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Departments of Obstetrics and Gynecology (L.E., J.J.P.) and Cell Biology (J.J.P.), University of Connecticut Health Center, Farmington, Connecticut 06030; Faculty of Clinical Medicine (R.L., M.W.), Mannheim Institute of Clinical Pharmacology, University of Heidelberg, D-68135 Mannheim, Germany; and Medicine/Experimental Medicine (M.W.), AstraZeneca R&D, S-48183 Molndal, Sweden
Address all correspondence and requests for reprints to: John J. Peluso, Ph.D., Department of Cell Biology, University of Connecticut Health Center, Farmington, Connecticut 06030. E-mail: peloso{at}nsoz.uchc.edu.
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Abstract |
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Objective: Our objective was to assess the role of the nuclear P4 receptor (PGR) and PGR membrane component 1 (PGRMC1) in mediating P4’s antiapoptotic action in human granulosa/luteal cells.
Design, Setting, and Patients: In vitro laboratory studies were designed in which human granulosa/luteal cells were harvested from in vitro fertilization patients from 2004–2006.
Main Outcome Measure: Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assays and DNA staining. Protein expression was observed by Western blot and immunocytochemistry.
Results: PGR was detected in 20% of the human granulosa/luteal cells, and 25 and 50 µM RU486 induced at least 70% of the cells to undergo apoptosis. Five micromolar RU486 neither induced apoptosis nor attenuated the antiapoptotic action of 1 µM P4. PGRMC1 and its binding partner, plasminogen activator inhibitor RNA-binding protein-1 (PAIRBP1), were detected in human granulosa/luteal cells. Antibodies to either PGRMC1 or PAIRBP1 completely attenuated P4’s action.
Conclusions: PGR does not exclusively mediate P4’s action because 1) 5 µM RU486 should have been able to override the antiapoptotic action of 1 µM P4 because RU486 binds to the PGR at a greater affinity than P4; 2) 25 and 50 µM RU486 induce three to four times more cells to undergo apoptosis than express PGR; 3) P4 must be continuously present to prevent apoptosis, which implies a rapid, possibly membrane-initiated mechanism of action; and 4) expression and blocking antibody studies suggest that PGRMC1 and PAIRBP1 account in part for P4’s action in human granulosa/luteal cells.
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Introduction |
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These observations clearly support a role for P4 in preventing human luteal cell apoptosis and imply that P4’s action is mediated through the PGR. However, because the PGR has not been ablated in luteal cells derived from either humans or nonhuman primates, a definitive role for these receptors in regulating luteal cell viability cannot be ascribed. Furthermore, PGR knockout mice do not ovulate and form CL, so these mice cannot be used to assess the role of the PGRs in regulating luteal cell survival (7).
Interestingly, neither rat granulosa cells before the LH surge nor rat luteal cells isolated from cycling, psuedopregnant, and pregnant animals express PGR (8, 9, 10, 11). In these rat cells, P4’s antiapoptotic action appears to be mediated by a P4 membrane receptor complex composed of two proteins: PGR membrane component-1 (PGRMC1) and its binding partner, plasminogen activator inhibitor RNA-binding protein-1 (PAIRBP1) (12, 13). PAIRBP1 is also known as RDA288 or SERBP1 (14). These findings in rat ovarian cells raise the possibility that the PGRMC1-PAIRBP1 complex plays a similar role in P4’s antiapoptotic action in human luteal cells. To test this hypothesis, a human luteal cell model system was used as outlined by VandeVoort et al. (15). In this model, luteinizing granulosa cells are isolated 36 h after hCG and induced to differentiate (i.e. luteinize) in vitro in the presence of serum and hCG. As a result, the granulosa cells differentiate into luteal cells with large cytoplasmic to nuclear ratios, express high levels of steroidogenic enzymes, and possess the capacity to secrete both estradiol and P4.
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Patients and Methods |
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Granulosa/luteal cells were obtained by follicular aspiration from women with various infertility diagnoses undergoing in vitro fertilization under a protocol approved by the Institutional Review Board of the University of Connecticut Health Center. Briefly, patients were treated with a GnRH analog (Lupron) during the luteal phase to suppress ovarian function (i.e. estradiol levels of <75 pg/ml and no follicles of >10 mm). Once ovarian function was suppressed, then the patients were treated with recombinant FSH (Gonal F; Serono, Inc., Rockland, MA) (16). When two or three leading follicles were at least 18 mm in diameter, hCG was administered sc and transvaginal ultrasound-directed oocyte retrieval was performed approximately 36 h after hCG administration. Only follicular fluid from patients with adequate ovarian response was collected, and patients with a poor response to ovarian stimulation were excluded from the study. A poor ovarian response was defined as a peak serum estradiol level of 850 pg/ml or less and/or four or fewer preovulatory follicles greater than 15 mm in diameter on the day of hCG administration (17).
