Biol Reprod Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Peluso, J.J.
Right arrow Articles by White, B.A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Peluso, J.J.
Right arrow Articles by White, B.A.
Agricola
Right arrow Articles by Peluso, J.J.
Right arrow Articles by White, B.A.
Biology of Reproduction 67, 379-385 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Membrane-Initiated Events Account for Progesterone's Ability to Regulate Intracellular Free Calcium Levels and Inhibit Rat Granulosa Cell Mitosis1

J.J. Peluso2,,a,b, G. Fernandeza, A. Pappalardoa, and B.A. Whitea

a Departments of Physiology and b Obstetrics and Gynecology, University of Connecticut Health Center, Farmington, Connecticut 06030


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It has been proposed that the antimitogenic action of progesterone (P4) is mediated through a membrane receptor that has GABAA receptor-like characteristics. To test this hypothesis, studies were designed to compare the antimitogenic effects of P4 with its gamma amino butyric acidA (GABAA) receptor-activating metabolite, 5{alpha}-pregnane-3{alpha}–21-diol-20-one (5{alpha}3{alpha}). These studies revealed that P4 was more effective than 5{alpha}3{alpha} in blocking mitogen-dependent mitosis of both small granulosa cells (GCs) and spontaneously immortalized granulosa cells (SIGCs). Ligand binding studies illustrated that P4 bound to SIGCs with an apparent dissociation constant (Kd) of 0.32 ± 0.09 µM, whereas 5{alpha}3{alpha} bound with an apparent Kd of 40 ± 19 µM. Further, the GABAA antagonist, bicuculline, did not attenuate P4's antimitotic action in SIGCs. Finally, reverse transcriptase-polymerase chain reaction (RT-PCR) studies demonstrated that none of the 6 known {alpha} chains of the GABAA receptors to which bicuculline binds were detected in SIGCs. Taken together, these studies suggest that P4 does not mediate its action via a GABAA-like receptor. Additional studies revealed that P4 regulated intracellular free calcium levels ([Ca2+]i) as part of its antimitotic action. Specifically, P4 maintained a basal [Ca2+]i level that was slightly lower than normal. Increasing extracellular calcium not only increased basal [Ca2+]i but also attenuated P4's antimitogenic effect. P4's actions appeared to be initiated at the membrane, since horseradish peroxidase conjugated-P4 (HP-P4), which is cell impermeable, was as effective in blocking mitosis as P4. Progesterone receptor (PR) mRNA was not detected in SIGCs by RT-PCR analysis, which is consistent with the findings in GCs. However, a 60-kDa protein was detected within crude membrane fractions of both GCs and SIGCs using an antibody directed against the ligand binding domain of the PR (C-262). This antibody was also used in immunocytochemical studies to detect a protein that was associated with the plasma membrane of SIGCs. It is proposed that this 60-kDa protein mediates P4's membrane-initiated actions.

calcium, granulosa cells, progesterone, progesterone receptor


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Progesterone (P4) is a major steroid that is synthesized and secreted by the ovary [1, 2]. Although luteal cells have the greatest capacity to synthesize and secrete this steroid, granulosa cells (GCs) of preantral follicles also synthesize P4 [3, 4]. In addition, the ability of GCs to synthesize P4 increases throughout the course of follicular development [3, 4]. As a result, P4 concentrations in preovulatory rat follicles can reach micromolar levels prior to the ovulatory gonadotropin surge [3].

Work from several laboratories suggests that P4 can directly influence GC function. It is clear that P4 is involved in the ovulatory process, and this action appears to be mediated through the nuclear P4 receptor (PR), which is induced by the ovulatory gonadotropin surge [2]. The expression of the PR is transient such that PR is only detected from 4 to 6 h after the gonadotropin surge [5–8]. However, P4 can modulate the function of GCs isolated prior to the gonadotropin surge. For example, in GCs isolated from immature rats, P4 can inhibit FSH-induced estrogen secretion [9] and mitogen-dependent mitosis [10, 11]. Although these GCs do not express the PR [5, 6], P4's ability to suppress GC mitosis is steroid-specific, dose-dependent, and reversed by 100-fold molar excess of RU 486 [10–12].

Ligand binding studies, conducted in the late 1970s, demonstrate that GCs of immature rats specifically bind radiolabeled progestins [13–15]. More recent studies using spontaneously immortalized granulosa cells (SIGCs) reveal that these cells express a single high-capacity, low-affinity binding site for P4 [16]. This binding site could function as a membrane receptor for P4. Support for this concept comes from several different experimental lines of evidence. First, FITC-BSA-conjugated P4 binds to the plasma membrane in a steroid-specific manner [17]. Second, Western blot analysis using an antibody directed against the ligand binding domain of the PR (C-262) detects a 60-kDa protein that is present in the plasma membrane of both GCs and SIGCs [16]. Third, this 60-kDa protein binds P4 [16]. Fourth, both FITC-BSA-conjugated P4 binding and P4's antimitotic action are inhibited by C-262 [12, 17]. Collectively, these observations are consistent with the concept that this 60-kDa protein functions as a plasma membrane receptor for P4.

Several studies have shown that P4's action in both GCs [12] and sperm [18, 19] is influenced by the GABAA receptor antagonist, bicuculline. Given the GABAA receptor pharmacology and the fact that P4 can modulate GABAA receptor activity [20, 21], it is possible that P4's actions are mediated through a GABAA-like receptor. Interestingly, P4's metabolite, 5{alpha}-pregnane-3{alpha}-21-diol-20-one (5{alpha}3{alpha}) is more effective in activating the GABAA receptor than P4 [20], whereas 5{alpha}-pregnane-3ß-21-diol-20-one (5{alpha}3ß) does not effect the GABAA receptor activity [22]. Thus the first series of studies compared the antimitotic effects of 5{alpha}3{alpha} and 5{alpha}3ß to that of P4. Subsequent studies focused on the mechanism through which P4 mediates its antimitotic action.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Reagents

Immature female Wistar rats (22 days of age) were obtained from Charles River Laboratory (Wilmington, MA) and housed under controlled conditions of temperature, humidity, and photoperiod (12L:12D; lights-on at 0700 h). On the day of the experiment, immature animals were 23 or 24 days of age. The rats were killed by cervical dislocation between 0930 and 1000 h and the ovaries were removed. This protocol was approved by the Animal Care Committee of the University of Connecticut Health Center.

All reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated. Insulin, progesterone (P4), 5{alpha}-pregnane-3{alpha}–21-diol-20-one (5{alpha}3{alpha}), 5{alpha}-pregnane-3ß-21-diol-20-one (5{alpha}3ß), horseradish peroxidase-conjugated P4 (HP-P4), and bicuculline were added to cultures at various concentrations depending on the experimental design. The monoclonal antibody to the ligand binding domain of the PR (C-262) was purchased from StressGen (Victoria, BC, Canada). The PR cDNA was provided by Dr. O.K. Park-Sarge (University of Kentucky, Lexington, KY). The basic fibroblast growth factor (bFGF) was purchased from R&D Labs (Minneapolis, MN).

Granulosa Cell Isolation and Culture

Since small but not large GCs undergo insulin-dependent mitosis in vitro [23], only small GCs were used in these studies. This population of GCs was isolated according to the procedure of Lederer et al. [23]. The small GCs were collected from fractions 3 and 4, washed and then suspended in RPMI-1640. Small GCs were plated at a density of 1.5–2 x 105 cells/ml of RPMI-1640 and incubated at 37°C in 5% CO2 in air with RPMI-1640 supplemented with the mitogen, insulin (5 µg/ml), and other reagents as indicated. After 2 h, the media were replaced to remove any nonattached GCs. The number of remaining GCs was counted within 8 different 160-µm2 grids within the 35-mm dish [11]. The grids were located at the ends of horizontal and vertical axes and at the center of each dish. After 24 h of culture, the number of GCs present in these same areas was determined. Cell proliferation was expressed as a fold increase in cell number over 2 h control values.

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]. For binding studies, cells were plated in 35-mm culture dishes. For the SIGC mitosis studies, the cells were plated at 1.25 x 104 cell/ml on plastic culture dishes in 2 ml of serum-containing medium that was supplemented with the vehicle (control), P4, 5{alpha}3{alpha}, or 5{alpha}3ß. After 6 h the number of cells within 4 grids were counted. The number of cells in these grids was counted again after 24 and/or 48 h of culture depending on the experimental design. The fold increase in cell number was then calculated as previously described.

In the studies involving mitosis and supplemental calcium, 1 x 106 cells were plated on 60-mm glass Petri dishes for 24 h in serum-containing medium. Then the medium with a 1 mM concentration of CaCl2 was supplemented with the vehicle (ethanol), P4, and/or CaCl2 such that the final concentrations were 1 µM and 1.5 mM for P4 and CaCl2, respectively. At 6 and 24 h, the cells were counted in a hematocytometer. The increase in cell number compared to the 6 h control was then calculated.

Intracellular Free Calcium ([Ca2+]i) Measurements

For these studies, SIGCs were plated on glass coverslips in serum-supplemented medium for 6 h and then cultured for 18 h in serum-free medium supplemented with bFGF (6 ng/ml). The cells were then loaded with Fluo-4 (Molecular Probes, Eugene, OR) for 30 min [25], rinsed in Kreb-Hepes buffer (pH 7.3) with 1 µM P4, and the Fluo-4 fluorescence were assessed under 3 experimental conditions. After a 3.5-min stabilization period, either P4 was removed, maintained, or CaCl2 was added to P4-supplemented Kreb-Hepes buffer such that extracellular calcium levels increased by 50%. Next, [Ca2+]i levels were determined at 30-sec intervals for 10 min of culture as previously described [25]. To determine the actual intracellular concentration of calcium, calcium ionophore, A23187 (50 µM; Sigma) was added after 10 min to determine maximum fluorescent intensity (Fmax). EGTA (7 mM; Sigma) was then added and the minimum fluorescent intensity determined (Fmin). The [Ca2+]i was estimated by the following equation: nM intracellular free calcium = 345 nM (F - Fmin)/(Fmax - F) [25].

Western Blot and Immunocytochemical Analysis

Small GCs and SIGCs were lysed in 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (20 mM MOPS, 5 mM sodium acetate, 0.7 mM EDTA, pH 7.0), which was supplemented with a complete protease inhibitor cocktail (Roche, Mannheim, Germany) and phosphatase inhibitor cocktail 1 (Sigma) and then centrifuged at 1000 x g at 4°C for 5 min. The supernatant was collected and centrifuged at 100 000 x g for 60 min at 4°C. The membrane pellets were stored at -20°C.

Levels of 60-kDa P4 binding protein were assessed by using the antibody C-262 in a Western blot analysis. Briefly, 20 µg of membrane preparation was run on a 10% acrylamide gel and transferred to nitrocellulose. The nitrocellulose was then incubated with 5% nonfat dry milk overnight at 4°C. The nitrocellulose blot was incubated with C-262 antibody at a dilution of 1:1000 (1.9 µg/ml) for 1 h at room temperature and processed for Western blot analysis as previously described [16].

Protein detected by C-262 was localized to the plasma membrane by staining living cells as outlined previously [26]. Briefly, cells were rinsed with PBS and then incubated in the presence of either a C-262 antibody (1:50 in 8% BSA/PBS) or IgG 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.

Ligand Binding Studies

The protocol used to assess the total cellular binding of 3H-P4 to SIGCs has been previously described [16, 27]. Briefly, SIGCs were plated at 3.6 x 105 cells/35-mm culture dish and cultured overnight in a serum-supplemented medium. The cells were washed twice in PBS and then incubated at 4°C in 500 µl of 0.1% digitonin in a 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) 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 the filters were counted in a scintillation counter. Displacement studies were conducted in which the effect of increasing concentrations of nonlabeled P4, 5{alpha}3{alpha}, and 5{alpha}3ß on 3H-P4 binding was determined. For each experiment, each concentration was tested in duplicate with the entire experiment repeated at least 3 times. The dissociation constant (Kd) for each steroid was calculated using the Ligand program provided by the National Institutes of Health.

