Dominant Role of Nuclear Progesterone Receptor in the Control of Rat Periovulatory Granulosa Cell Apoptosis 1

BOR Papers in Press. Published on February 4, 2009 as DOI:10.1095/biolreprod.108.073932 1 Dominant Role of Nuclear Progesterone Receptor in the Contr...
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BOR Papers in Press. Published on February 4, 2009 as DOI:10.1095/biolreprod.108.073932

1 Dominant Role of Nuclear Progesterone Receptor in the Control of Rat Periovulatory Granulosa Cell Apoptosis1 P. Anders Friberg, D. G. Joakim Larsson and Hakan Billig2 Department of Physiology/Endocrinology, Institute of Neuroscience and Physiology, the Sahlgrenska Academy at Gothenburg University, Gothenburg, Sweden Short title: Progesterone control of granulosa cell apoptosis Summary sentence: Apoptosis is regulated by progesterone acting primarily via PGR in periovulatory rat granulosa cells in vitro. Key words: Apoptosis, progesterone, progesterone receptor, granulosa cells, mechanisms of hormone action Abstract In this study, it was hypothesized that progesterone acts as a survival factor primarily by actions of the classical nuclear progesterone receptor (PGR) signaling pathway in rat periovulatory granulosa cells. Granulosa cells were isolated from immature female rats primed with eCG/hCG and treated in vitro with PGR antagonists. As little as 10 nanomolar of two different PGR antagonists (Org 31710 and RU 486) increased apoptosis measured as caspase 3/7 activity, which was reversed by co-treatment with the progestin R5020. Concurrently, progesterone synthesis was decreased. Inhibition of progesterone synthesis by cyanoketone similarly induced apoptosis but required a greater inhibition of progesterone synthesis than seen after treatment with PGR antagonists. Therefore, the induction of apoptosis by PGR antagonists cannot be explained by decreased progesterone synthesis alone. Low concentrations of R5020 also completely reversed the effects of cyanoketone. Inhibition of progesterone synthesis was more effective in inducing apoptosis than treatment with PGR antagonists. However, co-treatment with PGR antagonists protected the cells from the additional effects of cyanoketone, indicating partial agonist effects of the antagonists and a dominating role for the PGR in progesteronemediated regulation of apoptosis. The progesterone receptor membrane component 1 (PGRMC1) was expressed in granulosa cells; however, an anti-PGRMC1 antibody did not induce apoptosis in periovulatory granulosa cells. Neither anti-PGRMC1 nor progesterone or cyanoketone affected apoptosis of immature granulosa cells. In conclusion, we show that progesterone regulates apoptosis in periovulatory granulosa cells by acting via the classical nuclear receptor.

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Supported by grants 10380, 13550, 14807, 14572 and 14573 from the Swedish research council (medicine) and by grants from the Lars Hierta foundation, the Längmanska foundation, the Wilhelm and Martina Lundgren foundation, the Emil and Maria Palm foundation, the Hjalmar Svensson foundation and the Eva and Oscar Ahrén foundation.

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Correspondence and reprint requests: Håkan Billig, Department of Physiology/Endocrinology, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at Gothenburg University, P.O. Box 434, SE 40530 Gothenburg, Sweden. FAX: +46 31 7863512; e-mail: [email protected]

Copyright 2009 by The Society for the Study of Reproduction.