Cell preparation and culture
After the oocytes were removed, follicular aspirates were pooled and centrifuged at 250 x g for 10 min. The cell pellet was resuspended in PBS, layered on Histopaque-1077, and centrifuged for 30 min at 400 x g. After centrifugation, the opaque interface containing the granulosa/luteal cells was carefully aspirated and transferred into a 15-ml sterile conical centrifuge tube. The cells were then resuspended in 12 ml PBS and centrifuged at 250 x g for 10 min. This was repeated two additional times. The cell pellet was then resuspended in 1 ml of 0.25% trypsin-EDTA solution and incubated for 5 min to dissociate the cells. After trypsinization, 5 ml DMEM/F12 culture medium supplemented with 5% fetal bovine serum (FBS) (i.e. serum-supplemented medium) was added and the cells centrifuged at 250 x g for 10 min. The cells were then resuspended in DMEM/F12 culture medium supplemented with 5% FBS, counted in a hemocytometer, and resuspended to yield a final concentration 1 x 106 cells/ml.
Plastic eight-chamber Lab-Tek slides (BD Bioscience, Bedford, MA) that had been previously coated with growth factor reduced Matrigel matrix (BD Bioscience) were plated at 6 x 104 cells per well in 0.5 ml DMEM/F12 culture medium supplemented with 5% FBS medium and 2 IU/ml hCG (15). The medium was changed after 24 h to remove any remaining blood cells or nonattached granulosa/luteal cells and the cultures continued for two additional days. The cultures were then subjected to the various experimental treatments as outlined in Results.
Detection of apoptotic nuclei
Both terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) and in situ DNA staining were used to identify apoptotic nuclei. For the TUNEL assay, human granulosa/luteal cells were cultured for 5 h in serum-free medium and then fixed in 10% formalin. The cells were stained using the Apoptag peroxidase in situ kit staining according to the manufacturer’s instructions (Chemicon, Temecula, CA). In situ DNA staining was done by adding hydroethidine directly to the culture medium at a final concentration of 3.5 µg/ml (18). The cultures were incubated for 15 min at room temperature in the dark. After staining, the cells were observed under epifluorescent optics. Under these conditions, only cells with condensed or fragmented nuclei were stained intensely with hydroethidine. These cells were considered to be apoptotic (18). At least 100 cells per culture well were counted and the percentage of apoptotic nuclei in each well determined. The values obtained from the in situ DNA staining were used to determine the percentage of apoptotic human granulosa/luteal cells as illustrated in the graphs.
Immunocytochemical and Western blot analysis
To localize PGR, cells were fixed with 10% formalin and incubated with 0.1% Triton X-100. Endogenous peroxidase activity was blocked by incubating the cells in 0.3% peroxidase in methanol for 30 min at room temperature. To reduce nonspecific staining, the slides were incubated with Powerblock (Biogenex, San Roman, CA) and then incubated overnight with a 1:50 dilution of PGR antibody (Ab-8; Lab Vision/Neomarker, Fremont, CA). The epitope for this antibody is the N-terminal half of the human PGR. It is a mouse monoclonal antibody that detects both PGRA and PGRB and cross-reacts with human, equine, ovine, and porcine tissues. After incubation with the PGR antibody, the cells were then incubated with biotinylated goat antimouse IgG followed by a 30-min incubation with ABC reagent (Vector Laboratories, Burlingame, CA). The slides were developed using a diaminobenzidine-peroxidase substrate for 5 min followed by light counterstaining with Methyl Green. The presence of PGR was revealed by the presence of a reddish-brown precipitate.
