laboratory (Wilmington, MA) and housed under controlled conditions of temperature, humidity, and photoperiod (12L:12D, lights-on at 0700 h). On the day of the experiment, immature animals were 23 or 24 days of age. The rats were killed by cervical dislocation between 0930 and 1000 h and the ovaries were removed. In some experiments granulosa cells were isolated from eCG-primed immature rats [15]. This protocol was approved by the Animal Care Committee of the University of Connecticut Health Center.
All reagents were purchased from Sigma Chemical Company (St. Louis, MO) unless otherwise stated. The antibodies to the P4 (C-262) and IP3 receptor (Ab-R3) were purchased from StressGen (Victoria, BC, Canada) and Transduction Laboratories (Lexington, KY), respectively. PD 98059 (Calbiochem, San Diego, CA) was used at a final concentration of 50 µM. Mifepristone (RU 486) and onapristone (ZK98299) were gifts from Dr. Gary Hodgen (The Jones Institute, Norfolk, VA) and Dr. Klaus Stockemann (Schering AG, Berlin, Germany).
SIGC Culture
Spontaneously immortalized granulosa cells were generously provided by Dr. Robert Burghardt of Texas A&M University (College Station, TX) and cultured as previously described [24]. SIGCs were routinely maintained in Falcon T-flasks (Becton Dickinson Labware, Lincoln Park, NJ). For experiments involving Western blots, SIGCs were plated in 100-mm glass (Kimax) culture dishes at a density of 4 x 105 cells/ml in 5 ml of medium. In experiments in which apoptosis was assessed by YOPRO-1 staining, SIGCs were plated in 0.4 ml of medium at 1.25 x 105 cells/ml in 8-chamber glass Lab-Tek slides (Nunc Inc., Naperville, IL). Regardless of the culture vessel, the cells were initially cultured in DMEM/F-12 supplemented with 5% fetal bovine serum for 24 h. The serum-supplemented medium was removed and the cells were cultured in serum-free DMEM/F-12 for up to 5 additional hours.
Western Blot Analysis
Preparation of cell lysates Ovarian lysate was prepared as previously described [23]. SIGC lysate was prepared by collecting the cells by centrifugation and discarding the supernatant. One milliliter of boiling lysate buffer (125 nM Tris pH 6.8; 4% SDS, 10% glycerol, 0.006% bromphenol blue, and 2% mercaptoethanol) was added to each cell preparation. The cells were then passed through a 26-gauge needle several times to reduce viscosity. The samples were then boiled for 5 min and subsequently centrifuged for 5 min at 13 000 x g at 4°C to remove insoluble material. For each experiment a parallel culture was extracted with a boiling lysate buffer that contained 1% SDS, 1.0 mM sodium ortho-vanadate, and 10 mM Tris (pH 7.4). Protein determinations were made on the samples from the parallel cultures using the BCA method (Pierce, Rockford, IL).
Isolation of cytoplasmic and membrane fractions Ovarian cytoplasmic and membrane fractions were prepared as previously described [23]. Crude membrane fractions from SIGCs were isolated by centrifuging the lysate at 15 000 x g for 20 min at 4°C. The pellet was discarded and the supernatant centrifuged at 100 000 x g for 90 min at 4°C. The cytosol was in the supernatant, whereas a crude membrane preparation remained in the pellet. Both fractions were stored at -20°C. Ten micrograms of each preparation was loaded onto a gel and subsequently used in a Western blot analysis.
Biotinylation of membrane-associated proteins To determine whether the 60-kDa protein was associated with plasma membrane, the membrane proteins of freshly isolated granulosa cells from eCG-primed immature rats [15] and SIGCs were biotinylated using the EZ-link sulfo-NHS-LC-LC-Biotin reagent and protocol provided by Pierce [25]. Once these proteins were biotinylated, cell lysates were prepared. A sample of this lysate was assessed for the total amount of 60 kDa by Western blot. To isolate the plasma membrane proteins, the remainder of the sample was affinity-purified using Ultralink Immobilized streptavidin and the protocol provided by Pierce. Twenty micrograms of the affinity-purified sample was then run on a 10% acrylamide gel and the amount of 60 kDa that was present at the plasma membrane was determined by Western blot.
