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BOR - Papers in Press, published online ahead of print April 6, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.041061
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BIOLOGY OF REPRODUCTION 73, 261–270 (2005)
DOI: 10.1095/biolreprod.105.041061
© 2005 by the Society for the Study of Reproduction, Inc.

Expression and Function of PAIRBP1 Within Gonadotropin-Primed Immature Rat Ovaries: PAIRBP1 Regulation of Granulosa and Luteal Cell Viability1

John J. Peluso 2 3 , Anna Pappalardo 2, Ralf Losel 4, and Martin Wehling 4,5 

Departments of Cell Biology2 Obstetrics and Gynecology,3 University of Connecticut Health Center, Farmington, Connecticut 06030 Faculty of Clinical Medicine Mannheim,4 Institute of Clinical Pharmacology, University of Heidelberg, Mannheim, D-68167 Germany Medicine/Experimental Medicine,5 AstraZeneca R&D, S48183 Molndal, Sweden


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The protein PAIRBP1, which was initially referred to as RDA288, is involved in mediating the antiapoptotic action of progesterone (P4) in spontaneously immortalized granulosa cells (SIGCs). The present studies were designed to assess the expression and function of PAIRBP1 in the different cell types within the immature rat ovary. Western blot analysis detected PAIRBP1 within whole-cell lysates of immature rat ovaries. Equine gonadotropin (eCG) induced a 3-fold increase in ovarian levels of PAIRBP1. Moreover, human chorionic gonadotropin (hCG), given 48 h after eCG, maintained these elevated levels for up to 4 days. Immunohistochemical analysis confirmed this and further demonstrated that interstitial, thecal, and surface epithelial cells also expressed PAIRBP1. The level of PAIRBP1 in these cells was not influenced by gonadotropin treatment. In contrast, eCG stimulated an increase in PAIRBP1 within the granulosa cells of the developing follicles. Treatment with hCG induced ovulation and ultimately the formation of corpora lutea (CL). High levels of PAIRBP1 expression were also observed within the luteal cells. Immunocytochemical studies on living, nonpermeabilized granulosa and luteal cells revealed that some PAIRBP1 localized to the extracellular surface of these cells. The presence of PAIRBP1 on the extracellular surface was consistent with the observation that an antibody to PAIRBP1 attenuated P4's antiapoptotic action in both granulosa and luteal cells. Although the PAIRBP1 antibody attenuated P4's action, it did not reduce the capacity of cells to specifically bind 3H-P4. Immunoprecipitation with the PAIRBP1 antibody pulled down the membrane P4 binding protein known as progesterone receptor membrane complex-1 (PGRMC1; rat homolog accession number AJ005837). Taken together, these findings suggest that gonadotropins regulate the expression of PAIRBP1 in granulosa and luteal cells and that PAIRBP1 plays an important role in mediating P4's antiapoptotic action in these ovarian cell types. The exact mechanism of PAIRBP1's action remains to be elucidated, but it may involve an interaction with PGRMC1.

apoptosis, corpus luteum, granulosa cells, luteal cell, progesterone, signal transduction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Progesterone (P4) plays an important role in regulating the viability of both granulosa [1] and luteal cells [2–4]. While part of P4's actions could be due to its ability to regulate gonadotropin levels [5, 6], P4 also acts directly on these ovarian cells to maintain their structural integrity and steroidogenic capacity. Rothchild was the first to propose this intraovarian site of action for P4 [3]. In his 1981 review, he outlined a series of experiments that suggested that P4 directly enhances luteal cell function [3]. Since then, several labs have provided mechanistic data to support this concept. For example, studies on primate luteal cells have demonstrated that these cells 1) express the nuclear progesterone receptors (nPRs) [7]; 2) show a decrease in nPR expression coincident with luteal regression [7–9]; 3) bind P4 with high affinity [2] 4) are prevented from undergoing apoptosis by P4 at nanomolar doses [10], consistent with the observed kd for P4 binding [2] and 5) undergo apoptosis in the presence of a large molar excess of RU486, a nPR antagonist [10, 11]. It is important to appreciate that, although these observations are consistent with a nPR mediation of P4's action, they do not conclusively demonstrate that nPRs are essential for P4's actions.

Support for an alternative to a nPR-mediation of P4's actions comes from studies of rat corpora lutea (CL). For example, P4 reduces the rate of apoptosis in the CL of pregnancy [4]. In addition, injections of RU486 promote the demise of the CL of pregnancy [12]. Like the studies involving primate luteal cells, these observations are consistent with an involvement of nPR. However, the rat luteal cells do not express nPRs [13, 14]. These findings call into question the mechanism through which P4 promotes luteal cell viability.

Discrepancies in mechanism of P4's antiapoptotic action also exist with regard to granulosa cells. P4 prevents apoptosis of granulosa cell isolated from follicles before [1, 15, 16] as well as after the LH surge [17, 18]. Importantly, only granulosa cells isolated after the LH surge express nPR [14, 18]. Therefore, in granulosa cells isolated before the LH surge, P4 must be able to activate an antiapoptotic pathway that is independent of the nPRs.