Expression and localization of PGRMC1 and PAIRBP1 were assessed by Western blot and immunocytochemistry, respectively. For Western blot studies, human granulosa/luteal cells were lysed in RIPA buffer (50 mM Tris, 150 mM sodium chloride, 1.0 mM EDTA, 1% Nonidet P40, and 0.25% sodium-deoxycholate, pH 7.0), which was supplemented with complete protease inhibitor cocktail (Roche, Mannheim, Germany) and phosphatase inhibitor cocktail 1 (Sigma Chemical Co., St Louis, MO). The lysate was then centrifuged at 1000 x g at 4 C for 5 min. The supernatant was collected and centrifuged at 100,000 x g at 4 C for 1 h. Twenty micrograms of this membrane preparation were run on a 12% acrylamide gel and transferred to nitrocellulose. The nitrocellulose was then incubated with 5% nonfat dry milk overnight at 4 C. The nitrocellulose blot was then incubated with either the chicken PAIRBP1 antibody at a dilution of 1:2000 (12) or the rabbit PGRMC1-NT antibody (1:2000) (13) for 1 h at room temperature.
The PGRMC1 antibody is a rabbit antibody directed against 15 N-terminal amino acids of porcine PGRMC1 (19). This amino acid sequence is exactly conserved in human, rat, and porcine PGRMC1. Moreover, the antibody has been shown to detect 28- and 56-kDa proteins in human sperm (20). The PAIRBP1 antibody is a chicken antibody built against the amino acid sequence KQLRKESQKDR (21). This sequence is found in both human and rat PAIRBP1. The PAIRBP1 antibody has been shown to detect a 60-kDa protein in human sperm (22).
Western blots were processed using a horseradish peroxidase goat antichicken IgY (1:50,000; Aves Labs, Tigard, OR) or a horseradish peroxidase goat antirabbit antibody (1: 10,000). The KPL LumiGlo detection system was used to reveal the presence of both proteins. As a negative control, an immunodepleted antibody preparation or rabbit IgG was used in place of the PAIRBP1 antibody and PGRMC1 antibody, respectively.
For immunocytochemical studies, human granulosa/luteal cells were grown on glass coverslips in 35-mm culture dishes. After 3 d of culture, these cells were washed and then fixed in 10% formalin and permeabilized as previously described (13). The coverslips were then incubated overnight at 4 C with the antibodies to PAIRBP1 (1:50), PGRMC1 (1:50), or both. After a wash to remove the primary antibodies, the coverslips were incubated for 1 h at room temperature in the dark with Alexa Fluor 633-goat antichicken IgY (1:100) and Alexa Fluor 488-goat antirabbit IgG (1:100). The coverslips were again washed and observed under the confocal microscope. Negative controls were also processed as described above with the exception that the immunodepleted antibody preparation or IgG was used in place of the PAIRBP1 or PGRMC1 antibody, respectively.
PGRMC1 and PAIRBP1 blocking antibody study
Human granulosa/luteal cells were plated on eight-chamber Lab-Tek slides and cultured for 3 d as previously described. The cells were then washed in serum-free medium and cultured for 1 h with either serum-free medium supplemented with rabbit IgG (20 µg/ml), antibody to PGRMC1 (20 µg/ml), IgY (34 µg/ml), or an antibody to PAIRBP1 (34 µg/ml) in the presence or absence of P4 (0.1 µM). After culture, the cells were rinsed in Krebs/HEPES buffer and stained to detect apoptotic nuclei (18). One hundred cells in each chamber were counted and the percentage of apoptotic nuclei determined as previously described.
Statistical analysis
All experiments were repeated at least four times. When appropriate, the data were pooled to generate means ± SE and analyzed by either a Student’s t test when an experiment consisted of two treatment groups or a one-way ANOVA followed by a Student-Newman-Keuls test if more than two treatments groups were being compared. P values of <0.05 were considered to be significant.
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Results |
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). About 10% of the human granulosa/luteal cells maintained in serum-supplemented medium for 3 d were considered to be apoptotic. This percentage increased to about 30% within 5 h of serum withdrawal (P < 0.05; Fig. 2A). Time-course studies revealed that a similar increase in apoptosis was observed after 1 h in serum-free medium and that this increase was suppressed by P4 (Fig. 2B). Moreover, P4 at all doses tested suppressed apoptosis. The lowest effective dose was 10 nM (Fig. 2C).
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). Moreover, 25- and 50-µM doses of the PGR antagonist RU486 increased the percentage of apoptotic nuclei to greater than 70% (Fig. 3B). P4 at 1 µM could not override the effect of RU486 at these high concentrations (Fig. 3B). Interestingly, 5 µM RU486 did not increase the percentage of apoptotic nuclei compared with control, and 1 µM P4 was still capable of suppressing serum-withdrawal-induced apoptosis (Fig. 3B). Finally, when human granulosa/luteal cells were deprived of P4 for 15 or 30 min, P4’s ability to prevent apoptosis was lost (Fig. 3C).