Reverse Transcriptase-Polymerase Chain Reaction Detection of the PR and Alpha Chains of the GABAA Receptor

RNA from SIGCs was isolated using the RNAzol isolation system following the manufacturer's instructions (Biotecx, Houston, TX). For reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, 2 µg of total RNA was converted to first-strand cDNA with SuperScript II RNaseH- reverse transcriptase and oligo-dT primer (Life Technologies, Gaithersburg, MD). PCR was performed using 20% of the RT reaction product as template with primers specific for the ligand binding domain of the nuclear PR. The sense primer was identical to that described by Park and Mayo [28] (5'-GATGACCAGATAACTCTCAT-3') and the antisense primer (5'-GTGAAAGAGAGAGGCTTC-3') ends at position 2763 (accession L16922; [6]). The alpha chains of the GABAA receptor were detected using the primer pairs and protocol of Liu and Burt [29].

The RT-PCR protocol to assess the expression of the PR in SIGCs was conducted as follows. In addition to SIGC cDNA, the PCR reaction also contained 1X PCR buffer (Life Technologies), 1.5 mM MgCl2, 1 pmol/µl of each primer, 200 µM of each deoxynucleotide (dATP, dCTP, dGTP, and dTTP; Promega, Madison, WI), and 2.5 U of recombinant Taq polymerase (Life Technologies). Amplification of primer products by 30 cycles of PCR in a thermal cycler followed this profile: 94°C denaturation, 1 min; 72°C primer hybridization, 1 min; 72°C extension, 1 min; 72°C final extension, 10 min. Forty-five nanograms of PR cDNA, which was provided by Dr. O.K. Park-Sarge (University of Kentucky), was also included as a positive control. An aliquot of each PCR reaction was separated on a 1.5% agarose gel containing ethidium bromide (0.5 µg/ml) and visualized under UV light.

The RT-PCR reaction to detect moesin was also run as a positive control. Rat moesin primers were designed from positions 1831 to 2075 (accession AF004811; sense: 5'-AGTGGGCGCGCAGCCGTTAGGGAC-3'; antisense: 5'-GCTATGTTGAATGAGTGTGACAAAG-3').

Statistical Analysis

All experiments were repeated at least 2–3 times with each experiment yielding essentially identical results. When appropriate, the data were pooled and analyzed by 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
 
P4 suppressed insulin-dependent GC mitosis in a dose-dependent manner (Fig. 1). Although 5{alpha}3{alpha} was effective, 5{alpha}3ß was ineffective in suppressing insulin-dependent GC mitosis. Compared to P4, higher doses of 5{alpha}3{alpha} were required to attenuate the mitogenic effect of insulin.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 1. The effect of P4, 5{alpha}3{alpha}, and 5{alpha}3ß on insulin-dependent GC mitosis. In this and subsequent graphs, the data is expressed as a mean fold increase ± 1 SEM. SEMs are not shown for the different steroid concentrations to improve graphic clarity. The lowest concentration that significantly suppressed mitosis is 40 nM for P4 and 80 nM for 5{alpha}3{alpha}. An * indicates a value that is different from the control group (P < 0.05)

The ability of P4 to regulate SIGC mitosis was also evaluated. In these studies, 10% bovine fetal serum was used as a mitogen. In the presence of serum the number of SIGCs increased 2- to 3-fold by 48 h of culture (Fig. 2A). Similar to GC studies, serum-dependent growth was inhibited by P4 (Fig. 2, A–C) and 5{alpha}3{alpha} (Fig. 2C), but higher doses of 5{alpha}3{alpha} were required to inhibit mitosis. Serum-dependent mitosis was not inhibited by 5{alpha}3ß (Fig. 2C).



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 2. The effect of P4, 5{alpha}3{alpha}, and 5{alpha}3ß on serum-dependent SIGC mitosis. A) The effect of time of culture and P4 (1 µM) are shown. B) the effect on increasing P4 concentrations on SIGC proliferation after 48 h of culture. The effects of 5{alpha}3{alpha} and 5{alpha}3ß on SIGC proliferation are shown in C. An * indicates a value that is different from the control group (P < 0.05)

3H-P4 binding studies were conducted on SIGCs in order to correlate mitotic effects of P4 and its metabolites with their ability to displace 3H-P4. Nonlabeled P4 displaced 3H-P4 binding in a dose-dependent manner with an apparent Kd of 0.32 ± 0.09 µM. 5{alpha}3{alpha} also suppressed 3H-P4 binding in a dose-dependent manner but with a higher apparent Kd of 40 ± 19 µM (Fig. 3). 5{alpha}3ß was ineffective in displacing 3H-P4 binding at concentrations less than 10-5 M (Fig. 3).



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 3. The effect of P4, 5{alpha}3{alpha}, and 5{alpha}3ß on 3H-progesterone binding in SIGCs. Values represent a mean of 6–12 observations per point

In GCs, a GABAA receptor antagonist, bicuculline, partially attenuates P4's antimitotic effects [12]. Bicuculline alone did not significantly alter serum-induced SIGC proliferation (P > 0.05; Fig. 4A). In contrast, P4 alone or in the presence of bicuculline reduced the fold increase in the number of cells compared to both the serum control group (P < 0.05) and bicuculline group (P < 0.05). Since bicuculline acts by binding to the alpha chain of the GABAA receptor [21], the expression of the 6 known alpha chains was assessed in SIGCs by RT-PCR. Although each alpha chain was detected within RNA isolated from rat brain (i.e., a positive control), none of the alpha chains were detected in RNA isolated from SIGCs (Fig. 4B).