The mediator of P4's action in immature granulosa cells and spontaneously immortalized granulosa cells (SIGCs) is unknown. We have recently suggested that the protein, referred to as either RDA288 or PAI-1 mRNA binding protein 1 (PAIRBP1; accession number XM_216160), might be involved [19]. PAIRBP1 has been implicated by the observations that 1) overexpression of PAIRBP1 in SIGCs increases 3H-P4 binding and responsiveness to P4 and 2) an antibody to PAIRBP1 ablates P4's antiapoptotic action in these cells [19]. Therefore, the present studies were designed to assess the expression and function of PAIRBP1 within granulosa and luteal cells of immature gonadotropin-primed rats.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Ovarian Cell Cultures

Immature female Wistar rats (21 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). To monitor the effect of gonadotropins on PAIRBP1 expression, 23- to 25-day-old immature rats were injected with eCG (20 IU given i.p.) and hCG (10 IU given i.p.) as previously described [20]. Groups of three rats were autopsied at selected times after gonadotropin treatment, and one ovary was removed, trimmed of fat, and fixed in formalin for immunohistochemical analysis of PAIRBP1 expression. Whole-cell lysate was prepared from the remaining ovary and used for analysis of PAIRBP1 expression by Western blot [21].

For the culture studies, granulosa cells were obtained from antral follicles of immature animals that were 23 or 25 days of age, as previously described [22], with the exception that the granulosa cells were not separated into small and large granulosa cells by Percoll-gradient centrifugation. The granulosa cells were plated in 0.5 ml of medium at 1.25 x 105 cells/ml in eight-chamber glass lab-tek slides (Nunc Inc., Naperville, IL). The cells were initially cultured in Dulbecco modified Eagle medium (DMEM)/F-12 supplemented with 5% fetal bovine serum for 24 h. The serum-supplemented medium was removed and the cells rinsed with three changes of serum-free medium and then cultured in serum-free DMEM/ F-12 with various reagents for 5 additional h.

Luteal cells were isolated from eCG/hCG-treated rats 96 h after hCG using a modification of the protocol published by Quirk et al. [23]. Briefly, ovaries containing the CL were punctured several times with 25-gauge needles and then incubated in a 0.25% Trypsin/1 mM EGTA solution for 15 min at 37°C in the 5% CO2 atmosphere to remove the ovarian surface epithelial cells [23]. The CL within the ovaries were teased away from the ovarian connective tissue using 25-gauge needles, incubated with a DNase (0.8 mg/ml)/Collagenase (0.01 g/ml) solution for 1 h at 37°C in the 5% CO2 atmosphere, and sequentially incubated in EGTA and EGTA/ sucrose [22]. The CL were then pressed with a cell scraper to release the isolated luteal cells. Most of the cells were considered to be luteal cells based on their morphological and Nile Red-staining characteristics. 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 rinsed with three changes of serum-free medium and then cultured in serum-free DMEM/F-12 with various reagents for an additional 5 h. In some studies, the media was collected after the 5-h culture period, frozen, and then stored at –20°C until assayed for P4 using a RIA kit provided by Diagnostics Products Corp (Los Angeles, CA). The P4 values are expressed as a mean nM ± SEM (n = 4–6). These protocols were approved by the Animal Care Committee of the University of Connecticut Health Center.

Spontaneously Immortalized Granulosa Cell (SIGC) Culture

SIGCs were generously provided by Dr. Robert Burghardt of Texas A & M University (College Station, TX) and cultured as previously described [24]. Prior to use, the serum-supplemented medium was removed and the cells were rinsed with three changes of serum-free medium and then whole cell lysates prepared for Western blot analysis [21].

Detection of Apoptotic Cells

The percentage of cells with apoptotic nuclei was assessed by in situ staining using the nuclear dye YOPRO-1 [24]. To stain granulosa 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 fluorescein isothiocyanate (FITC) filter set. The number of green fluorescent cells (i.e., apoptotic cells) in a field was counted. The total number of cells in that field was also counted under phase optics. A total of 100 cells per well were counted. The percentage of apoptotic cells was then calculated.

A similar protocol was used to assess luteal cells. The only modification was that the cells were stained with YOPRO-1 and Nile Red (5 µg/ ml) [25–27]. Nile Red stained the lipid droplets, allowing for the identification of well-differentiated luteal cells [25, 26]. Most of the cells isolated from eCG/hCG-treated rats were well-differentiated luteal cells, as judged by phase microscopy and Nile Red staining. A total of 100 Nile Red-stained cells per well were counted. The percentage of apoptotic cells was then calculated.

Western Blot Analysis of Ovarian PAIRBP1 Levels

Ovarian lysates were prepared as previously described [21]. Granulosa cells, luteal cells, and SIGCs were lysed in RIPA buffer (50 mM TRIS, 150 mM sodium chloride, 1.0 mM EDTA, 1% Nonidet P40, and 0.25% sodium-deoxycolate; 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) and then centrifuged at 16 000 relative centrifugal force at 4°C for 5 min. Protein concentration was determined using a Bio-Rad protein determination kit.