Ligand Blot Analysis
To determine whether the 60-kDa protein bound P4, a ligand blot assay was used as described by Luconi et al. [26]. Briefly, 100–150 µg of cell lysate was mixed with 400 µl of distilled water, 500 µl of 2x immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium vanadate, 0.2 mM PMSF, and 0.5% NP-40) and 10 µg of C-262 antibody. As a control, C-262 antibody was replaced with 10 µg of IP3 receptor antibody (Ab-R3). The mixture was vortexed and placed on ice for 1 h. Rabbit anti-mouse immunoglobulin G (IgG; 5 µg) was added and the mixture vortexed. The incubation was continued for an additional 30 min. Finally, 50 µl of a solution of 10% Protein A sepharose in 0.1 M phosphate buffer (pH 7.4) was added and the mixture vortexed and incubated for 30 min in an ice bath with agitation. This mixture was then centrifuged at 13 000 x g for 5 min and washed three times with 1x immunoprecipitation buffer. The pellet was resuspended in 20 µl of loading buffer, boiled for 5 min, centrifuged, and loaded onto a 10% polyacrylamide gel. The proteins were then transferred to nitrocellulose and probed with the horseradish peroxidase-conjugated P4 (HP-P4; 0.5 µM) in the presence (10 µM) or absence of P4. The blot was then rinsed and processed as described for the Western blot procedure.
Ligand Binding Studies
SIGCs were plated at 3.6 x 105 cells/35-mm culture dish and cultured overnight in serum-supplemented medium. The cells were washed twice in PBS and then incubated at 4°C in 500 µl of 0.1% digitonin in TEMGD buffer as described by Ambhaikar and Puri [27]. After 30 min, 1,2,6,7-3H-progesterone (1 nM 3H-P4, 50 000 cpm, SA = 86 Ci/mmol; Amersham, Arlington Heights, IL) was added and the incubation continued for an additional 60 min. The cells were then washed several times, harvested, and filtered through Whatman Glass Microfiber filters (GF/F) (Fisher Scientific Inc., Pittsburgh, PA). The filters were rinsed twice with 1 ml cold PBS and then filter-counted in a scintillation counter. Dissociation constant (Kd) and Bmax were calculated using the Ligand program provided by the National Institutes of Health. Displacement studies were also conducted in which the effect of increasing concentrations of dexamethasone, mifepristone (RU 486), or onapristone (ZK98299) on 3H-P4 binding was determined. For each experiment, each concentration was tested in duplicate with the entire experiment repeated at least three times.
Identification of Apoptotic Cells
Apoptosis was assessed by in situ staining using the impermeant nuclear dye, YOPRO-1 [28, 29]. To stain apoptotic cells, YOPRO-1 was added directly into each culture chamber at a final concentration of 10 µM. The cells were incubated for 10 min at 37°C and then observed at a magnification of 200x under fluorescent optics using the FITC filter set. The number of fluorescent cells (i.e., apoptotic cells) in a field was counted. The total number of cells in that field was counted under phase optics. A total of 100–200 cells per well were counted. The percentage of apoptotic cells was then calculated.
Assessment of MEK 1/2 Activity
After treatment, cell lysates were prepared as previously described. Protein determinations were made using the BCA method (Pierce). Typically, 10 µg of lysate was loaded onto each lane and the sample electrophoresed on a 10% polyacrylamide gel at 100 V. Proteins were then transferred to nitrocellulose and incubated with 5% nonfat milk (Nestlé Food Company, Glendale, CA) in Tris-buffered saline with 0.1% Tween-20 (TBS-T) for 1 h with agitation at room temperature. The level and activity MEK 1/2; was assessed using reagents and protocols provided by Cell Signaling Technology (Beverly, MA). Briefly, the total amount of MEK was detected using a monoclonal antibody, whereas the amount of activated MEK was determined using a monoclonal antibody that selectively recognizes active MEK (i.e., phosphorylated MEK at Ser217/221). The antibodies used in these studies detected both MEK 1 and 2.
