| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Ovary |
a Departments of Physiology
b Obstetrics and Gynecology, University of Connecticut Health Center, Farmington, Connecticut 06030
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
apoptosis, granulosa cells, progesterone, progesterone receptor, signal transduction
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
While P4's role in the periovulatory period is well accepted, the ability of P4 to influence granulosa cells isolated prior to ovulation remains somewhat controversial. This controversy is due to the observation that granulosa cells isolated prior to the ovulatory gonadotropin surge do not express the PR [6, 7]. In spite of this, P4 acts directly on granulosa cells of immature rats to modulate their steroidogenic capacity [10, 11] as well as their rate of mitosis [12, 13] and apoptosis [12, 14]. Specifically, granulosa cell mitosis and apoptosis is inhibited by P4 in a steroid-specific, dose-dependent manner, which can be inhibited by a 100-fold molar excess of the PR antagonist, RU 486 [12, 14]. Similarly, P4 inhibits mitosis [15] and apoptosis of spontaneously immortalized granulosa cells (SIGCs) [14], which do not express the PR [15].
The mechanism through which P4 regulates mitosis and apoptosis of granulosa cells of immature rats is ill-defined. In the late 1970s [16, 17] and early 1980s [18], ligand binding studies revealed that immature rat ovaries possessed a P4-binding protein. Recently, 3H-P4 has also been shown to bind to SIGCs in a steroid-specific manner [14]. The KD for this single binding site is approximately 360 nM [14]. Because bicuculline, a gamma aminobutyric acidA (GABAA) receptor antagonist, blocks P4's actions in granulosa cells [12] and P4 modulates GABAA receptor activity [19, 20], it has been proposed that P4 could mediate its actions through the GABAA-like receptor [12]. However, this does not appear to be the case given that a GABAA receptor-activating metabolite of P4 neither displaces 3H-P4 binding nor inhibits granulosa cell and SIGC mitosis as effectively as P4 [15]. In addition, muscimol, a GABAA receptor activator, but not P4 increases cAMP response element binding protein (CREB) phosphorylation in granulosa cells [12].
It is also possible that P4 functions by activating the glucocorticoid receptor (GR). The GR is expressed by granulosa cells [21, 22] and P4 can activate this receptor [21, 22]. This possibility is unlikely, however, because dexamethasone, a GR agonist, does not displace 3H-P4 binding [15] or mimic P4's antiapoptotic [23] and antimitotic action [13].
Interestingly, fluorescein isothiocynate (FITC)-BSA-P4 has been shown to bind to the plasma membrane of granulosa cells [23]. This binding is steroid specific and is displaced by both P4 and an antibody directed against the ligand binding domain of the PR (i.e., C-262) [23]. The C-262 antibody also attenuates P4's antimitotic and antiapoptotic action in granulosa cells [23]. Further, C-262 antibody detects a 60-kDa protein within the membrane fraction of granulosa cells and SIGCs [14]. This protein has been isolated by immunoprecipitation using C-262 and has been shown to bind horseradish peroxidase-conjugated P4 [14]. Moreover, the capacity of horseradish peroxidase-conjugated P4 to bind to this 60-kDa protein is reduced by P4 [14]. Thus, this 60-kDa P4-binding protein (P4BP) could function as a membrane receptor for P4. To gain further insight into this possibility, studies were designed 1) to determine the expression of this 60-kDa P4BP during follicular growth and atresia and 2) to assess the signal transduction pathway(s) that are regulated by P4.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Immature female Wistar rats (22 day 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). At 23 days of age, rats were injected i.p. with either eCG (20 mIU; Sigma Chemical Co., St. Louis, MO) or 0.2 ml of PBS. The rats were killed by cervical dislocation between 0930 and 1000 h at either 0, 2, or 5 days after injection. The ovaries were removed and either fixed in 10% buffered formalin for immunohistochemical evaluation or prepared for Western blot analysis. This protocol was approved by the Animal Care Committee of the University of Connecticut Health Center.