2 Introduction Progesterone (P4) is best known as a hormone produced by the corpus luteum and the placenta, with the main function to prepare the uterine endometrium for implantation of a fertilized oocyte and to maintain pregnancy. However, P4 is also involved in ovulation and luteinization of the post-ovulatory follicle. Working in an auto/paracrine manner, P4 takes on an orchestrating role in the transcriptional changes of the luteinizing granulosa cells after the ovulation-inducing surge of LH [1]. During follicular development, granulosa cells are subjected to a strong selective pressure based on apoptosis, ultimately leading to atresia of more than 99.9% of all growing follicles [2-4]. A multitude of factors take part in the regulation of proliferation and apoptosis of granulosa cells. P4 is one of several factors that play an anti-apoptotic role in the regulation of granulosa and luteal cell survival in most species studied [5-10]. Traditionally, P4 was thought to mediate all of its effects via the nuclear progesterone receptor (PGR). Expression of PGR in granulosa cells is induced transiently by LH in rodents [8, 11, 12], in contrast to humans, where PGR remains expressed in the corpus luteum [13, 14]. However, rapid action of P4 in sperm activation, for example, has lead to the identification of several other candidate progesterone receptors or receptor groups mediating cellular actions in a non-genomic manner [15, 16]. The involvement of the classical PGR in apoptosis regulation of granulosa cells has previously been studied by our group [6, 8, 17-20] and others [5, 7, 9]. However, the biological relevance of previous studies using PGR antagonists is controversial since the antagonist concentrations used were several potencies higher than the local concentration of P4 [21]. Indeed, since high concentrations of PGR antagonists decrease P4 synthesis [20], the possibility has been raised that the apoptotic effects of specific PGR antagonists could be an indirect effect mediated via decreased stimulation of other progesterone receptors [21]. Importantly, P4 has been implicated in apoptosis regulation in several stages of granulosa cell development, including early preovulatory granulosa cells lacking expression of PGR [22]. The effects of P4 in cells lacking PGR could be attributed to recently characterized membrane progestin receptors or binding proteins. One group of proteins reported to function in rapid, membrane-initiated P4 signaling has been studied in the laboratories of Peluso and Wehling [21, 23-26]. In granulosa cells, these proteins (termed progesterone receptor membrane component 1 (PGRMC1) and serpine1 mRNA binding protein 1 (SERBP1)) interact as a functional complex binding P4 [23, 24]. Interestingly, several studies report that these proteins are involved in the regulation of apoptosis in early differentiated granulosa cells, mainly based on experiments with spontaneously immortalized rat granulosa cells (SIGC) [23-25] as well as luteinized human granulosa cells [21]. In summary, P4 is well established as a survival factor in granulosa cells. However, the mechanism by which P4 acts to inhibit apoptosis of periovulatory granulosa cells is still controversial. This controversy warrants a careful investigation of the involvement of the PGR. It is hypothesized that PGR is involved in apoptosis regulation of rat periovulatory granulosa cells and furthermore that this involvement cannot be attributed to mediatory roles of other P4 signaling systems. This study was designed to 1) examine the dose-dependency and specificity of