[Ca2+]i Measurements
SIGCs were plated in serum-supplemented medium on round cover glass for 24 h and then 24 h with 10 ng/ml of basic fibroblast growth factor (bFGF; R&D Systems, Minneapolis, MN). The cells were then loaded at room temperature with Fluo-4 AM, a calcium dye indicator, according to the protocol provided by Molecular Probes in the presence of Pluronic F-127, sulfinpyrazone, and bFGF. After loading, the cover glass was placed in a coverslip clamp culture chamber (ALA Scientific Instruments, Inc., Westbury, NY). The cells were rinsed and then incubated at room temperature in 0.5 ml of Krebs-HEPES buffer supplemented with bFGF (10 ng/ml). A field of cells was selected based on their phase image. Fluorescent (i.e., Fluo-4) images were then captured at 30-sec intervals. To allow the cells to establish a baseline level, the first seven images (i.e., first 3.5 min) were discarded. The Krebs-HEPES buffer was replaced with Krebs-HEPES buffer with or without P4 (640 nM). Fluorescent images were collected from cells in the presence or absence of P4. The intensity of the Fluo-4 fluorescence was assessed in each cell using IP Lab Spectrum (Signal Analytics Corp., Vienna, VA). Intracellular free calcium levels were expressed as a fold change compared to the 3.5-min value.
Similar experiments were conducted in which bFGF was present. In this study, either P4 or vehicle was added after 3.5 min and images captured every 0.5 sec for a 70-sec time period. Intracellular free calcium levels were determined as outlined above.
Statistical Analysis
All experiments were repeated at least two to three times with each experiment yielding essentially identical results. Each experiment in which apoptosis was assessed by YOPRO-1 staining was done in quadruplicate. These data were pooled and analyzed by a one-way or two-way ANOVA followed by a Student-Newman-Keuls test, when appropriate. P values of less than 0.05 were considered to be significant.
RESULTS
Western blot analysis confirmed that immature rat ovaries express a 60-kDa protein that is detected by an antibody directed against the P4 binding domain of the nuclear P4 receptor (C-262). Not only was this protein more abundant in the ovarian membrane fraction, it was virtually absent in the ovarian cytoplasm (Fig. 1, A and B). In addition, granulosa cells isolated from eCG-primed rats also expressed this 60-kDa protein. The membrane localization of this protein in granulosa cells was demonstrated by detecting it among the membrane proteins that were biotinylated and isolated by affinity purification (Fig. 1C).
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To determine whether this 60-kDa protein binds P4, a ligand blot assay was conducted. As a control, ovarian lysate was immunoprecipitated with either an unrelated antibody (i.e., an antibody to IP3 receptors, Ab-R3) or C-262. Both immunoprecipitates were electrophoresed, blotted, and then probed with C-262. As expected, the 60-kDa protein was detected only after immunoprecipitation withC-262 and not with Ab-R3 (Fig. 2A). Probing the blots obtained after C-262 immunoprecipitation with a HP-P4 revealed the presence of a 60-kDa protein. Less of this protein was detected when P4 was included in the HP-P4 incubation (Fig. 2B). Taken together, these observations indicate that the 60-kDa protein can bind P4.
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A protein with similar characteristics was also detected in SIGCs. Specifically, a 60-kDa protein was selectively detected in the membrane fraction of SIGCs (Fig. 3A). Its plasma membrane localization was confirmed because it was detected after the surface membranes were biotinylated and isolated by affinity purification (Fig. 3B). Finally, ligand plot assay revealed that this 60-kDa protein detected in SIGCs binds P4 as does the 60-kDa protein that is detected in ovarian lysates (Fig. 3C).