After all the ovarian samples were collected, equal amounts of protein were loaded onto gels. For this study, there were 15 samples (five time points with three animals/point). Aliquots of each sample were used to assess for one of four end points (PAIRBP1 Western; PAIRBP1 negative control; ß-actin Western; and ß-actin negative control). Because all 15 samples could not be loaded on one 10-lane gel, this experiment required a total of eight mini-gels. The samples for the PAIRBP1 (two gels) and PAIRBP1 negative control (two gels) were run on 10% acrylamide gels at the same time in two electrophoresis chambers and then transferred to nitrocellulose.

The nitrocellulose was incubated with 5% nonfat dry milk overnight at 4°C and then incubated with PAIRBP1-1 antibody at a dilution of 1:2000 for 1 h at room temperature. This antibody was generated in chickens by Aves Labs (Tigard, OR) against the peptide sequence KQLRKESQKDRKN (amino acids #68–80). This antibody detects a single band that corresponds to the PAIRBP1 protein [19]. After exposure to PAIRBP1-1, the nitrocellulose blot was processed for Western blot analysis using a horseradish peroxidase goat anti-chicken IgY (1:50 000; Aves Labs) and KPL LumiGlo detection system. As recommended by Aves Labs, an immunodeplete antibody preparation was used in place of the PAIRBP1-1 antibody and served as a negative control. This preparation is the effluent obtained as a consequence of purifying the PAIRBP1-1 antibody by affinity column chromatography. Thus, it has all of the proteins that are present in the PAIRBP1-1 preparation except the PAIRBP1-1 antibody. IgY was also used to replace PAIRBP1-1 antibody and yielded similar results to that obtained with the immunodeplete antibody preparation. To ensure equal loading, the level of ß-actin was assessed in an aliquot from each ovarian sample. In this Western blot, ß-actin antibody (Clone AC-15; Sigma Chemical Co.) was used at a dilution of 1:1000.

It is important to appreciate that the blots were processed at the same time under identical conditions and then placed in the same cassette. As a result, the films generated for the PAIRBP1 and the PAIRBP1 negative controls were processed and exposed exactly the same. Because the processing and exposure were exactly the same, valid comparisons between treatment groups can be made. An identical approach was used to assess ß-actin.

To assess the effect of gonadotropins on the level of PAIRBP1 protein, the Western blots were analyzed in a semiquantitative manner. Specifically, the films were scanned into the computer. The average intensity (grayscale value/pixel) of each band that corresponded to PAIRBP1 was determined using IPGel software (Scanalytics, Vienna, VA). The average intensity of areas adjacent to the PAIRBP1 bands was also determined and these background values were subtracted from the PAIRBP1 band intensity. Only films with band intensities within the linear range were assessed (i.e., grayscale values of 250 to 0). The mean intensity of the PAIRBP1 bands ± one SEM for each treatment group was calculated.

Immunochemical Localization of PAIRBP1

For immunohistochemical assessments, rat ovaries were sectioned at 5 µm and mounted on glass slides. Representative slides from each ovary were stained at the same time, as outlined in the following protocol. Endogenous peroxidase activity was quenched by incubating the slides in 0.3% hydrogen peroxide in methanol for 30 min at room temperature. Slides were then incubated in BlokHen (Aves Labs) for 1 h at room temperature to reduce nonspecific staining and then incubated overnight at 4°C with PAIRBP1-1 antibody at 1:500 dilution (3.4 µg/ml). The slides were then incubated with biotinylated goat-anti-chicken IgY for 30 min at room temperature, washed in PBS, and incubated with ABC reagent for 30 min at room temperature. The slides were developed using a diaminobenzidine-peroxidase substrate for 5 min. Finally, the slides were counterstained with Methyl Green for 10 sec, rinsed in distilled water, dehydrated, cleared, and mounted. As a negative control, an immunodeplete antibody preparation replaced PAIRBP1-1 antibody in this immunohistochemical protocol. The presence of PAIRBP1 was revealed by the presence of a reddish-brown precipitate.

To determine whether PAIRBP1 was localized to the extracellular surface of the plasma membrane, granulosa and luteal cells were cultured for 24 h as previously described. Then these living, nonpermeabilized, nonfixed cells were stained as outlined by Peluso et al. [28]. Briefly, the cells were rinsed with PBS and then incubated in the presence of either 34 µg/ ml of PAIRBP1-1 antibody (in 8% BSA/PBS) or immunodeplete antibody preparation for 15 min at room temperature. After this incubation, the cells were washed in PBS and incubated with FITC-IgG (1:100 in 8% BSA/ PBS) for 15 min in the dark at room temperature. The cells were then washed with PBS and observed under phase and standard epifluorescent optics using a FITC filter set. This approach takes advantage of the fact that antibodies cannot enter living cells; thus, any staining detects proteins localized to the extracellular surface of the plasma membrane.