3 effects of PGR antagonists on apoptosis in vitro in primary periovulatory granulosa cells and 2) relate the effects of PGR antagonists to other possible pathways mediating effects of P4. Materials and Methods Animals Immature female rats (Sprague-Dawley, 20 days of age) were purchased from B&K Universal, Sollentuna, Sweden or from Taconic, Ejby, Denmark. After arrival, the animals were acclimatized for 6 days before onset of experiments. The rats were given free access to food and water and were kept in a controlled environment with a 12:12 h photoperiod. All animal experiments were approved by the local animal ethics committee in Gothenburg, Sweden. Granulosa cell isolation At 26 days of age, rats were injected subcutaneously with eCG (10 IU, Sigma, St. Louis, MO) to induce follicle growth and development. After 48 hours, an intraperitoneal injection of hCG (50 IU, Organon, Oss, The Netherlands) was given to induce luteinization of preovulatory follicles according to previously established protocols [6, 17, 19]. A subset of rats was used as untreated immature controls. The rats were killed by cervical dislocation 7 h after hCG-treatment, ovaries were isolated and granulosa cells were collected by follicle puncture. The granulosa cell isolation time was chosen to approximately match the peak expression of PGR [8, 11]. Responding follicles were selected based on the degree of vascularization and size. Isolation of granulosa cells was done in Minimum Essential Medium (MEM) with Earle salts and L-glutamine (Invitrogen, Carlsbad, CA) supplemented with sodium hydrogen carbonate, 0.0375‰ (w/v). Cells were pelleted by centrifugation for five minutes at 200g, re-suspended in Eagle MEM with Glutamax-I, Earle salts and Hepes (Invitrogen) supplemented with penicillin (100 IU/ml, Invitrogen), streptomycin (100 μg/ml, Invitrogen) and BSA, fraction V (0.1%, Sigma) and counted in a Bürker chamber after Trypan blue staining. Granulosa cell incubation Isolated granulosa cells were incubated in Eagle MEM (supplemented as above) with 106 cells/ml in 0.5 or 1.0 ml in culture tubes (Falcon 12×75 mm, Becton Dickinson, NJ) for P4 assay and immunoblotting purposes, or 20,000 cells in 100 μl in 96-well plates (96F Nuclon delta white microwell S1, Nunc, Roskilde, Denmark) for caspase activity assays. The incubations were carried out with or without addition of the nuclear PGR antagonists Org 31710 (10 nM – 10 μM, Organon, Oss, The Netherlands) or RU 486 (10 nM – 10 μM, Exelgyn. Paris, France), the progestin R5020 (1 nM – 1 μM, PerkinElmer, Waltham, MA), progesterone (10 nM – 1 μM, Sigma), the irreversible hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid deltaisomerase cluster (HSD3B) -inhibitor cyanoketone (3 nM – 1 μM, Sterling Drugs Inc., Rensselaer, NY) or antibodies against PGRMC1 (1 – 50 μg/ml, kindly provided by Ralf Lösel, Dept. of Clinical Pharmacology, University of Heidelberg, Mennheim, Germany) [27]. Cyanoketone was dissolved as a stock solution in DMSO, whereas all other drugs were dissolved as stock solutions in ethanol and the lyophilized PGRMC1 antibody was diluted directly in culture medium. The final concentration of solvent in the incubation medium did not exceed 0.1%, and this concentration had no effect on the measured variables (data not shown). Incubations were carried out at 37°C in 5% CO2 and 95% humidified air for 22 h. Serum-free incubation is commonly used to induce apoptosis in isolated granulosa cells [28]. The periovulatory interval is defined as the time from the onset of the LH surge or hCG

4 administration to follicle rupture [1]. Here, we use the term periovulatory for preovulatory granulosa cells, isolated from eCG/hCG primed immature rats and cultured in vitro for up to 22 h [6, 17, 19]. Apoptosis measurements Caspase 3/7 activity was measured using the Caspase-Glo 3/7 Assay kit (Promega, Madison, WI) according to the manufacturer’s instructions. The assay is based on the cleavage of a proluminescent caspase substrate containing the tetrapeptide sequence DEVD. The amount of luminescent product generated is proportional to the caspase 3/7 activity in the sample. Luminescence was detected 30 minutes after addition of substrate using a Fluostar platereader (BMG Labtechnologies, Offenburg, Germany). Progesterone measurements P4 in spent medium was analyzed using dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) [29] (PerkinElmer, Åbo, Finland) according to the manufacturer’s instructions. Immunoblot Analysis After incubation, cells to be used for immunoblotting were pelleted at 10,000g at 4°C and the pellets were washed in ice cold PBS and frozen at −70°C. Intact ovaries were washed in PBS and snap-frozen in liquid nitrogen immediately after dissection. Cells and tissues were homogenized at 4°C in lysing buffer (RIPA buffer: 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM NaF, 1% Nonidet P-40, 10 µg/ml phenylmethylsulfonyl fluoride, 1 mM Na3VO4 and a cocktail of protease inhibitors (Roche Diagnostics GmbH, Mannheim, Germany)) and incubated on ice for 30 min. Subsequently, insoluble material was removed by centrifugation at 10,000g for 30 min at 4°C. The protein content of the extracts was determined using a bicinchoninic acid protein assay (Pierce, Rockford, IL) with BSA as the standard. Thirty micrograms of protein per sample were heated at 70°C for 10 min with reducing agents and loaded onto 4–12% Bis-Tris gels (Novex, San Diego, CA) using a MOPS-SDS running system. Nonspecific protein-binding to the polyvinylidene difluoride membranes (Amersham International, Buckinghamshire, UK) was prevented by pre-incubation of the membrane with 5% nonfat milk in a Tris-buffered saline (TBS)-Tween 20 buffer (10 mM Tris, 150 mM NaCl and 0.1% Tween 20, pH 8.0) for 2 h. Antibodies against PGRMC1 were kindly provided by Ralf Lösel (Dept. of Clinical Pharmacology, University of Heidelberg, Mennheim, Germany) as lyophilized serum. The PGRMC1 antibody is a rabbit polyclonal antibody directed against 15 N-terminal amino acids of the porcine PGRMC1 [27]. This sequence is identical in porcine, human and rat PGRMC1. The membranes were incubated with primary antibody at a dilution of 1:1,000 in blocking buffer overnight at 4°C, followed by incubation with the appropriate goat-anti-rabbit alkaline phosphatase-linked secondary antibody (Tropix, Bedford, MA) at a 1:40,000 (v/v) dilution for 2 h. The immunosignal-CDP-Star substrate for the alkaline phosphatase system (Tropix, Bedford, MA) was used to visualize protein bands. ECL films of the western blots were captured with an image scanner (EPSON perfection 2450 photo). All steps were carried out at room temperature unless otherwise stated.