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and 5). Unlike PGR expression, virtually all the human granulosa/luteal cells expressed PGRMC1 and PAIRBP1 (Fig. 5). These proteins colocalized near the plasma membrane as well as to a fibrous network within the cytoplasm (Fig. 5). Although these proteins were often colocalized, PGRMC1 did not appear to associate with PAIRBP1 at the points of cell-cell contact or in the nucleus (Fig. 5). The nuclear localization of PGRMC1 was particularly intense.
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Discussion |
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It is possible that P4’s antiapoptotic action is not exclusively transduced through the PGRs. Support for this concept is provided by a careful review of the RU486 studies. For example, in human granulosa/luteal cells, RU486 must be in at least a 10-M excess to attenuate P4’s antiapoptotic action (5, 6). In contrast, a 5-nM dose of RU486 completely inhibits the effects of 50 nM of the progestin R5020 in breast cancer cells (T47D) (25). Given that RU486 binds the PGR at a higher affinity than either P4 or R5020 (26), the requirement for such a high molar excess of RU486 to disrupt P4’s antiapoptotic effect is inconsistent with a PGR-mediated mechanism of action in human granulosa/luteal cells. Finally, after 3 d of culture, only about 20% of the granulosa/luteal cells express PGR, but high doses (i.e. 25 and 50 µM) of RU486 induce over 70% of the cells to undergo apoptosis. A similar response to RU486 has also been observed when freshly isolated human granulosa/luteal cells were examined (5). These observations make it unlikely that antagonizing PGR is the sole mechanism that accounts for RU486’s apoptotic effects.
If not by inhibiting the action of PGR, how might RU486 induce granulosa/luteal cell apoptosis? One explanation could be related to RU486’s ability to suppress P4 synthesis. Previous work by our lab (27) and the Billig laboratory (6) has shown that P4 levels in human granulosa/luteal cell cultures are between 100 and 300 nM after 1 h of culture. RU486 induces a dose-dependent decrease in the amount of P4 secreted over a 24-h period (6). In fact, RU486 induces apoptosis only at concentrations that suppress P4 secretion (28). RU486 appears to inhibit P4 synthesis by directly inhibiting the activity of two steroidogenic enzymes, cytochrome P450scc and 3ß-hydroxysteroid dehydrogenase, and this effect may not involve an interaction with PGR (29, 30, 31). As a result, the net effect of RU486 treatment is to reduce intracellular P4 levels rapidly. This in turn would reduce the amount of P4 available to bind and activate a yet-to-be-identified receptor that directly transduces P4’s antiapoptotic action. Importantly, this mechanism for RU486 would explain its ability to induce apoptosis in human granulosa/luteal cells that do not express PGR.
There are several potential receptors that could mediate P4’s antiapoptotic action. These potential receptors include the glucocorticoid (32) and GABAA (33) receptors because P4 promiscuously binds and activates these receptors. Although there are reports that the glucocorticoid dexamethasone inhibits human granulosa/luteal cell apoptosis in vitro (34, 35), studies that compare the same doses of dexamethasone and P4 failed to show an antiapoptotic effect of dexamethasone (5). Similarly, GABAA agonists do not mimic P4’s antiapoptotic actions in human granulosa/luteal cells (6).
In rat granulosa and luteal cells, P4’s antiapoptotic action is mediated through a membrane P4 receptor complex composed of two proteins, PAIRBP1 and PGRMC1 (12, 13). The present Western blot analysis demonstrates for the first time that these two proteins are expressed in human granulosa/luteal cells. This is consistent with the microarray study that detected the mRNA that encodes PGRMC1 in cultured human granulosa/luteal cells (36). In addition, the confocal study revealed that PAIRBP1 and PGRMC1 often colocalize near the plasma membrane. In rat spontaneously immortalized granulosa cells (SIGCs), these two proteins also colocalize at the plasma membrane and project to the extracellular surface as revealed by biochemical analysis (13). Furthermore, the two proteins coimmunoprecipitate, thereby demonstrating that they interact with each other in vivo (13). It is possible then that the PAIRBP1-PGRMC1 complex functions as a P4 membrane receptor complex in human granulosa/luteal cells. When ligand activated, this receptor complex is likely to trigger a signal transduction pathway that prevents human granulosa/luteal cell apoptosis. This concept is supported by the blocking antibody study, which revealed a role for each protein in P4’s antiapoptotic mechanism of action.