Immunoprecipitation Using PAIRBP1-1 Antibody

The immunoprecipitation protocol using the PAIRBP1-1 antibody was done according to the instructions provided by Aves Labs. Briefly, approximately 2 mg of whole-cell lysate was prepared from SIGCs and incubated with gentle agitation for 30 min with 50 µl of PrecipHen (Aves Labs). The suspension was then centrifuged to remove any nonspecifically bound proteins. The precleared lysates were then incubated for 1 h at 4°C with either 5 µg of PAIRBP1-1 antibody or 5 µg of PAIRBP1 immunodeplete antibody preparation. Then 100 µl of PrecipHen was added to each preparation and incubation continued at 4°C for an additional 18 h. The incubation tubes were centrifuged for 5 min at 4°C and the supernatant discarded. The pellet was washed twice and then Laemmli buffer added to the pellet. The suspension was then boiled for 5 min and then half of the suspension loaded onto one of two 12% acrylamide gels. After electrophoresis, the proteins in the gel were transferred to nitrocellulose. The blots were then probed with either the PAIRBP1-1 or a 1:2000 dilution of PGRMC1 antibody [29]. The PGRMC1 antibody was a rabbit antibody directed against a 16 amino acid peptide (MAAEDVAATGADPSEL) with C-terminal cysteines conjugated to keyhole limpet hemocyanine. This sequence represents amino acids #53–66 and is part of the extracellular domain of PGRMC1. PAIRBP1 and PGRMC1 were detected as previously described for Western blot analysis.

Ligand Binding Studies

The protocol used to assess total and specific binding of 3H-P4 to SIGCs has been previously described [21]. For this procedure, 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 (10 mM TRIS-HCl, pH 7.4, 1.5 mM EDTA, 10% glycerol, 25 mM sodium molybdate, and 1 mM dithiothreitol). After 30 min, 1,2,6,7-3H-progesterone (1 nM 3H-P4, 50 000 cpm, SA = 86 Ci/mmol; Amersham, Arlington Heights, IL) and either vehicle or 1 mM P4 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), rinsed twice with 1 ml cold PBS, and then the filter counted in a scintillation counter. Specific binding was determined by subtracting the dpm obtained in the presence of 1 mM P4 from the dpm obtained in the absence of P4. Means ± one SEM of each P4 binding parameter was calculated for each treatment group (n = 5–8/group).

Statistical Analysis

All experiments were repeated at least three times, with each experiment yielding essentially identical results. For studies involving apoptosis, an attempt was made to get enough granulosa and luteal cells so that quadruplicate cultures for each treatment could be run on each day. However, for some experimental replicates, only duplicate cultures for each treatment were run. Regardless of the number of replicates assessed per treatment, the entire experiment was conducted at least three times. The data from each experiment were pooled and analyzed by either a two-way or a one-way ANOVA followed by a Student-Newman-Keuls test. P values of less than 0.05 were considered to be significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of PAIRBP1 within the Gonadotropin-Primed Immature Rat Ovary

Western blot analysis confirmed that PAIRBP1 was present in immature rat ovaries (Fig. 1A). Moreover, eCG treatment induced a nearly 3-fold increase in ovarian PAIRBP1 levels within 24 h (P < 0.05). PAIRBP1 levels continued to increase in response to both eCG and hCG (Fig. 1, B and C). Unlike PAIRBP1, ß-actin levels did not change in response to gonadotropin treatment (Fig. 1B). In a second experiment, 23-day-old rats were either not treated or treated with eCG and hCG and autopsied 96 h after hCG (i.e., when the rats were 29 days of age). As expected, the PAIRBP1 levels within gonadotropin-treated ovaries was several-fold greater than that observed within the ovaries of nontreated 29-day-old rats (data not shown).



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  		FIG. 1. The effect of equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) on ovarian levels of PAIRBP1. For this study, immature female rats 23 days of age were treated with eCG (20 IU i.p.) and then either autopsied at 0, 24, and 48 h after eCG or injected with hCG (10 IU i.p.) 48 h after eCG. The eCG-hCG-treated rats were autopsied at 24 or 96 h after hCG. A) A representative Western blot from a nontreated immature rat ovary is shown demonstrating that the PAIRBP1-1 antibody (+) detects a single band of approximately 55 kDa that corresponds to PAIRBP1. A negative control (–) is also shown. B) A representative Western blots for PAIRBP1 and ß-actin for each gonadotropin treatment. C) Bar graph depicting the relative level of ovarian PAIRBP1 in each treatment group. Values are expressed as a mean ± one SEM (n = 3/group). Asterisks indicate a value that is significantly greater than the 0-h control group

Immunohistochemical analysis revealed that PAIRBP1 was detected in interstitial, thecal, and surface epithelial cells (compare Fig. 2A with 2, B and C). However, the level of PAIRBP1 expression in these cells was not influenced by gonadotropin treatment. In contrast, PAIRBP1 expression in granulosa cells was regulated by gonadotropins. PAIRBP1 was barely detectable within granulosa cells of preantral follicles (Fig. 2D) but readily detectable within the cytoplasm of granulosa cells of nonatretic antral follicles (Fig. 2E). The level of PAIRBP1 expression continued to increase in granulosa cells throughout eCG-induced follicular development (Fig. 2F). However, PAIRBP1 was only detected within a few granulosa cells of atretic antral follicles (Fig. 2E).