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Statistical analysis All quantitative experiments were performed at least three times with typically n=6–8 for each treatment in each experiment. Combined data from multiple experiments are presented graphically as normalized to the control group in each experiment, unless stated otherwise. All statistical analyses were made using SPSS statistical software (version 15, SPSS, Chicago, IL). Differences between group means were analyzed using univariate general linear model (GLM) including terms for treatment and experiment. Post-hoc tests were applied where appropriate, including Dunnet’s test for multiple comparisons to a control or Bonferroni’s test for comparisons between groups. Single- or double-sided tests were used where appropriate based on hypotheses and indications from previous data. Residual analyses were used to check assumptions of normality and equality of variances. Data were transformed into natural logarithms as appropriate after residual analyses. The caspase-activity assays used here occasionally results in outlier values due to cell clumping and well debris. Outliers were therefore detected and omitted objectively, based on studentized residuals with P < 0.01 [30]. Data are presented as mean ± SEM. P-values < 0.05 were considered statistically significant. Results PGR antagonists Org 31710 and RU486 induce apoptosis To characterize the importance of PGR in granulosa cell apoptosis, cells were isolated from eCG and hCG-primed immature rats, 7 hours after the hCG injection. The selective PGR antagonist Org 31710 as well as the more commonly used RU 486 were used to block cellular effects mediated via PGR. Both antagonists dose-dependently induced apoptosis as measured by caspase 3/7 activity assay (Fig. 1A–B). The lowest effective concentration tested was 10 nM (P < 0.01) for both antagonists. The caspase activity reached an apparent plateau at an approximate 22-32% increase compared with controls at concentrations of antagonists between 10–30 nM up to 1,000–3,000 nM. However, higher micromolar concentrations resulted in a second, much more potent increase in caspase activity. To verify that the cause of the induced caspase activity at nanomolar concentrations of antagonists was a result of competitive antagonism of the PGR, granulosa cells were co-treated with Org 31710 and the progestin R5020 (Promegestone). Increasing concentrations of R5020 completely reversed apoptosis induced by Org (100 nM, Fig. 1C). In contrast, the increase in apoptosis resulting from Org 31710 at concentrations above 1 μM could not be reversed by R5020 (data not shown). By itself, R5020 significantly reduced caspase activity to 86% of control (P < 0.001, Fig. 2). Inhibition of progesterone synthesis increases apoptosis P4 has been suggested to control its own synthesis by regulating the activity of steroidogenic enzymes. Accumulation of P4 after culturing cells for 22 h was therefore measured. Treatment of granulosa cells with Org 31710 (100 nM) significantly decreased the amount of P4 to approximately 76% of control levels in spent medium, from 51 ± 1.2 nM to 39 ± 0.75 nM (P < 0.001). It has been suggested that PGR antagonists could induce apoptosis indirectly by their ability to decrease P4 synthesis. If alternative progesterone receptors are involved and play a mediatory role, they would be dependent on the availability of the ligand. To investigate if this inhibition of P4 synthesis resulting from Org 31710-treatment could explain the increase in apoptosis, granulosa cells were treated with the irreversible HSD3B-inhibitor cyanoketone. As expected, cyanoketone decreased P4 accumulation dose-dependently with a consistent significant