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To determine the binding characteristics of this P4 binding protein, ligand binding studies were conducted. As shown in the upper panel of Figure 4, specific 3H-P4 binding increased and then came to equilibrium by 1 h of incubation at 4°C. This binding was reduced by increasing doses of P4 (Fig. 4, middle panel). Scatchard plot analysis revealed that P4 bound to a single site (i.e., single protein) with an apparent Kd of 360 nM (Fig. 4, lower panel). The number of binding sites (Bmax) was estimated to be 200 pmol/mg. 3H-Progesterone binding was not reduced by increasing concentrations of dexamethasone, mifepristone (RU 486), or onapristone (ZK98299) (Fig. 5, A and B).
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Because P4 prevents granulosa cells from undergoing apoptosis [11, 15], a study was conducted to determine whether P4 has the same effect on SIGCs. As can be seen in Figure 6, the percentage of apoptotic nuclei increased after serum withdrawal. This increase was attenuated by P4 in a dose-dependent manner. This antiapoptotic action of P4 was not inhibited by P4 receptor antagonists, mifepristone (RU 486) or onapristone (ZK98299) when P4 and either antagonist were administered at equal molar concentrations (Fig. 7).
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Because MEK is a central component of the MAP kinase cascade that regulates cell viability [30, 31], the ability of P4 to maintain MEK activity in SIGCs was assessed. As shown in Figure 8, the activity, but not the amount of MEK, decreased within 5 h of serum withdrawal. Progesterone prevented this decrease in MEK activity. Conversely, an inhibitor of MEK, PD 98059, blocked the antiapoptotic action of P4 (Fig. 9).
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In granulosa cells, P4 regulates cell viability in part by maintaining calcium homeostasis [15]. The final series of studies were conducted to determine whether P4 has the same effect in SIGCs. In SIGCs, [Ca2+]i levels gradually increased, reaching a twofold increase by 5 min after serum withdrawal. Progesterone prevented this increase (Fig. 10A). Because P4 maintained relatively constant levels of [Ca2+]i, it was difficult to detect any rapid response to P4. To resolve this problem, SIGCs were cultured with bFGF, which maintains constant [Ca2+]i levels [29]. Under these conditions, P4 decreased [Ca2+]i levels by 15%–20% within the first minute of exposure (Fig. 10B).
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DISCUSSION
Previous studies have shown that rat granulosa cells as well as cells derived from these cells (i.e., SIGCs) express a 60-kDa protein that is detected using an antibody directed against the P4 binding domain of the nuclear P4 receptor (C-262) [23]. The present studies confirm that this 60-kDa protein is a membrane protein because it is selectively detected in the membrane fraction and is among the membrane proteins that were biotinylated and subsequently isolated by streptavidin affinity purification. To determine whether this 60-kDa membrane protein binds P4, both ovarian cell and SIGC lysates were immunoprecipitated with C-262 and then the immunoprecipitate was electrophoresed and probed with a HP-P4. These studies indicate that this 60-kDa membrane protein binds P4.
Recent studies have shown that FITC-BSA-conjugated P4 binds to granulosa cell membranes with its binding being reduced by P4 but not by other steroids, including estradiol-17ß, testosterone, and dexamethasone [32]. In addition, FITC-BSA-conjugated P4 binding is inhibited by C-262 [32]. Scatchard plot analysis of 3H-P4 binding reveals a single binding site (i.e., single protein) in SIGCs that is relatively abundant (200 pmol/mg) and has an apparent Kd of 360 nM. These data are consistent with the possibility that the 60-kDa P4 binding protein is a low-affinity, high-capacity membrane receptor for P4. Similar low-affinity P4 binding proteins with Kd of 
300 nM have been described for sperm [26] and Leydig tumor cells [33]. Another membrane P4 binding protein that is expressed by bovine luteal cells has a Kd of 80 nM [34]. As such there may exist a family of membrane proteins in gonadal tissue that bind P4 and function as P4 receptors.