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  		FIG. 2. PAIRBP1 expression within the ovary of immature and gonadotropin-primed rats. A) The image was stained using the immunodeplete antibody preparation and counterstained with methyl green (negative control). B–F, H–J, L) Images are stained using the PAIRBP1-1 antibody and counterstained with methyl green. G, K) Images were stained with hematoxylin-eosin. A) A negative control section of an immature rat ovary. Note that that the oocytes show nonspecific staining while none of the other ovarian cells types are stained in the absence of the PAIRBP1-1 antibody. B) An adjacent section to the one shown in A that was stained with PAIRBP1-1 antibody. PAIRBP1 staining was most intense in the interstitial tissue and thecal cell layers. C) The surface epithelial cells also stained intensely for PAIRBP1. D) A nonatretic preantral follicle. Note the intense PAIRBP1 staining within the thecal cells with very minimal staining in a few granulosa cells. E) A nonatretic (left side) and an atretic (right side) antral follicle within an immature rat ovary. The atretic follicle was identified by the numerous pyknotic nuclei within the antrum and membrane granulosa cell layers. Some pyknotic nuclei are marked with arrowheads. Most membrane granulosa cells of the atretic follicle were not stained for PAIRBP1. In contrast, the membrane granulosa cells of the nonatretic follicle stained for PAIRBP1. The thecal cells associated with both nonatretic and atretic follicles were intensely stained for PAIRBP1. F) PAIRBP1 staining in the membrane granulosa cell layers of an antral follicle 24 h after treatment with eCG. G) A hematoxylin-eosin-stained section of a recently ovulated follicle within an eCG-primed rat ovary 24 h after an hCG injection. Note the site of follicular rupture. H) An adjacent section of the ovulated follicle shown in G. The cells within this follicle are intensely stained for PAIRBP1 although they are small and morphologically similar to granulosa cells of antral follicles. I) Corpora lutea observed 96 h after hCG treatment. These corpora lutea are completely luteinized with the luteal cells most intensely stained for PAIRBP1 localized to the periphery. J) A higher magnification of the image shown in I. This image shows that there are intensely stained luteal cells within the central area but that their frequency is reduced compared with the peripheral area of these corpora lutea. Also note the intense PAIRBP1 staining of the surface epithelial cells. K, L) Adjacent sections stained with either hematoxylin-eosin (K) or PAIRBP1-1 antibody (L). The luteal cells in K possess a large cytoplasmic-to-nuclear ratio. The cytoplasm is very eosinophilic and stains pink. In L, the cytoplasm of the well-differentiated luteal cells stain intensely for PAIRBP1. Bar = 400 µm for A, B; 25 µm for C; 20 µm for D, F, H, K, L; 40 µm for E; 100 µm for G; 200 µm for I; 50 µm for J

An injection of hCG induced the preovulatory follicles that were present 48 h after eCG to ovulate (Fig. 2G). In these ovulating follicles, PAIRBP1 expression was detected in nearly all of the granulosa cells, but the highest expression was associated with the cells nearest the antrum (Fig. 2H). Similarly, most cells within mature CL that were present 4 days after hCG expressed PAIRBP1 (Fig. 2I). Interestingly, the cells that expressed the highest levels of PAIRBP1 were localized to the periphery of the luteal structure (Fig. 2J). There was also considerable variability in the level of expression between luteal cells within these mature corpus lutea (Fig. 2, K and L).

Regardless of the level of expression, PAIRBP1 localized to the cytoplasm and not the nucleus of the granulosa (Fig. 2, E and F) and luteal cells (Fig. 2L). Moreover, at least some of the PAIRBP1 was localized to the extracellular surface of both granulosa and luteal cells (Fig. 3). This was demonstrated by the immunocytochemical detection of PAIRBP1 on the extracellular surface of living nonpermeabilized granulosa and luteal cells.



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  		FIG. 3. Localization of PAIRBP1 to the extracellular surface of granulosa (A, B) and luteal (C–F) cells. In this and other figures, granulosa cells were isolated from immature rats and luteal cells from eCG- and hCG-treated rats 96 h after hCG. Phase images are shown in A, C, and E and respective fluorescent images in B, D, and F. The images in B and D indicate that PAIRBP1 localizes to the extracellular surface of both granulosa and luteal cells. Phase and fluorescent images of a negative control are shown in E and F, respectively. Bar = 20 µm

Involvement of PAIRBP1 in P4's Antiapoptotic Action

P4 at a concentration of 10 nM was sufficient to suppress granulosa cell apoptosis (Fig. 4A). Treatment with PAIRBP1-1 antibody but not IgG attenuated the antiapoptotic action of P4 in granulosa cells (Fig. 4B). If the dose of P4 was increased to 1 µM, then P4 overrode the effects of the PAIRBP1-1 antibody (Fig. 4C).