6 effect at a lowest concentration of 10 nM (Fig 3A). Treatment with cyanoketone concurrently induced apoptosis (Fig. 3B). The lowest effective concentration was 3 nM (P < 0.001), which increased apoptosis by 5.1%. At a concentration of 30 nM the degree of apoptosis induced was comparable to PGR antagonists at 100 nM. Since inhibition of HSD3B has broad effects on general steroid synthesis, granulosa cells were co-treated with cyanoketone and R5020 to determine if the effects were caused by decreased P4 synthesis specifically. The increase in caspase activity induced by cyanoketone (1,000 nM) was completely reversible by R5020 (5 nM, Fig. 3C). PGR antagonists protect cells from the effects of cyanoketone It can be assumed that removal of P4 should have the same effect on induction of apoptosis as treatment with PGR antagonists, providing that that P4 regulates apoptosis solely via the actions of PGR. To determine whether cyanoketone has additional effects in comparison to Org 31710, a set of experiments was carried out to compare the treatments. Indeed, the effect of cyanoketone was significantly greater than the effects of both Org 31710 and RU 486 (Fig. 4). However, when granulosa cells were co-treated with cyanoketone and Org 31710 or RU 486, the additional effect of cyanoketone was abolished, implying that these PGR antagonists can protect the cells from cyanoketone-induced apoptosis. Blocking antibody against PGRMC1 does not induce apoptosis Studies in rat and human granulosa and luteal cells suggest an involvement of PGRMC1 and SERBP1 in apoptosis regulation by P4. Antibodies against PGRMC1 have been shown to block the anti-apoptotic effects of P4 in rat SIGCs and human luteal cells. Therefore, to investigate the involvement of PGRMC1 in rat periovulatory granulosa cells, western blotting was performed to determine the presence of PGRMC1 in the granulosa cell cultures and to confirm the integrity of the antibody (Fig. 5A). A clear band of approximately 26 kDa was detected in isolated periovulatory granulosa cells as well as in whole ovary. To test if PGRMC1 is involved in control of apoptosis induction in periovulatory rat granulosa cells, anti-PGRMC1 antibodies were included in the culture medium. None of the concentrations used resulted in an increased caspase-activity (Fig. 5B). On the contrary, there was a significant decrease in apoptosis in cells treated with antibodies as well as in cells treated with dilutions of pre-immunized rabbit serum (data not shown), which indicates an unspecific beneficial effect of serum on cell survival. To validate the blocking activity of the anti-PGRMC1 antibody, its effect on immature granulosa cells was investigated. In contrast to previously published data, inclusion of anti-PGRMC1 suppressed rather than induced apoptosis, regardless of presence of exogenous P4 in the medium (Fig. 5C). A similar dose-dependent decrease in caspase activity was seen after treatment with pre-immunized rabbit serum in immature cells (data not shown). Since anti-PGRMC1 has only been described to reverse an anti-apoptotic effect of P4, the effect of P4 on immature granulosa cells was investigated. In contrast to previously published data, P4 (10–1,000 nM) had no beneficial effect on the survival of immature granulosa cells (Fig. 5D). To investigate if the lack of effect of added P4 was due to a low intrinsic production of P4, the effect of cyanoketone (1 μM) was determined. Cyanoketone did not affect the degree of apoptosis and the effect was not affected by addition of P4 (Fig. 5E). To control for a possible presence of PGR or unspecific effects of Org 31710, immature granulosa cells were treated with 100 nM or 10 μM Org 31710.