Immunohistochemical and Western Blot Analysis Using C-262
To detect the 60-kDa P4BP, an antibody directed against the ligand binding domain of the nuclear PR was used (i.e., C-262; StressGen, Victoria, BC, Canada). For immunohistochemistry, the ovaries were embedded in paraffin and sectioned at 5 µ. For each ovary, a series of four adjacent sections were taken at two different levels, one level at approximately 25% and another at approximately 50% through the ovary. The first section in each series was stained with hematoxylin and eosin. The second section was stained for apoptotic nuclei using a TUNEL assay (i.e., Apoptag, Intergen Co., Purchase, NY) according to the manufacturer's instructions. The third section was used to localize a C-262 detectable protein using immunohistochemistry, while the fourth section served as a negative control for the immunohistochemical protocol.
For the immunohistochemistry, sections were incubated in 0.3% hydrogen peroxide in methanol for 30 min to quench endogenous peroxidase activity. The sections were then incubated with protease (0.5 mg/ml) for 10 min, rinsed in PBS, and placed in a Target Retrieval Solution (DAKO Corp., Carpinteria, CA) at 95°C for 20 min. The sections were exposed to normal horse serum (1:200 dilution) for 20 min, rinsed, and incubated overnight in a 1:100 dilution of either C-262 (section 3) or IgG (section 4). The C-262 identifiable protein was detected using the Vectastain Elite ABC kit (Vector Labs, Burlingame, CA) with 3,3' diaminobenzidine as a substrate. The presence of a C-262 detectable protein was revealed by a brown precipitate. One ovary from each of the three rats per time point was evaluated with a total of 127 preantral follicles, 62 midsize antral follicles (300–500 µ in diameter), and 35 preovulatory follicles (
500 µ in diameter) examined.
For Western blot analysis, cell lysates were prepared from either whole ovaries or granulosa cells collected from isolated follicles. Whole ovaries were placed in 20 mM 3-[N-morpholino] propanesulfonic acid (MOPS), 5 mM sodium acetate, 0.7 mM EDTA buffer supplemented with the complete protease inhibitor cocktail (Roche Molecular Products, Mannheim, Germany) and homogenized at 4°C. To collect granulosa cells from healthy and atretic follicles, the largest antral follicles were dissected from the ovary under a dissecting microscope. The follicles were then incubated in a 0.04% solution of trypan blue for 5 min at room temperature. The follicles were then observed at a magnification of x40 and atretic follicles identified by the presence of a blue stain within the granulosa cell compartment of the follicle. Once the follicles were classified as either healthy or atretic, the follicles were punctured with 26-gauge needles to release the granulosa cells. Cell lysate was prepared from isolated cells as previously described [14].
Levels of 60-kDa P4 binding protein were assessed by using the C-262 antibody in a Western blot protocol. Briefly, 10–20 µg of protein 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 for 1 h at room temperature and processed for Western blot analysis [14].
Isolation of Membrane-Associated Proteins
To determine whether the 60-kDa P4BP was associated with plasma membrane, the granulosa cells of either healthy or atretic follicles were isolated as outlined above. The membrane proteins were biotinylated using the EZ-link sulfo-NHS-LC-Biotin reagent and protocol provided by Pierce (Rockford, IL; [24]). Once these proteins were biotinylated, cell lysates were prepared as previously described. A sample of this lysate was assessed for the total amount of 60-kDa P4BP by Western blot. To isolate the plasma membrane proteins, the remainder of the sample was affinity-purified using Ultralink Immobilized streptavidin [24]. Twenty micrograms of the affinity-purified sample were then run on a 10% acrylamide gel and the amount of 60 kDa that was present at the plasma membrane determined by Western blot. This protocol was also used to assess the membrane levels of this 60-kDa P4BP in viable SIGCs or SIGCs undergoing apoptosis after serum or P4 withdrawal.
Spontaneously Immortalized Granulosa Cell (SIGC) Culture and Detection of Apoptotic Nuclei
SIGCs were generously provided by Dr. Robert Burghardt of Texas A&M University (College Station, TX) and cultured as previously described [25]. For these experiments, SIGCs were cultured with P4 (1 µM), C-262 (20 µg/ml), IgG (20 µg/ml), or PD58059 at 25 µM as per each experimental design. These reagents were purchased from Sigma Chemical Co. In some experiments, SIGCs were loaded with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) prior to treatment as previously published [26].