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  		FIG. 4. The effect of PAIRBP1-1 antibody (34 µg/ml) or IgG (34 µg/ml) on the ability of progesterone (P4) to inhibit granulosa cell apoptosis. The effect of increasing concentrations of P4 on granulosa cell viability is shown in A. The effect of PAIRBP1-1 antibody on the ability of low (10 and 100 nM) and high (1 µM) doses of P4 to promote granulosa cell viability are shown in panels B and C, respectively. Values are expressed as a mean ± one SEM. The * identifies a value that is different from the control group. In B, ** indicates values are different from their respective P4+IgG control

Similarly, the PAIRBP1-1 antibody blocked P4's antiapoptotic action in luteal cells, but these studies were complicated by two factors. First, luteal cells cultured in serum-free media for 5 h secreted about 10 nM of P4 (Fig. 5A). This amount of P4 was sufficient to inhibit apoptosis. This issue was addressed by supplementing the culture media with aminoglutethamide. In the presence of aminoglutethamide, the amount of P4 secreted in both granulosa and luteal cell cultures was reduced (P < 0.05) with P4 levels in luteal cell cultures being less than 5 nM (Fig. 5A). The second factor related to identifying apoptotic luteal cells. This was resolved by costaining the cells with Nile Red, which detects lipids that are characteristic of luteal cells, and YOPRO-1, which detects apoptotic nuclei (Fig. 5B). As can be seen in Figure 5C, aminoglutethamide nearly doubled the percentage of apoptotic luteal cells cultured for 5 h compared with controls (Fig. 5C). In the presence of aminoglutethamide, P4 reduced luteal cell apoptosis in a dose-dependent manner, with 10 nM being a minimal effective dose (Fig. 6A). Treatment with PAIRBP1-1 antibody attenuated the antiapoptotic action of P4 in luteal cells (P < 0.05) (Fig. 6B). As with granulosa cells, 1 µM P4 ablated the effect of PAIRBP1-1 antibody treatment on the viability of luteal cells (Fig. 6C).



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  		FIG. 5. The effect of aminoglutethamide (AG, 0.2 µM) on progesterone (P4) secretion by granulosa (Gran) and luteal cells over a 5-h culture period (A) and apoptosis (B, C). Two-way ANOVA indicated that luteal cells secreted more P4 than granulosa cells (P < 0.05) and that aminoglutethamide significantly suppressed P4 secretion (P < 0.05). B) A luteal cell preparation stained with Nile Red and YOPRO-1. The cells were observed under a FITC filter set. The luteal cells are identified by the presence of Nile Red-stained lipid droplets, which fluoresce orange under the FITC filter set. The apoptotic nuclei are revealed by the YOPRO-1 staining, which fluoresces green. Bar = 100 µm. C) The effect of AG on the rate of luteal cell apoptosis. * indicates a value greater than control (cont)



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  		FIG. 6. The effect of aminoglutethamide (AG; 0.2 µM; A) and PAIRBP1-1 antibody (B) on P4's antiapoptotic action in rat luteal cells. The PAIRBP1-1 antibody and IgG were used at 34 µg/ml. A) The effect of AG and increasing doses of P4 on luteal cell apoptosis is shown. Values are means ± one SEM. In this panel, * indicates a value that is less than control. The effect of low (10 and 100 nM) and high (1 µM) P4 in the presence or absence of PAIRBP1-1 antibody is shown in B and C, respectively. The * identifies a value that is different from both the IgG and PAIRBP1-1 antibody control. The ** indicates the value is different from the IgG+P4 treatment

Although PAIRBP1-1 antibody was capable of preventing P4's action, it failed to reduce the amount of 3H-P4 that was bound to SIGCs. This inability to affect 3H-P4 was observed for both total and specific 3H-P4 binding (Fig. 7). This observation suggested that PAIRBP1 may not be the P4 binding protein but rather might be interacting with an unknown P4 binding protein. To test this, SIGC lysate was immunoprecipitated using the PAIRBP1-1 antibody. As would be predicted, PAIRBP1-1 selectively immunoprecipitated PAIRBP1 (Fig. 8A). Probing the PAIRBP1 immunoprecipitate with the PGRMC1 antibody revealed a 28-kDa protein, which corresponded to PGRMC1. This protein was not detected in the immunoprecipitates obtained using the immunodeplete antibody preparation (Fig. 8B). The PGRMC1 antibody did detect higher molecular-weight proteins in the immunoprecipitates obtained from both the PAIRBP1-1 antibody and the immunodeplete antibody preparations. Some of these higher molecular-weight proteins most likely corresponded to the heavy and light chains of IgG, which are present in both immunoprecipitates in great abundance.



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  		FIG. 7. The effect of PAIRBP1-1 antibody on total (open bars), nonspecific (black bars), and specific (shaded bars) binding to SIGCs. The PAIRBP1-1 antibody and IgG were used at 34 µg/ml. Nonspecific binding was estimated from cells treated with 1 mM P4 and specific binding was calculated by subtracting nonspecific binding from total 3H-P4 binding