7 As expected, the low dose of Org 31710 had no effect on caspase activity, while the high dose significantly induced apoptosis. Discussion In this study, it was hypothesized that PGR is involved in regulating rat periovulatory granulosa cell apoptosis. The results presented demonstrate that PGR antagonists specifically and reversibly induce apoptosis without involving other possible signaling pathways of P4. In contrast, no support for the involvement of the proposed progesterone-binding protein PGRMC1 could be found. RU 486 is a well-characterized antiprogestin that also possesses antiglucocorticoid actions [31], whereas Org 31710 on the other hand is more specific for the PGR [32]. Both antagonists have been used previously to study granulosa cell apoptosis [6, 7, 20, 33]. However, the physiological relevance of previous findings have been questioned as the concentrations used have been in the micromolar range [21]. The expression of PGR in cultured periovulatory granulosa cells has been examined using microarrays and real-time PCR (P.A. Friberg et al, unpublished data). A high level of Pgr mRNA is present at the beginning of culture but the level declines sharply after 22 h. In addition, Org 31710 causes a slight but significant suppression of Pgr mRNA early in the culture. Here, Org 31710 and RU 486 were applied in a wide range of concentrations to examine if PGR plays a physiologically plausible role in apoptosis regulation. Low concentrations of the PGR antagonists, comparable to the culture levels of P4 (approximately 35–50 nM), indeed induced apoptosis. Supporting a specific effect on PGR by RU 486 and Org 31710, the induced apoptosis was fully reversible by the addition of the progestin R5020. To the best of our knowledge, the effects of nanomolar concentrations of PGR antagonists on apoptosis and a complete reversal by adding a progestin have not been demonstrated previously in periovulatory rat granulosa cells. The massive apoptosis induced by micromolar concentrations of the PGR antagonists was not reversed by R5020 indicating unspecific or toxic effects. In addition, while Org 31710 at 100 nM as expected had no effect in immature granulosa cells, there was a modest but significant effect of the micromolar concentrations. In addition to the classical transcriptional effects of PGR, P4 can also act via rapid non-genomic signaling pathways. Such rapid effects can be mediated by the recently characterized membrane progesterone receptor family discovered in the Thomas laboratory [34, 35]. Comprising three members now named progestin and adipoQ receptor family member (PAQR) 7 (formerly progestin membrane receptor α), PAQR8 (progestin membrane receptor β) and PAQR5 (progestin membrane receptor γ), these receptors are involved in diverse reproductive functions in mammals as well as in other vertebrates [16]. Current understanding of the PAQR family does not, however, include a role in apoptosis regulation, and a functional role for these proteins has not been investigated here. In the context of apoptosis, the most interesting candidates for rapid responses to P4 are the PGRMC1 and SERBP1 proteins. The functions, ligands and binding partners of PGRMC1 have not been clearly defined but a proposed hypothetical model states that PGRMC1 functions as a ligand and/or kinase-regulated signaling adapter molecule involved in membrane trafficking [26,