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 4. The effect of the GABAA receptor antagonist, bicuculline (Bic) and P4 on SIGC proliferation (A). An * indicates a value that is different from the control group (P < 0.05). RT-PCR analysis of the known alpha chains of the GABAA receptor is shown in B. For each alpha chain, the results for an SIGC RNA preparation (first lane) and a rat brain RNA preparation (second lane) are shown. Both SIGC and rat brain RNA were assessed for moesin as a positive control. This analysis was conducted on RNAs that were isolated from 3 different SIGC cultures and 2 rat brains

To assess whether P4's antimitotic actions were initiated at the membrane, GCs and SIGCs were cultured with insulin or serum, respectively, in the presence of P4 or a cell-impermeable P4 conjugate (i.e., P4 conjugated with horseradish peroxidase, HP-P4). As shown in Figure 5, A and B, both P4 and HP-P4 were equally effective in attenuating mitogen-dependent GC and SIGC mitosis.



View larger version (13K):
[in this window]
[in a new window]
  		FIG. 5. The effect of P4 and HP-P4 on insulin-dependent GC mitosis (A) and serum-dependent SIGC mitosis (B). The P4 concentration was 160 nM and 1 µM for the experiments shown in A and B, respectively. Since there was approximately 1 molecule of P4 conjugated to each molecule of horseradish peroxidase, HP-P4 was used at the same molarity as P4. An * indicates a value that is different from the control group (P < 0.05)

Previous studies have shown that [Ca2+]i levels in normal healthy SIGCs are approximately 100 nM [25], and that P4 suppresses [Ca2+]i [16]. Given that P4 suppresses [Ca2+]i levels, studies were conducted to determine whether P4's ability to lower [Ca2+]i was related to its antimitogenic effects. In the presence of P4, [Ca2+]i levels were relatively constant with the [Ca2+]i level of 6 separate experiments averaging 69 ± 2 nM. Increasing the extracellular calcium level by 50% increased [Ca2+]i levels by an average of 26% ± 3% to approximately 87 nM (Fig. 6A). Similarly, P4's ability to inhibit mitosis was abrogated by increasing extracellular calcium by 50% (Fig. 6B).



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 6. The effect of P4 and increased extracellular calcium on [Ca2+]i (A) and serum-dependent SIGC mitosis (B). CaCl2 was added to increase the extracellular calcium concentration by 50%. A) Mean values of at least 16 cells per treatment are shown. The SEM of all time points is 0.03. B) An * indicates a value that is different from the control group (P < 0.05)

Although previous studies have shown that the PR is not expressed in primary GCs [5, 6], PR expression has not been assessed in SIGCs. Using RT-PCR, RNA that encodes the PR was not detected within SIGC preparations, although a specific and appropriate-sized RT-PCR product was detected in the presence of PR cDNA (Fig. 7A). Moesin was readily detectable by RT-PCR in these SIGC RNA extracts (Fig. 7A).



View larger version (27K):
[in this window]
[in a new window]
  		FIG. 7. RT-PCR analysis of PR and moesin expression in RNA isolated from 2 different SIGC cultures. The PR cDNA was run as a positive control for PR RNA (A). B) A Western blot analysis of a crude membrane preparation of GCs and SIGCs is shown. In this Western blot the antibody to the ligand binding domain of the PR (C-262) was used and revealed a 60-kDa band in both GC and SIGC preparations. A negative control (Neg) in which the primary antibody was omitted from the Western blot protocol is also shown in the figure

As seen in Figure 7B, the PR antibody (C-262) detects a 60-kDa protein within crude membrane preparations of GCs and SIGCs. Further, C-262 specifically bound to the surface membrane of living SIGCs (Fig. 8).



View larger version (128K):
[in this window]
[in a new window]
  		FIG. 8. Immunocytochemical localization of the 60-kDa protein at the surface membrane of living SIGCs. Living SIGCs were incubated with either C-262 (A, C) or IgG (B, D) and then FITC-conjugated secondary antibody. Phase images are shown in A and B, whereas specific fluorescence associated with C-262 is shown in C. Nonspecific fluorescence is shown in D. All images are shown at the same magnification (approximately x500)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Several studies have shown that P4 can directly influence the mitogenic [10, 12, 17] and steroidogenic [9, 30] activity of GCs isolated from immature rats. Because these cells do not express the PR [5, 6], the mechanism through which P4 mediates its actions in these GCs remains to be defined. One of the limiting factors in elucidating P4's antimitotic mechanism is that large numbers of GCs are required to conduct these studies. The present studies resolve this limitation by demonstrating that P4 inhibits the proliferation of spontaneously immortalized cells that were derived from primary GCs (i.e., SIGCs) [24]. Therefore, studies can now be conducted on both primary GCs and SIGCs to more fully elucidate the antimitotic action of P4.

It has been suggested that P4's antimitotic mechanism involves a GABAA-like receptor [12]. The rat ovary has high levels of GABA [31–33], and primary GCs express GABAA receptors [33, 34]. Therefore it is likely that these cells possess a basal GABAA receptor activity level that is maintained by endogenous levels of both P4, which can activate the GABAA receptor [20], and GABA. This endogenous basal GABAA receptor activity may suppress GC mitosis, since the GABAA receptor antagonist, bicuculline, attenuates in part P4's antimitotic action in GCs [12].

Although P4-GABAA receptor interaction may have a role in regulating GC mitosis, it does not appear to be the dominant mechanism through which P4 inhibits proliferation. This conclusion is based on the following observations. First, if a GABAA-like receptor mediates P4's actions, then the anesthetic, GABAA-activating metabolite of P4, 5{alpha}3{alpha}, would be more effective than P4 in inhibiting GC mitosis, since 5{alpha}3{alpha} is 12 times more potent than P4 in activating GABAA receptors [20]. Conversely, 5{alpha}3ß, a nonanesthetic, non-GABAA-activating isomer of 5{alpha}3{alpha} [22], would not inhibit GC mitosis. Although 5{alpha}3{alpha} inhibits and 5{alpha}3ß does not effect GC and SIGCs mitosis, 5{alpha}3{alpha} is less effective than P4. This reduced ability of 5{alpha}3{alpha} and 5{alpha}3ß to prevent GC and SIGC proliferation correlates with their reduced capacity to displace 3H-P4 binding to SIGCs. This could indicate that both P4 and 5{alpha}3{alpha} bind to the same receptor to inhibit mitosis (see subsequent discussion). Second, bicuculline does not attenuate P4's antimitotic action in SIGCs. Bicuculline interferes with GABAA receptor function by interacting with the alpha chain of the GABAA receptor [21]. Since none of the known alpha chains of the GABAA receptor are expressed by SIGCs, this accounts for the inability of bicuculline to effect SIGC mitosis. Finally, P4 inhibits both GCs and SIGCs within the same dose response range even though the bicuculline-sensitive antimitotic mechanism is not present in SIGCs. Taken together, these observations suggest the existence of a non-GABAA receptor mechanism through which P4 prevents GC and SIGC mitosis.