After various treatments, the percentage of cells with apoptotic nuclei was assessed by in situ staining using the nuclear dye YOPRO-1 [26]. To stain apoptotic cells, YOPRO-1 was added directly into each culture chamber at a final concentration of 10 µM. The cells were incubated for 10 min at 37°C and then observed at a magnification of x200 under fluorescent optics using the FITC filter set. The number of fluorescent cells (i.e., apoptotic cells) in a field was counted. The total number of cells in that field was counted under phase optics. A total of 100–200 cells per well were counted. The percentage of apoptotic cells was then calculated.
Kinase Screen
The effect of P4 on the level of various kinases was initially assessed through the use of a kinase screen, which was done by Kinexus Bioinformatics Corp. (Vancouver, BC, Canada). For this screen, cell lysates were prepared from SIGCs cultured for 5 and 10 min at room temperature in serum-free P4-free medium or for 10 min in serum-free P4 (1 µM)-supplemented medium. Cell lysates were prepared as outlined in the Kinexus protocol. Details regarding this screen can be found at the URL www.kinexus.ca. Briefly, cells were collected by centrifugation and then suspended in a buffer containing 20 mM MOPS, 2 mM EGTA, 5 mM EDTA, 30 nM sodium fluoride, 10 mM sodium pyrophosphate, 5 µM pepstatin A, 10 µM leupeptin, and 0.5% NP-40 (pH 7.0). The cells were then sonicated twice for 15 sec. This mixture was then centrifuged at 100 000 x g for 30 min. The protein concentration of each lysate was determined by a BCA Assay (Pierce). Then 4x SDS-PAGE buffer (125 mM Tris-HCl, 4% SDS, 50% glycerol, 5% ß-mercaptoethanol, and 0.08% bromophenol blue) was added to yield a final concentration of approximately 600 µg/ml. This lysate was boiled for 4 min and then stored at -20°C until shipped to Kinexus Bioinformatics Corp. for analysis.
Assessment of the Total Amount and Activity of MEK 1/2 and Erk 1 and 2 Kinases
After treatment, cell lysates were prepared either as previously described for the kinase screen or according to the instructions provide by Cell Signaling Inc. (Beverly, MA). Protein determinations were made using the BCA method (Pierce). Typically, 10 µg of lysate was loaded onto each lane and the sample electrophoresed on a 10% polyacrylamide gel at 100 V. Proteins were then transferred to nitrocellulose and incubated with 5% nonfat milk (Nestlé Food Company, Glendale, CA) in Tris-buffered saline with 0.1% Tween-20 (TBS-T) for 1 h with agitation at room temperature. The total amount and activity of mitogen-activated protein kinase kinase (MEK) 1/2 and Erk 1 and 2 was assessed using reagents and protocols provided by Cell Signaling Inc.
To estimate the amount and activity of each kinase, the film from each Western blot was scanned into the computer. The average intensity (grayscale value/pixel) of each band that corresponded to a given kinase was determined using IPGel software (Scanalytics, Vienna, VA). Average intensity of areas adjacent to the bands was also determined and these background values were subtracted from the band intensity. Only films with band intensities within the linear range were assessed (i.e., grayscale values of 250 to 0). Mean intensities of each band ± standard errors for each treatment group were calculated.
Statistical Analysis
All experiments were repeated at least two to three 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 |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
|
To confirm these immunohistochemical observations, granulosa cells were isolated from nonatretic and atretic antral follicles, which were collected 2 and 5 days after eCG injection. As seen in Figure 3A, the same amount of the 60-kDa P4BP was present in lysates of granulosa cells of nonatretic and atretic follicles. However, the amount of this 60-kDa protein that was observed to be at the surface membrane of granulosa cells of nonatretic follicles was considerably greater than that observed in granulosa cells of atretic follicles (Fig. 3B).