The signal transduction pathway that is induced after PAIRBP1-PGRMC1 activation is just beginning to be elucidated. Earlier work has shown that P4 increases cGMP levels in human granulosa cells (37), which is known to activate protein kinase G (PKG) (38, 39). In mouse granulosa cells and SIGCs, P4 mediates its antiapoptotic action in part by increasing PKG activity (18). PKG activation in turn suppresses basal intracellular ATP concentrations (Peluso, J. J., unpublished). Moreover, early in the apoptotic cascade, there is a transient increase in intracellular ATP that is required for SIGCs (Peluso, J. J., unpublished) and human granulosa/luteal cells (40) as well as other cells (41) to undergo apoptosis. This increase is followed by a decrease in intracellular ATP (40), which is associated with a decrease in mitochondrial potential as observed in human granulosa/luteal cells by Makino et al. (28). Taken together, these studies suggest that P4 acting via the PGRMC1-PAIRBP1 membrane complex increases cGMP, which in turn activates PKG and thereby maintains basal intracellular ATP levels. Consistent with this hypothesis is the observation that exogenous ATP induces human granulosa/luteal cells to undergo apoptosis (40, 42). However, many more studies must be conducted to confirm this putative mechanism of action.
The observation that PAIRBP1 and PGRMC1 colocalize near the plasma membrane is supportive of the proposed membrane-initiated mechanism of action. However, the role of PGRMC1 in regulating human granulosa/luteal cell function may be more complex than proposed. For example, the confocal studies demonstrate that PGRMC1 but not PAIRBP1 localizes to the points of cell-cell contact. Previous studies with human granulosa/luteal cells (43) as well as rat granulosa cells indicate that N-cadherin-mediated cell contact per se activates the survival pathway (44, 45). The localization of PGRMC1 suggests a role in regulating cell survival through a cell-contact-mediated mechanism. Similarly, PGRMC1 localizes to the nuclei of human granulosa/luteal cells. PGRMC1 has been shown to localize to the nucleus of rat luteal cells but not granulosa cells (13). Moreover, a phosphorylated form of PGRMC1 has recently been detected within the nucleus of Hela cells (46). Based on these findings, it is temping to speculate that PGRMC1 may function to regulate gene transcription in luteal cells. However, the role, if any, that PGRMC1 plays in regulating cell contact and/or gene transcription remains to be determined experimentally.
In summary, the present data provide evidence that argues against the concept that P4 activates the PGR exclusively to mediate its antiapoptotic action in human granulosa/luteal cells. Rather, the present data support the hypothesis that P4’s antiapoptotic action may be mediated in part through a novel membrane P4 receptor complex that is composed of two proteins, PGRMC1 and PAIRBP1. The data that support this concept include the observations that these proteins are expressed in 100% of the human granulosa/luteal cells and that antibodies to each protein attenuate P4’s antiapoptotic action. More detailed biochemical and molecular biological studies must be conducted to elucidate the precise roles of PGR, PGRMC1, and PAIRBP1 in mediating P4’s antiapoptotic action.
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Acknowledgments |
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Footnotes |
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J.J.P, L.E., R.L., and M.W. have nothing to declare. M.W. is currently employed by AstraZeneca R&D Molndal.
First Published Online September 19, 2006
Abbreviations: CL, Corpus luteum; FBS, fetal bovine serum; hCG, human chorionic gonadotropin; P4, progesterone; PAIRBP1, plasminogen activator inhibitor RNA-binding protein-1; PGR, P4 receptor; PGRMC1, PGR membrane component 1; PKG, protein kinase G; SIGC, spontaneously immortalized granulosa cell; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling.
Received May 25, 2006.
Accepted September 11, 2006.
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, values that are different from the no-RU486 treatment (P < 0.05). C, Time-dependent nature of P4’s antiapoptotic effect. In this study, P4 was either not added (serum-free control) or added to the culture medium 0, 15, or 30 min after serum withdrawal. Values in this graph are means ± SE of 16 replicate cultures taken from four patients. *, Value that is different from the serum-free control (P < 0.05). The rate of apoptosis for studies shown in B and C was assessed after 1 h of culture.