This non-GABAA receptor pathway may be mediated by a membrane receptor for P4. Three lines of evidence support this concept. First, FITC-BSA-P4 specifically binds to the surface membranes of GCs [17]. Second, the cell-impermeable P4, HP-P4, inhibits mitosis at the same concentrations as P4. Third, P4 induces a 15%–20% decrease in [Ca2+]i levels within seconds [16]. Although this rapid change in [Ca2+]i is consistent with a membrane-initiated event, it is conceivable that an intracellular site of action is also responsible for changes in [Ca2+]i.

The ability of P4 to suppress basal [Ca2+]i appears to be related to its antimitotic effect. In normal healthy porcine [35], human [36], and rat GCs [37] as well as SIGCs [25], basal [Ca2+]i levels are approximately 100 nM. As indicated, P4 suppresses [Ca2+]i levels. In the present studies, P4 maintained [Ca2+]i at approximately 70 nM. As predicted from the studies of rat hepatocytes [38], increasing extracellular CaCl2 in SIGCs by 50% increased [Ca2+]i levels by approximately 25%. As a result, [Ca2+]i increased to that of normal healthy SIGCs. This elevation in [Ca2+]i overcomes the antimitotic effect of P4. In a number of different cells, mitogens induce transient increases in [Ca2+]i levels that occur at specific stages of the cell cycle [39–41]. These increases in [Ca2+]i are sufficient to allow the transition from G0/G1 to the S phase of the cell cycle [39]. If these calcium transits are buffered by the intracellular calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid (BAPTA), then the cells do not traverse the cell cycle [40]. Based on the essential role of calcium transits in stimulating the cell cycle, P4 could prevent the cell-cycle-dependent calcium transits from occurring, and thereby prevent GC mitosis. Alternatively, mitogens could induce cell-cycle-dependent calcium transits, but since the basal [Ca2+]i levels would be lower in P4-treated cells, the calcium transits may not be able to increase [Ca2+]i to the level required to induce the subsequent cell-cycle events.

The molecular mechanism by which [Ca2+]i levels regulate early events of the cell cycle is just beginning to be understood. In GCs, insulin induces the expression of 2 immediate/early genes, c-fos and c-jun, which are required for GCs to undergo insulin-dependent mitosis [11]. Further, P4 blocks the insulin-dependent expression of c-fos and c-jun [11]. Recent studies have shown that a transient increase in [Ca2+]i precedes the mitogen-dependent increase in c-fos and c-jun expression [42, 43]. If this increase in [Ca2+]i is abrogated by BAPTA, then c-fos and c-jun levels are not increased [42]. Similarly, tumor cells, which overexpress c-fos and c-jun, have higher levels of [Ca2+]i than do the nontransformed cells [43]. Lowering [Ca2+]i by BAPTA treatment also reduces c-fos and c-jun levels [43]. Since in other cells increases in [Ca2+]i play an important role in inducing c-fos and c-jun expression, the ability of P4 to suppress [Ca2+]i could account for its ability to inhibit insulin-dependent expression of c-fos and c-jun and subsequently GC mitosis.

Although it is likely that increased extracellular calcium attenuates P4 antimitotic action by increasing [Ca2+]i, it is also possible that increases in extracellular calcium promote mitosis independently of its actions on [Ca2+]i levels. In ovarian surface epithelial cells, increasing the extracellular calcium concentration from 0.8 mM to either 1.4 or 1.8 mM induces a 2-fold increase in 3H-thymidine incorporation within 16 h [44]. A similar fold increase in cell number is observed by 48 h after increasing extracellular calcium [44]. The increase in cell proliferation in ovarian surface epithelial cells appears to be mediated through a calcium-sensing receptor, which has been shown to activate the mitogen-activated protein (MAP) kinase cascade [44, 45]. In the present studies, extracellular calcium was increased from 1 to 1.5 mM. However, SIGCs cultured with serum and calcium supplementation do not proliferate at a greater rate than those cells cultured with serum alone. Since serum could mask any mitogenic action of extracellular calcium, SIGCs were cultured with bFGF, which maintains cell viability without promoting mitosis [46]. In the presence of bFGF, increasing extracellular calcium was not mitogenic (1.37 ± 0.08-fold increase in cell number for bFGF verses 1.23 ± 0.07-fold increase in cell number for bFGF plus supplemental CaCl2; n = 6, P > 0.05; unpublished results). Although the present studies do not support the concept that increasing extracellular calcium promotes mitosis by activating calcium-sensing receptors, detailed molecular studies are required to completely rule out this possibility.