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  		FIG. 8. The interaction between PAIRBP1 and PGRMC1 as revealed by coimmunoprecipitation. In this study, lysates were prepared from SIGCs and immunoprecipitated using the PAIRBP1-1 antibody. As a control, the immunoprecipitation protocol was conducted using an immunodeplete antibody preparation as described in the text. The immunoprecipitates were run on a 12% acrylamide gels and then the proteins transferred to nitrocellulose. The blots were then probed with either the PAIRBP1-1 antibody or PGRMC1 antibody. As can be seen in A, immunoprecipitation with the PAIRBP1-1 antibody dramatically increases in the amount of PAIRBP1 that can be detected as marked by the arrow. B) The PGRMC1 antibody specifically detects a 28-kDa protein, which is the appropriate size of PGRMC1 (arrowhead)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our previous studies on the expression of PAIRBP1 have been limited to SIGCs and granulosa cells isolated from immature rat ovaries [19]. The present studies confirm and extend these findings in two important ways. First, the present studies demonstrate that not only granulosa cells but also luteal, thecal, interstitial, and surface epithelial cells express PAIRBP1. The immunohistochemical studies indicate that these ovarian cells express more PAIRBP1 than do granulosa cells. Thecal and interstitial cells rarely undergo apoptosis [30]. In fact, the thecal cells of atretic follicles are thought to ultimately form the interstitial cell clusters, which remain within the ovary for extended periods of time [31]. Like thecal/interstitial cells, ovarian surface epithelial cells do not frequently apoptose. Rather, only those surface epithelial cells associated with the site of follicular rupture at the time of ovulation have been shown to undergo apoptosis at any appreciable rate [26, 32]. It is possible that the high levels of PAIRBP1 within these cells could be involved in maintaining their viability because PAIRBP1 mediates P4's antiapoptotic action in granulosa and luteal cells (see subsequent discussion). However, the role, if any, that PAIRBP1 plays in regulating the viability of thecal, interstitial, and surface epithelial cells remains to be demonstrated.

The second important finding from the expression studies is that gonadotropins induce a large increase in ovarian levels of PAIRBP1. Based on immunohistochemical staining intensities, it appears that initial increase induced by eCG is associated with the granulosa cells of developing follicles. This is interesting in that granulosa cell apoptosis occurs most frequently in small antral follicles [33], when PAIRBP1 levels are low or nondetectable. As the follicles develop into preovulatory follicles in response to eCG, the rate of granulosa cell apoptosis decreases [17, 30]. This lower incidence of apoptosis observed in eCG-induced developing follicles correlates with the increased expression of PAIRBP1. In addition, follicular fluid levels of P4 are approximately 5 µM in small antral follicles and increase to about 12 µM in preovulatory follicles [34]. Moreover, the present in vitro studies clearly demonstrate that 1) P4 at doses well below the micromolar range suppresses granulosa cell apoptosis and 2) P4's antiapoptotic action is attenuated by PAIRBP1-1 antibody treatment. These observations strongly support the hypothesis that the simultaneous increase in P4 and PAIRBP1 account in part for the reduced frequency of apoptotic granulosa cells within developing antral follicles. In fact, the limiting factor does not appear to be the amount of P4 that is present but rather the amount of PAIRBP1 that is expressed within the granulosa cell layers of the developing follicle. This concept is consistent with the observations that few granulosa cells of atretic follicles express PAIRBP1.

Our previous studies have shown that P4 prevents apoptosis of granulosa cells isolated from antral follicles of immature rats [35]. It is important to appreciate that granulosa cells of antral and preovulatory follicles do not express the nPR. The nPRs are expressed after exposure to LH (hCG) but only transiently for a period of about 2–4 h during the periovulatory period [14, 17, 18, 36, 37]. During this time, the granulosa cells become more resistant to apoptosis [17, 18]. Based on studies that used nPR antagonists, it appears that the endogenous P4 secreted by the granulosa cells of periovulatory follicles acts through the newly expressed nPRs to promote their survival [17, 18].

Our studies reveal that PAIRBP1 levels are elevated in granulosa cells of preovulatory follicles and are maintained at an elevated level during the transformation of the preovulatory follicles into CL. It is possible that, during the periovulatory period (i.e., ≤12 h after hCG), P4 could act through both nPR- and PAIRBP1-dependent pathways to maintain the viability of granulosa cells. These dual pathways for P4's actions could account for the increased resistance to apoptosis and provide a fail-safe mechanism, which would ensure that granulosa cells ultimately develop into viable luteal cells. While this dual pathway concept is attractive, it is also possible that PAIRBP1 levels within the periovulatory follicles are transiently suppressed at the time when the nPR levels are elevated. If so, P4-regulated viability of the granulosa cells during the periovulatory period would be solely mediated through the nPR. Additional studies on the expression and function of PAIRBP1 and nPR throughout the periovulatory period must be conducted to resolve this issue.

The present studies also demonstrate that P4 inhibits apoptosis of rat luteal cells obtained from eCG-hCG-primed immature rats. This conclusion is supported by the observations that 1) aminoglutethamide reduces luteal cell P4 secretion below 10 nM, the minimum dose known to prevent apoptosis; and 2) aminoglutethamide also increases the percentage of luteal cells that undergo apoptosis; and 3) supplemental P4 reduces the percentage of apoptotic luteal cells cultured with aminoglutethamide compared with that observed in the absence of aminoglutethamide.