8 36]. PGRMC1/SERBP1 have been suggested to control apoptosis in granulosa cells of many developmental stages and have been proposed to provide an explanation as to why P4 can regulate apoptosis in cells where the PGR is not expressed [21, 23-25]. In this study, an additional objective was, therefore, to determine the possibility that the effects of PGR antagonists on apoptosis could be mediated indirectly via PGRMC1/SERBP1. PGR antagonists have been suggested to affect the production of P4 in luteal cells, possibly by decreasing the activity of the key steroidogenic enzyme HSD3B [37-39]. This decrease in available ligand for P4 membrane receptors has been proposed to explain the apoptosis induced by PGR antagonists [21]. In the primary cultured periovulatory granulosa cells used here, accumulation of P4 in spent medium was indeed decreased after treatment with Org 31710 (100 nM). However, the rather modest decrease in P4 synthesis is not necessarily enough to cause apoptosis, and it could in fact be the result of the increase in, rather than the cause of apoptosis. When cyanoketone was used to block P4 synthesis, apoptosis was indeed induced concurrently. However, the increase in caspase activity only reached comparable levels to those seen after treatment with the PGR antagonists when accumulated P4 was strongly reduced to below 5 nM. Therefore, the apoptosis induced by PGR antagonists cannot be explained by inhibition of P4 synthesis alone. Even at the 1 μM concentration of cyanoketone, the increase in apoptosis was fully reversible by addition of R5020 (5 nM), indicating a P4-specific action. Importantly, 5 nM of R5020 was enough to fully provide a P4-mediated anti-apoptotic response, which is reasonable considering that the KD of R5020 for PGR has been reported to be in the 1–4 nM range [40, 41]. In fact, specific phosphorylation of PGR can render the receptor even more sensitive to progestins, below 1 nM [42]. In comparison, the reported P4 binding sites of PGRMC1 have KD values of 11 nM and 286 nM for P4, respectively [43], while affinity for R5020 has not been reported. The PAQR family members have demonstrated KD values of 5 nM for P4, but they have much lower affinity for R5020 and RU 486 with values in the micromolar range, at least regarding PAQR7 [16, 44]. It follows that apoptosis induced by cyanoketonemediated removal of P4 is unlikely mediated via PGRMC1 or the PAQR family. The results presented here (Fig. 4) clearly show that cyanoketone more potently induces apoptosis than either Org 31710 or RU 486. These data apparently speak in favor of the effects of P4 that are mediated by other receptors in addition to the PGR. However, the additional effect of cyanoketone was surprisingly completely abolished when co-treated with the PGR antagonists Org 31710 or RU 486. A possible explanation for this apparent anomaly is that the anti-apoptotic effect of PGR antagonists in combination with cyanoketone could be explained by their partial agonistic properties [45-47], which only become apparent in the absence of the natural ligand (P4). The effect of cyanoketone could thus be interpreted as the response to a complete lack of PGR signaling, whereas the effect of PGR antagonists is only a partial reduction of the normal PGR response. Partial agonism of RU 486 is only mediated via the PGR-B form and not via PGR-A [45, 46] and could involve cross-talk with other transcription factors, which is often independent of whether the PGR ligand is an agonist or antagonist [48-51]. Interestingly, if this theory holds true, it strengthens the hypothesis that PGR is the dominant mediator of P4regulated apoptosis.