The present study also demonstrates that the cell-impermeable P4 conjugate, HP-P4, prevents mitogen-dependent mitosis in both GCs and SIGCs. This implies that P4 inhibits mitosis by regulating membrane-initiated events. A membrane-initiated mechanism accounts for P4's actions in both sperm [47, 48] and oocytes [49]. Recent studies have shown that PR mRNA can be detected in these gametes [50–52]. Further, small amounts of the PR can localize to the plasma membrane of oocytes and function as membrane receptors for P4 [51, 52]. Moreover, PR interacts with SH3 domains and activates Src family tyrosine kinases and has also been associated with P42 MAP kinase and phosphatidylinositol 3-kinase, thereby linking PR to signal transduction pathways that are initiated at the plasma membrane [53, 54]. Collectively, these studies indicate that the PR can localize to the plasma membrane and function as a P4 membrane receptor. The localization of the PR to the membranes of GCs and SIGCs, however, does not provide a mechanism that explains P4's ability to regulate GC function. This conclusion is based on the failure of RT-PCR protocols to detect PR in both primary GCs [6] and SIGCs (present study). As previously demonstrated, an antibody to the ligand binding domain of the PR (i.e., C-262) has detected a 60-kDa protein within crude membrane preparations of GCs and SIGCs [16]. Immunocytochemical studies using C-262 detect this protein at the surface membrane of SIGCs. Finally, blocking antibody studies have demonstrated that C-262 abrogates the binding of FITC-BSA-conjugated P4 to GCs [17] as well as P4's antimitotic effects [12]. Taken together, these data seem to indicate that this 60-kDa protein, which is detected by C-262, is as a novel membrane receptor for P4. The identity of the putative membrane receptor is presently under investigation.


   ACKNOWLEDGMENTS
 
The authors would like to thank Dr. Robert Burghardt of Texas A&M University for providing the SIGC cells. We would also like to thank Dr. O. K. Park-Sarge for the PR cDNA.


   FOOTNOTES
 
First decision: 5 February 2002.

1 Supported by NIH grant HD 34383. Back

2 Correspondence: John J. Peluso, Department of Physiology, University of Connecticut Health Center, Farmington, CT 06030. FAX: 860 679 1269; peluso{at}nso2.uchc.edu Back

Accepted: February 27, 2002.