The P4 binding characteristics observed in the present study are in marked contrast to those obtained in the initial P4 binding studies conducted in the 1980s [19–22]. These early studies were conducted on cytosol fractions isolated from either whole rat ovaries or granulosa cells. Using 3H-R5020 as a ligand, these studies detected a P4 binding protein with a Kd between 1.5 and 14 nM and a binding capacity of between 50 and 232 fmol/mg of cytosol [19–22]. It is interesting that mifepristone (RU 486) was more effective than P4 in displacing 3H-R5020 binding [22]. Because both P4 and RU 486 bind to the ovarian glucocorticoid receptor [35], it is possible that the studies performed on ovarian cytosol detected glucocorticoid and not progesterone receptors. Further support for this concept comes from molecular studies that demonstrate that the granulosa cells of immature rats express the glucocorticoid [36] but not the classic nuclear progesterone receptor [6, 12–14].
In contrast to the previously published P4 binding studies, the present binding studies were conducted on intact cells at 4°C. Because FITC-BSA conjugated P4 binds to the plasma membrane and is not incorporated into the cytoplasm for up to 45 min of incubation at room temperature [32], it is likely that the present studies detect a P4 binding protein within the plasma membrane. These studies show that 3H-P4 binding is not reduced by dexamethasone, mifepristone (RU 486), or onapristone (ZK98299). These characteristics clearly distinguish the 60-kDa P4 binding protein from both the nuclear and membrane glucocorticoid receptors. It is important to note that this putative P4 binding protein would not have been detected in the earlier studies because they used ovarian cytosol and not the membrane fraction [12, 20–22].
Not only does P4 bind to this 60-kDa membrane protein, it also prevents SIGCs (present study) and granulosa cells [11, 15, 23] from undergoing apoptosis. That the 60-kDa protein mediates the antiapoptotic action of P4 is supported by the observation that C-262 blocks the ability of P4 to prevent granulosa cell apoptosis [23]. In both SIGCs and granulosa cells, P4 inhibits apoptosis at an effective dose of 320 nM. This is consistent with the observed Kd of the putative membrane receptor. It is interesting that at concentrations of 640 nM, neither mifepristone (RU 486) nor onapristone (ZK98299) abrogates the antiapoptotic action of P4. This is consistent with the ligand binding studies in which neither antagonist reduces 3H-P4 binding.
The signal transduction pathway through which P4 promotes granulosa cell viability is not clearly defined but appears to involve MEK. MEK is a central component of an important kinase cascade in which ras activates raf-1 [30, 31]. Raf-1 in turn phosphorylates MEK, with MEK subsequently phosphorylating MAP kinase [30, 31]. The importance of MEK in SIGC survival is illustrated by the observation that the MEK inhibitor, PD 98059, abrogates the ability of P4 to prevent apoptosis. A similar role for MEK has also been demonstrated for human granulosa/luteal cells [37]. Further, the activity and/or levels of several components of this kinase cascade are reduced in granulosa cells of atretic follicles compared with nonatretic follicles [38]. It is interesting that P4 can stimulate the MAP kinase cascade in MCF7 cells [39]. In these cells P4 activates a transcriptionally inactive P4 receptor, causing it to interact with the estrogen receptor. This interaction is required for P4 to activate the MAP kinase pathway. This finding provides a possible mechanism through which P4 can activate the MAP kinase cascade and subsequently maintain cell viability in the absence of the nuclear P4 receptor. Whether this mechanism is operative in SIGCs remains to be determined.