A similar antiapoptotic effect of P4 has been demonstrated for rat CL of pregnancy. Importantly, these rat luteal cells do not express nPRs [36]. How then might P4 mediate its antiapoptotic action? There are at least three possibilities. First, P4 could be converted into androgen and androgen could act through its receptors to prevent apoptosis [13, 38, 39]. Second, P4 could act through the glucocorticoid receptor [40]. Finally, there could be an alternative, undefined receptor through which P4 mediates its antiapoptotic action [2, 41]. Which, if any, of these possible mechanisms is actually involved in transducing P4's antiapoptotic action in rat luteal cells remains to be definitively determined. However, the present studies demonstrate that 1) rat luteal cells express PAIRBP1 at very high levels and 2) the PAIRBP1-1 antibody blocks P4's action in rat luteal cells. In addition, a high dose of P4 can over ride PAIRBP1-1 antibody treatment, indicating that the PAIRBP1-1 antibody is not toxic to the luteal cells. Taken together, these findings argue that PAIRBP1 is part of the mechanism through which P4 prevents rat luteal cell apoptosis.

Like rat luteal cells, P4 maintains the viability of bovine and primate luteal cells. Although the bovine and human luteal cells express the nPRs, the fact that P4 prevents apoptosis of rat luteal cells that do not express nPRs raises the possibility that nPRs may not be exclusive mediators of P4's antiapoptotic action in bovine and human luteal cells. Support for this hypothesis is provided by a careful review of the studies involving the nPR antagonist, RU486. For example, in human luteal cells, RU486 must be in 10– 100 molar excess to attenuate P4's antiapoptotic action [10, 11]. In contrast, a 5 nM dose of RU486 completely inhibits the effects of 50 nM of the progestin, R5020, in breast cancer cells (T47D) [42]. Given that RU486 binds to the nPR with a higher affinity than either P4 or R5020 [43, 44], the high molar excess of RU486 required to inhibit P4's action in human luteal cells is inconsistent with a nPR-mediated mechanism of action. Finally, the human granulosa and luteal cells isolated from patients undergoing in vitro fertilization treatment express a PAIRBP1-like protein (Peluso, unpublished results). This could indicate that P4 acts through a PAIRBP1-dependent pathway to maintain the viability of human granulosa and luteal cells.

Another major issue is the mechanism through which P4 regulates cell survival. In granulosa cells isolated from immature rats and SIGCs, P4 binds to the plasma membrane and rapidly initiates membrane-based signaling events [21, 45–47]. This membrane interaction results in the suppression of intracellular free calcium levels within seconds [46] and the activation of protein kinase G by 10 min [48]. Importantly, both of these rapid actions are required for P4 to prevent apoptosis [46, 48]. This membrane site of action is also consistent with PAIRBP1 being localized to the extracellular surface of both granulosa and luteal cells and the fact that treatment with the PAIRBP1-1 antibody blocks P4's antiapoptotic action.

Because PAIRBP1 does not possess a transmembrane domain, it has been proposed that PAIRBP1 binds to a transmembrane protein to form a P4 receptor-membrane complex [19]. The observation that the PAIRBP1-1 antibody immunoprecipitates a previously described membrane P4 binding protein, PGRMC1 [49–51], supports this concept. The interaction between PAIRBP1 and PGRMC1 could also explain the failure of PAIRBP1 antibody to block 3H-P4 binding because it is likely that PGRMC1 and not PAIRBP1 directly binds P4. Moreover, it is possible that the PAIRBP1-1 antibody treatment disrupts the interaction between PGRMC1 and PAIRBP1, thereby interfering with P4's ability to activate an intracellular signal cascade. While more studies are required to elucidate the functional relationship between PAIRBP1 and PGRMC1, it is important to appreciate that PGRMC1 has recently been detected by gene-expression arrays in granulosa cells within mouse pre- and periovulatory follicles [52], the porcine ovary [53], and human granulosa/luteal cells [54]. These expression studies together with the present studies suggest that both PAIRBP1 and PGRMC1 play important physiological roles in regulating ovarian function.

In summary, the present paper reveals that thecal, interstitial, ovarian surface epithelial and luteal cells, as well as granulosa cells, express PAIRBP1. PAIRBP1 levels are induced by gonadotropins and PAIRBP1 localizes to the extracellular surface of granulosa and luteal cells. The PAIRBP1-1 antibody treatment also attenuates P4's antiapoptotic action in both granulosa and luteal cells, thereby placing PAIRBP1 in the P4 signal transduction pathway that preserves the viability of these ovarian cell types. Finally, the mechanism by which PAIRBP1 mediates P4's antiapoptotic action remains ill defined but may involve an interaction with PGRMC1, thereby resulting in the formation a functional P4 membrane receptor complex.


   ACKNOWLEDGMENTS
 
The authors would like to thank Dr. Robert Burghardt of Texas A&M University for providing the SIGC cells.


   FOOTNOTES
 
1 Supported by NIH grant HD 34383 and funds from the University of Connecticut Health Center. Back

2 Correspondences: Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06030. FAX: 860 6791269; peluso{at}nso2.uchc.edu Back

Received: 16 February 2005.

First decision: 28 February 2005.

Accepted: 23 March 2005.


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 RESULTS
 DISCUSSION
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