9 There is a possibility that there are parallel effects of P4 mediated both via PGR and via PGRMC1/SERBP1. Several reports from the laboratories of Peluso and Wheling have shown that an anti-PGRMC1 antibody specifically detects the PGRMC1 protein and that it completely blocks the anti-apoptotic effects of P4 in their models [21, 24]. In our hands, this antibody clearly detected a single band of the expected size, demonstrating its functional integrity and protein expression in our model system. The expression of both Pgrmc1 and Serbp1 has also been investigated (P.A. Friberg et al, unpublished observations), including mRNA data from microarray as well as real-time PCR, where the levels of both transcripts were high and unaffected by treatment with Org 31710. In spite of an apparently functional antibody, we saw no induction of apoptosis at any of the concentrations tested. In fact, the degree of apoptosis was decreased to approximately 90% of control, similar to the effect of pre-immunized rabbit serum, indicating an unspecific effect of serum proteins. Similar results were also obtained using immature rat granulosa cells. Interestingly, while previous studies have reported an anti-apoptotic effect of P4 in immature granulosa cells [33] and SIGCs [52], in our hands, addition of P4 had no effect on caspase activity in immature cells. This was not due to presence of P4 in the medium as cyanoketone, in contrast with previous studies [33], also failed to induce apoptosis. To summarize, we found no support for a role of PGRMC1 in the regulation of periovulatory or immature granulosa cell apoptosis, as we were neither able to induce apoptosis nor reverse an anti-apoptotic effect of P4 using anti-PGRMC1. Importantly, our data do not support an effect of P4 in immature granulosa cells, as has previously been suggested. In conclusion, the present study indicates that PGR is the primary mediator of P4-regulated apoptosis. Several lines of evidence support this conclusion: 1) the reversible induction of apoptosis by nanomolar concentrations of PGR antagonists, 2) the reversal of apoptosis induced by cyanoketone by 5 nM of R5020 in relation to reported affinities of R5020 to different P4 receptors, and 3) the ability of PGR antagonists to limit the effect of cyanoketone. In contrast, we were unable to find support for PGRMC1 as a regulator of apoptosis, or for P4 as a regulator of immature granulosa cell apoptosis. The role of PGRMC1 in this process is therefore uncertain. This work solidifies the role of P4 as a survival factor in the rat periovulatory interval, however, its biological significance in vivo and relative importance compared to other survival factors is still to be determined. Based on these data, future studies investigating the cellular events connecting progesterone to apoptosis in the rat periovulatory interval should concentrate on the transcriptome. Acknowledgements The authors are grateful to Ralf Lösel and Martin Wehling for sharing the antibody against PGRMC1, and to Ruijin Shao and Birgitta Weijdegård for providing assistance with immunoblotting and progesterone assays, respectively. The authors are also grateful to Erik Kristiansson for valuable statistical discussions. Org 31710 was generously provided by Organon, Oss, The Netherlands and RU 486 by Exelgyn, Paris, France. References 1. Chaffin CL, Stouffer RL. Local role of progesterone in the ovary during the periovulatory interval. Rev Endocr Metab Disord 2002; 3: 65-72.

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13 51. Kalkhoven E, Wissink S, van der Saag PT, van der Burg B. Negative interaction between the RelA(p65) subunit of NF-kappaB and the progesterone receptor. J Biol Chem 1996; 271: 62176224. 52. Peluso JJ, Fernandez G, Pappalardo A, White BA. Characterization of a putative membrane receptor for progesterone in rat granulosa cells. Biol Reprod 2001; 65: 94-101. Figure legends Figure 1. Effect of PGR antagonists Org 31710 (A) and RU 486 (B) on apoptosis measured as caspase 3/7 activity in rat periovulatory granulosa cells. (C) Reversibility of caspase 3/7 activity induced by Org 31710 through co-treatment with progestin R5020. Bars represent mean ± SEM. GLM followed by single-sided Dunnet’s post hoc test in (A) and (B). GLM followed by Bonferroni’s post hoc test in (C). **, P < 0.01, ***, P < 0.001. Different superscript letters in (C) represent significantly different treatment groups, P < 0.05. Figure 2. Effect of the progestin R5020 on apoptosis measured as caspase 3/7 activity in rat periovulatory granulosa cells. Bars represent mean ± SEM. GLM followed by single-sided Dunnet’s post hoc test. ***, P < 0.001. Figure 3. (A) Effect of HSD3B-inhibitor cyanoketone on accumulation of P4 in spent medium after 22 hours. The 30 and 100 nM concentrations were outside of the standard curve of the assay. a, P < 0.01 in only one out of three experiments. (B) Effect of cyanoketone on apoptosis measured as caspase 3/7 activity in rat periovulatory granulosa cells. (C) Reversibility of caspase 3/7 activity induced by cyanoketone by co-treatment with progestin R5020. Bars represent mean ± SEM. GLM followed by single-sided Dunnet’s post hoc test in (A) and (B). GLM followed by Bonferroni’s post hoc test in (C). ***, P < 0.001. Different superscript letters in (C) represent significantly different treatment groups, P < 0.001. Figure 4. Effect of HSD3B-inhibitor cyanoketone (Cyk) and PGR antagonists Org 31710 (A) or RU 486 (B), alone or in combination, on apoptosis measured as caspase 3/7 activity in rat periovulatory granulosa cells. Bars represent mean ± SEM. GLM followed by Bonferroni’s post hoc test. Different superscript letters represent significantly different treatment groups, P

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