Once activated, membrane receptors generally initiate very rapid responses. In the case of P4, [Ca2+]i levels are usually altered within seconds of P4 exposure. In sperm and oocytes, P4 induces a transient increase in [Ca2+]i (see review by Revelli et al. [40]). However, P4 does not always increase [Ca2+]i. For example, P4 appears to interact with calcium channels within the membrane of smooth muscle cells to reduce calcium influx, thereby suppressing [Ca2+]i [41–43]. Similarly, P4 suppresses the increase in [Ca2+]i that is induced by thapsigargin in T lymphocytes [44]. Although one study has shown that P4 acts on porcine granulosa cells to rapidly increase [Ca2+]i [45], our studies clearly demonstrate that P4 acts to maintain [Ca2+]i in rat granulosa cells [15] and SIGCs (present studies). Because P4 maintains calcium homeostasis and [Ca2+]i levels gradually increase in the absence of growth or survival factors, it is very difficult to detect rapid effects of P4 in SIGCs. To resolve this problem, SIGCs were cultured with bFGF, which maintains both stable [Ca2+]i levels and cell viability [29]. Under these conditions, the addition of P4 decreases [Ca2+]i by 15%–20% within 1 min. This action is consistent with the ability of P4 to suppress the rise in [Ca2+]i induced by growth factor withdrawal. This rapid response is another indication that P4 regulates granulosa cell function through a nongenomic, membrane-initiated mechanism.
Finally, it may be difficult to conceive of a membrane receptor with such a low affinity (Kd 360 nM) playing a physiological role in regulating granulosa cell function. It is well established that serum P4 levels fluctuate during the rat estrous cycle [46]. On the morning of estrus, P4 levels are about 6 nM then increase to about 22 nM by diestrus I. Serum P4 levels then declined to about 12 nM by diestrus II and remained at this level throughout the morning of proestrus. With the induction of the preovulatory gonadotropin surge serum P4 rapidly increases, peaking at 20 nM by the afternoon of proestrus. Whereas serum P4 levels are well below the Kd of the putative membrane receptor for P4, follicular fluid levels of P4 are considerably higher [46]. In general the concentration of P4 within the follicular fluid parallels that of serum P4. However, regardless of the stage of the estrous cycle, the follicular fluid concentration of P4 is always greater than 360 nM in antral follicles [46]. It is proposed then that in these antral follicles, P4 acts through this putative membrane receptor to regulate [Ca2+]i levels, the activity of the MAP kinase pathway and, ultimately, granulosa cell survival. Future studies must now be conducted to more completely elucidate the signal transduction pathways through which P4 mediates its action as well as determining the molecular identify of this putative P4 membrane receptor.
ACKNOWLEDGMENTS
The authors thank Dr. Robert Burghardt of Texas A&M University for providing the SIGC cells. We also thank Dr. Gary Hodgen (The Jones Institute, Norfolk, VA) and Dr. Klaus Stockemann (Schering AG, Berlin Germany) for the gifts of mifepristone (RU 486) and onapristone (ZK98299), respectively. We also acknowledge Dr. Terri Bermner for doing the biotinylation studies on rat granulosa cells.
FOOTNOTES
First decision: 26 December 2000.
1 Supported by National Institutes of Health grant HD 34383. 
2 Correspondence. FAX: 860 679 1269; peluso{at}nso2.uchc.edu
Accepted: February 13, 2001.
Received: November 20, 2000.
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J. J. Peluso, A. Pappalardo, G. Fernandez, and C. A. Wu Involvement of an Unnamed Protein, RDA288, in the Mechanism through which Progesterone Mediates Its Antiapoptotic Action in Spontaneously Immortalized Granulosa Cells Endocrinology, June 1, 2004; 145(6): 3014 - 3022. [Abstract] [Full Text] [PDF]
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P. Kezele and M. K. Skinner Regulation of Ovarian Primordial Follicle Assembly and Development by Estrogen and Progesterone: Endocrine Model of Follicle Assembly Endocrinology, August 1, 2003; 144(8): 3329 - 3337. [Abstract] [Full Text] [PDF]
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 R. M. LOSEL, E. FALKENSTEIN, M. FEURING, A. SCHULTZ, H.-C. TILLMANN, K. ROSSOL-HASEROTH, and M. WEHLING Nongenomic Steroid Action: Controversies, Questions, and Answers Physiol Rev, July 1, 2003; 83(3): 965 - 1016. [Abstract] [Full Text] [PDF]
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J.J. Peluso, T. Bremner, G. Fernandez, A. Pappalardo, and B.A. White 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, January 1, 2003; 68(1): 122 - 128. [Abstract] [Full Text] [PDF]
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