|
To determine whether this loss of membrane localization occurs early in the apoptotic cascade, studies were conducted on SIGCs. About 30% of these cells undergo apoptosis within 5 h of being deprived of serum [26]. As shown in Figure 4, serum deprivation did not alter the relative amount of the 60-kDa protein that was at the plasma membrane of SIGCs.
|
Although the membrane localization of this 60-kDa P4BP was not altered, it is possible that, within the first 5 h of initiating apoptosis (i.e., serum withdrawal), P4 becomes uncoupled from its antiapoptotic signal transduction mechanism, which is thought to be mediated through this 60-kDa P4BP. To test this concept, SIGCs were incubated for 15 min in PBS supplemented with either IgG or C-262. Then the IgG or C-262 was removed and the cells were cultured in serum-free medium in the presence or absence of P4. After 5 h, about 35% of the IgG-treated cells were apoptotic, and P4 reduced the percentage of apoptotic nuclei to about 7%. In contrast, pretreatment with C-262 attenuated P4's ability to prevent SIGCs from undergoing apoptosis (Fig. 5A). Interestingly, when SIGCs were not exposed to P4 for as little as 20 min, P4's ability to prevent apoptosis was almost completely lost (Fig. 5B).
|
These observations raised the question as to the nature of the signal transduction cascade that is activated by P4. To begin to address this issue, lysates were prepared from SIGCs, which were cultured in serum-free medium without P4 for 5 and 10 min. As a control, lysates were prepared from SIGCs that were maintained in serum-free P4-supplemented medium for 10 min. A kinase screen on these three lysates was conducted by Kinexus Bioinformatics. This screen revealed that a slightly higher molecular weight form of Erk-1 kinase was detected after 10 min without P4 (Fig. 6, upper panel). Similar changes were observed for Erk-2 (data not shown). Because this slightly higher molecular weight form of Erk-1 could represent activated (i.e., phosphorylated) Erk-1, these lysates were run in a standard Western blot assay. As can be seen in the middle panel of Figure 6, the relative levels of both Erk-1 and Erk-2 remain constant. However, the levels of phosphorylated Erk-1 and -2 were increased in the absence of P4 (Fig. 6, lower panel).
|
Subsequent Western blot studies revealed that, in the absence of P4, the phosphorylated levels of MEK 1/2, Erk-1, and Erk-2 increased in a time-dependent manner (Fig. 7A). This increase in the phosphorylation of these kinases occurred without a corresponding increase in the overall expression level of these kinases (Fig. 7A). Quantitative assessments of these images revealed that, by 10 and 25 min of P4 withdrawal, the amount of phosphorylated MEK 1/2 increased an average of 6- and 12-fold, respectively. Similarly, the level of phosphorylated Erk-1 changed dramatically, going from virtually nondetectable at the beginning of the culture period to a very readily detectable level in 10 min. The amount of phosphorylated Erk-1 then nearly doubled after 25 min compared with the 10-min level (Fig. 7B). A less dramatic increase in phosphorylated Erk-2 was observed, increasing nearly threefold after a 25-min period without P4 (Fig. 7B). Finally, the addition of a MEK inhibitor, PD98059, reduced the percentage of SIGCs that undergo apoptosis by about 50% compared with serum-free control levels (Fig. 8).
|
|
To determine whether P4 becomes uncoupled from its signaling mechanism, P4's ability to suppress MEK 1/2 and Erk-1 and -2 was monitored after 5 or 20 min in serum-free medium. Under these conditions, the total levels of these kinases did not change, but their phosphorylated levels were higher in the 25-min control group compared with the 10-min control group. P4 treatment did not alter the level of phosphorylated MEK 1/2 when administered after 5 or 20 min of serum-free culture. Interestingly, P4 suppressed the levels of phosphorylated Erk-1 when given after 5 but not 20 min of culture in serum-free medium (Fig. 9, A and B).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The developmental expression pattern of this 60-kDa P4BP coincides with an increase in the differentiated state of the follicle. Generally, differentiation is assessed in terms of an increased capacity of granulosa cells to synthesize and secrete steroids [5, 30, 31]. Along with enhanced steroidogenic function, these differentiated granulosa cells show a reduced mitogenic activity [32] and begin to express an endonuclease [33], which is thought to account for their ability to undergo apoptosis. Importantly, small granulosa cells are more mitogenic and undergo apoptosis in vitro less frequently than large granulosa cells [27]. Conversely, large granulosa cells do not seem to proliferate in vitro but undergo apoptosis in vitro [27]. Moreover, P4 inhibits both small granulosa cell mitosis and large granulosa cell apoptosis in vitro [12]. In this light, it is tempting to speculate that the coordinated expression of the 60-kDa P4BP and P4 synthesis sets in place in vivo conditions through which P4 functions to both suppress granulosa cell mitosis and apoptosis through autocrine and paracrine mechanisms.