Received: January 11, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Orly J. Molecular events defining follicular development and steroidogenesis in the ovary. Totowa, NJ: Human Press; 2000
  2. Robker RL, Russell DL, Yoshioka S, Sharma SC, Lydon JP, O'Malley BW, Espey LL, Richards JS. Ovulation: a multi-gene, multi-step process. Steroids 2000; 65:559-570[CrossRef][Medline]
  3. Fujii T, Hoover DJ, Channing CP. Changes in inhibin activity, and progesterone, oestrogen and androstenedione concentrations, in rat follicular fluid throughout the oestrus cycle. J Reprod Fertil 1983; 69::307-314[Abstract]
  4. Roy SK, Greenwald GS. In vitro steroidogenesis by primary to antral follicles in the hamster during the periovulatory period: effects of follicle-stimulating hormone, luteinizing hormone, and prolactin. Biol Reprod 1987; 37:39-46[Abstract]
  5. Natraj U, Richards JS. Hormonal regulation, localization, and functional activity of the progesterone receptor in granulosa cells of rat preovulatory follicles. Endocrinology 1993; 133:761-769[Abstract]
  6. Park-Sarge OK, Mayo KE. Regulation of the progesterone receptor gene by gonadotropins and cyclic adenosine 3',5'-monophosphate in rat granulosa cells. Endocrinology 1994; 134:709-718[Abstract]
  7. Park-Sarge OK, Sarge KD. Cis-regulatory elements conferring cyclic 3',5'-adenosine monophosphate responsiveness of the progesterone receptor gene in transfected rat granulosa cells. Endocrinology 1995; 136:5430-5437[Abstract]
  8. Park-Sarge OK, Parmer TG, Gu Y, Gibori G. Does the rat corpus luteum express the progesterone receptor gene?. Endocrinology 1995; 136:1537-1543[Abstract]
  9. Schreiber JR, Nakamura K, Erickson GF. Progestins inhibit FSH-stimulated granulosa estrogen production at a post-cAMP site. Mol Cell Endocrinol 1981; 21:161-170[CrossRef][Medline]
  10. Luciano AM, Peluso JJ. Effect of in vivo gonadotropin treatment on the ability of progesterone, estrogen, and cyclic adenosine 5'-monophosphate to inhibit insulin-dependent granulosa cell mitosis in vitro. Biol Reprod 1995; 53:664-669[Abstract]
  11. Peluso JJ, Luciano AM, Pappalardo A, White BA. Cellular and molecular mechanisms that mediate insulin-dependent rat granulosa cell mitosis. Biol Reprod 1995; 52:124-130[Abstract]
  12. Peluso JJ, Pappalardo A. Progesterone mediates its anti-mitogenic and anti-apoptotic actions in rat granulosa cells through a progesterone-binding protein with gamma aminobutyric acid A receptor-like features. Biol Reprod 1998; 58:1131-1137[Abstract/Free Full Text]
  13. Naess O. Characterization of cytoplasmic progesterone receptors in rat granulosa cells. Evidence for nuclear translocation. Acta Endocrinol 1981; 98:288-294
  14. Schreiber JR, Hsueh AJW. Progesterone "receptor" in rat ovary. Endocrinology 1979; 105:915-925[Abstract]
  15. Schreiber JR, Erickson GF. Progesterone receptor in the rat ovary: further characterization and localization in the granulosa cell. Steroids 1979; 34:459-469[CrossRef][Medline]
  16. 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[Abstract/Free Full Text]
  17. Peluso JJ, Pappalardo A. Progesterone maintains large rat granulosa cell viability indirectly by stimulating small granulosa cells to synthesize basic fibroblast growth factor. Biol Reprod 1999; 60:290-296[Abstract/Free Full Text]
  18. Calogero AE, Burrello N, Ferrara E, Hall J, Fishel S, D'Agata R. Gamma-aminobutyric acid (GABA) A and B receptors mediate the stimulatory effects of GABA on the human sperm acrosome reaction: interaction with progesterone. Fertil Steril 1999; 71:930-936[CrossRef][Medline]
  19. Shi QX, Roldan ER. Evidence that a GABAA-like receptor is involved in progesterone-induced acrosomal exocytosis in mouse spermatozoa. Biol Reprod 1995; 52:373-381[Abstract]
  20. Blackmore PF, Im WB, Bleasdale JE. The cell surface progesterone receptor which stimulates calcium influx in human sperm is unlike the A ring reduced steroid site on the GABAA receptor/chloride channel. Mol Cell Endocrinol 1994; 104:237-243[CrossRef][Medline]
  21. Mehta AK, Ticku MK. An update on GABAA receptors. Brain Res Rev 1999; 29:196-217[CrossRef][Medline]
  22. Wistrom CA, Meizel S. Evidence suggesting involvement of a unique human sperm steroid receptor/Cl- channel complex in the progesterone-initiated acrosome reaction. Dev Biol 1993; 159:679-690[CrossRef][Medline]
  23. Lederer KJ, Luciano AM, Pappalardo A, Peluso JJ. Proliferative and steroidogenic capabilities of rat granulosa cells of different sizes. J Reprod Fertil 1995; 103:47-54[Abstract]
  24. Stein LS, Stein DWJ, Echols J, Burghardt RC. Concomitant alterations of desmosomes, adhesiveness, and diffusion through gap junction channels in a rat ovarian transformation model system. Exp Cell Res 1993; 207:19-32[CrossRef][Medline]
  25. Peluso JJ, Pappalardo A, Fernandez G. Basic fibroblast growth factor maintains calcium homeostasis and granulosa cell viability by stimulating calcium efflux via a PKC delta-dependent pathway. Endocrinology 2001; 142:4203-4211[Abstract/Free Full Text]
  26. Peluso JJ, Pappalardo A, Fernandez G. E-cadherin-mediated cell contact prevents apoptosis of spontaneously immortalized granulosa cells by regulating Akt kinase activity. Biol Reprod 2001; 64:1183-1190[Abstract/Free Full Text]
  27. Ambhaikar M, Puri C. Cell surface binding sites for progesterone on human spermatozoa. Mol Hum Reprod 1998; 4:413-421[Abstract/Free Full Text]
  28. Park OK, Mayo KE. Transient expression of progesterone receptor messenger RNA in ovarian granulosa cells after the preovulatory luteinizing hormone surge. Mol Endocrinol 1991; 5:967-978[Abstract]
  29. Liu ZF, Burt DR. A synthetic standard for competitive RT-PCR quantitation of 13 GABA receptor type A subunit mRNAs in rats and mice. J Neurosci Methods 1998; 85:89-98[CrossRef][Medline]
  30. Schreiber JR, Nakamura K, Erickson GF. Progestins inhibit FSH-stimulated steroidogenesis in cultured rat granulosa cells. Mol Cell Endocrinol 1980; 19:165-173[CrossRef][Medline]
  31. Apud JA, Tappaz ML, Celotti F, Negri-Cesi P, Masotto C, Racagni G. Biochemical and immunochemical studies on the GABAergic system in the rat fallopian tube and ovary. J Neurochem 1984; 43:120-125[CrossRef][Medline]
  32. Martin del Rio R, Caballero AL. Presence of gamma-aminobutyric acid in rat ovary. J Neurochem 1980; 34:1584-1586[CrossRef][Medline]
  33. Schaeffer JM, Hsueh AJ. Identification of gamma-aminobutyric acid and its binding sites in the rat ovary. Life Sci 1982; 30:1599-1604[CrossRef][Medline]
  34. Erdo SL, Lapis E. Bicuculline-sensitive GABA receptors in rat ovary. Eur J Pharmacol 1982; 85:243-246[CrossRef][Medline]
  35. Lieberherr M, Grosse B, Machelon V. Phospholipase C-beta and ovarian sex steroids in pig granulosa cells. J Cell Biochem 1999; 74:50-60[CrossRef][Medline]
  36. Mayerhofer A, Sterzik K, Link H, Wiemann M, Gratzl M. Effect of oxytocin on free intracellular Ca2+ levels and progesterone release by human granulosa-lutein cells. J Clin Endocrinol Metab 1993; 77::1209-1214[Abstract]
  37. Wang J, Steele GL, Baimbridge KG, Rodway MR, Leung PC. Intracellular calcium and the signaling mechanism of luteinizing hormone-releasing hormone in rat granulosa cells. Am J Obstet Gynecol 1992; 167:541-547[Medline]
  38. Zhang BH, Farrell GC. Effects of extracellular Ca2+ and HCO3- on epidermal growth factor-induced DNA synthesis in cultured rat hepatocytes. Gastroenterology 1995; 108:477-486[CrossRef][Medline]
  39. Barbiero G, Munaron L, Antoniotti S, Baccino FM, Bonelli G, Lovisolo D. Role of mitogen-induced calcium influx in the control of the cell cycle in Balb-c 3T3 fibroblasts. Cell Calcium 1995; 18:542-556[CrossRef][Medline]
  40. Kao JP, Alderton JM, Tsien RY, Steinhardt RA. Active involvement of Ca2+ in mitotic progression of Swiss 3T3 fibroblasts. J Cell Biol 1990; 111:183-196[Abstract/Free Full Text]
  41. Preston SF, Sha'afi RI, Berlin RD. Regulation of Ca2+ influx during mitosis: Ca2+ influx and depletion of intracellular Ca2+ stores are coupled in interphase but not mitosis. Cell Regul 1991; 2:915-925[Medline]
  42. Chen J, Lin R, Hu ZW, Hoffman BB. Alpha1-adrenergic receptor activation of c-fos expression in transfected rat-1 fibroblasts: role of Ca2+. J Pharmacol Exp Ther 1999; 289:1376-1384[Abstract/Free Full Text]
  43. Joseph P, Muchnok TK, Klishis ML, Roberts JR, Antonini JM, Whong WZ, Ong T. Cadmium-induced cell transformation and tumorigenesis are associated with transcriptional activation of c-fos, c-jun, and c-myc proto-oncogenes: role of cellular calcium and reactive oxygen species. Toxicol Sci 2001; 61:295-303[Abstract/Free Full Text]
  44. McNeil L, Hobson S, Nipper V, Rodland KD. Functional calcium-sensing receptor expression in ovarian surface epithelial cells. Am J Obstet Gynecol 1998; 178:305-313[CrossRef][Medline]
  45. Brown EM, MacLeod RJ. Extracellular calcium sensing and extracellular calcium signaling. Physiol Rev 2001; 81:239-297[Abstract/Free Full Text]
  46. Lynch K, Fernandez G, Pappalardo A, Peluso JJ. Basic fibroblast growth factor inhibits apoptosis of spontaneously immortalized granulosa cells by regulating intracellular free calcium levels through a protein kinase c delta-dependent pathway. Endocrinology 2000; 141::4209-4217[Abstract/Free Full Text]
  47. Baldi E, Luconi M, Bonaccorsi L, Maggi M, Francavilla S, Gabriele A, Properzi G, Forti G. Nongenomic progesterone receptor on human spermatozoa: biochemical aspects and clinical implications. Steroids 1999; 64:143-148[CrossRef][Medline]
  48. Calogero AE, Burrello N, Barone N, Palermo I, Grasso U, D'Agata R. Effects of progesterone on sperm function: mechanisms of action. Hum Reprod 2000; 15:(suppl):28-45
  49. Morrill GA, Kostellow AB. Progesterone induces meiotic division in the amphibian oocyte by releasing lipid second messengers from the plasma membrane. Steroids 1999; 64:157-167[CrossRef][Medline]
  50. Sachdeva G, Shah CA, Kholkute SD, Puri CP. Detection of progesterone receptor transcript in human spermatozoa. Biol Reprod 2000; 62:1610-1614[Abstract/Free Full Text]
  51. Bayaa M, Booth RA, Sheng Y, Liu XJ. The classical progesterone receptor mediates Xenopus oocyte maturation through a nongenomic mechanism. Proc Natl Acad Sci USA 2000; 97:12607-12612[Abstract/Free Full Text]
  52. Tian J, Kim S, Heilig E, Ruderman JV. Identification of XPR-1, a progesterone receptor required for Xenopus oocyte activation. Proc Natl Acad Sci USA 2000; 97:14358-14363[Abstract/Free Full Text]
  53. Bagowski CP, Myers JW, Ferrell JE Jr. The classical progesterone receptor associates with p42 MAPK and is involved in phosphatidylinositol 3-kinase signaling in xenopus oocytes. J Biol Chem 2001; 276:37708-37714[Abstract/Free Full Text]
  54. Boonyaratanakornkit V, Scott MP, Ribon V, Sherman L, Anderson SM, Maller JL, Miller WT, Edwards DP. Progesterone receptor contains a proline-rich motif that directly interacts with SH3 domains and activates c-Src family tyrosine kinases. Mol Cell 2001; 8:269-280.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
S. Glaser, S. DeMorrow, H. Francis, Y. Ueno, E. Gaudio, S. Vaculin, J. Venter, A. Franchitto, P. Onori, B. Vaculin, et al.
Progesterone stimulates the proliferation of female and male cholangiocytes via autocrine/paracrine mechanisms
Am J Physiol Gastrointest Liver Physiol, July 1, 2008; 295(1): G124 - G136.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
J. J. Peluso, J. Romak, and X. Liu
Progesterone Receptor Membrane Component-1 (PGRMC1) Is the Mediator of Progesterone's Antiapoptotic Action in Spontaneously Immortalized Granulosa Cells As Revealed by PGRMC1 Small Interfering Ribonucleic Acid Treatment and Functional Analysis of PGRMC1 Mutations
Endocrinology, February 1, 2008; 149(2): 534 - 543.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
J. J. Peluso, X. Liu, and J. Romak
Progesterone Maintains Basal Intracellular Adenosine Triphosphate Levels and Viability of Spontaneously Immortalized Granulosa Cells by Promoting an Interaction between 14-3-3{sigma} and ATP Synthase{beta}/Precursor through a Protein Kinase G-Dependent Mechanism
Endocrinology, May 1, 2007; 148(5): 2037 - 2044.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
J. J. Peluso
Multiplicity of Progesterone's Actions and Receptors in the Mammalian Ovary
Biol Reprod, July 1, 2006; 75(1): 2 - 8.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
J. J. Peluso, A. Pappalardo, R. Losel, and M. Wehling
Progesterone Membrane Receptor Component 1 Expression in the Immature Rat Ovary and Its Role in Mediating Progesterone's Antiapoptotic Action
Endocrinology, June 1, 2006; 147(6): 3133 - 3140.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
Y. Wu, W. G Foster, and E. V Younglai
Rapid effects of pesticides on human granulosa-lutein cells
Reproduction, February 1, 2006; 131(2): 299 - 310.
[Abstract] [Full Text] [PDF]

Home page
 Reproductive SciencesHome page
T. M. Price, E. L. Hansen, and T. N. Oliver
Immunofluorescent Localization of a Novel Progesterone Receptor(s) in a T47D-Y Breast Cancer Cell Line Lacking Genomic Progesterone Receptor Expression
Reproductive Sciences, December 1, 2005; 12(8): 610 - 616.
[Abstract] [PDF]

Home page
 Hum ReprodHome page
E.V. Younglai, Y.J. Wu, T.K. Kwan, and C.-Y. Kwan
Non-genomic action of estradiol and progesterone on cytosolic calcium concentrations in primary cultures of human granulosa-lutein cells
Hum. Reprod., September 1, 2005; 20(9): 2383 - 2390.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
J. J. Peluso, A. Pappalardo, R. Losel, and M. Wehling
Expression and Function of PAIRBP1 Within Gonadotropin-Primed Immature Rat Ovaries: PAIRBP1 Regulation of Granulosa and Luteal Cell Viability
Biol Reprod, August 1, 2005; 73(2): 261 - 270.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
J.J. Peluso and A. Pappalardo
Progesterone Regulates Granulosa Cell Viability Through a Protein Kinase G-Dependent Mechanism That May Involve 14-3-3{sigma}
Biol Reprod, December 1, 2004; 71(6): 1870 - 1878.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
J. J. Peluso, A. Pappalardo, G. Fernandez, and C. A. Wu
Involvement of an Unnamed Protein, RDA288, in the Mechanism through which Progesterone Mediates Its Antiapoptotic Action in Spontaneously Immortalized Granulosa Cells
Endocrinology, June 1, 2004; 145(6): 3014 - 3022.
[Abstract] [Full Text] [PDF]