The ability of P4 to prevent both rat [34] and human [35] granulosa cell apoptosis in vitro is well documented. The observation that C-262 blocks P4's antiapoptotic action in granulosa cells [12] and SIGCs (present study) supports the concept that the 60-kDa P4BP mediates P4's antiapoptotic action. The finding that the membrane localization of the 60-kDa P4BP is lost or reduced in granulosa cells of atretic follicles is consistent with P4's role in maintaining the viability of antral follicles. However, the redistribution of this protein away from the plasma membrane is observed in granulosa cells of follicles that are already atretic. Because most of the granulosa cells of atretic follicles are either apoptotic or undergoing apoptosis, it is difficult to determine whether the loss of membrane localization of this 60-kDa P4BP is a result of or a cause of granulosa cell apoptosis.
To clarify this issue, SIGCs were studied because their viability is maintained by P4 and, in the absence of P4, they undergo apoptosis in a very precise time-dependent manner [26]. Further SIGCs do not express the PR but rather a 60-kDa P4BP [15]. This P4PB localizes to the plasma membrane and appears to be involved in mediating P4's antiapoptotic action, as indicated by the C-262 blocking antibody study. Interestingly, the membrane localization of C-262 is maintained for at least 5 h after the initiation of the apoptotic process (i.e., after withdrawal of serum). However, P4 must activate a survival signal within the first 20 min after initiation of apoptosis in order to prevent SIGCs from undergoing apoptosis. Taken together, these findings suggest that the loss of the 60-kDa P4BP's membrane localization is a consequence and not a cause of apoptosis.
Although the 60-kDa P4BP is present within the membrane of SIGCs undergoing apoptosis, P4's ability to "rescue" these cells from undergoing apoptosis is reduced by 20 min of initiating the apoptotic cascade. The inability to prevent granulosa cell apoptosis appears to be due to P4 becoming "uncoupled" from its antiapoptotic signal transduction cascade. This concept is supported by the findings that 1) continuous exposure to P4 suppresses the phosphorylation of Erk-1 and 2) P4 attenuates the increase in phospho-Erk-1 if administered after 5 min but not after 20 min of P4 deprivation (see subsequent discussion).
The time-dependent nature of P4's action raises the question as to the signal tranduction pathway that is controlled by P4. To begin to address this question, a kinase screen was conducted. This screen revealed that Erk-1 is activated within 10 min of SIGCs being deprived of P4. This finding was confirmed and expanded by standard Western blot analysis, which demonstrated that the phosphorylated levels of MEK 1/2, Erk-1 and -2 increase several fold within 10 min of P4 deprivation. Further, PD98059, a MEK inhibitor, significantly reduces the rate of apoptosis that is observed in the absence of P4. Because Erk-1 and -2 are phosphorylated by MEK 1/2 [36, 37], the PD98059 study implicates the activation of these MAP kinases in the apoptotic cascade.
The concept that SIGCs undergo apoptosis through a MAP kinase-dependent mechanism may appear to be in opposition to our own study [14] as well as others [38, 39]. The studies by Oliver et al. [38] demonstrate that complete inhibition of the Erk pathway by a 60-min pretreatment of human granulosa/luteal cells with a high concentration of PD98059 (100 µM) results in apoptosis. The study of Gebauer and associates [39] reveals that the activity of members of the Ras-MEK-Erk pathway is reduced in atretic follicles. Finally, our previous study demonstrates that PD98059 does not prevent SIGC apoptosis but rather abrogates P4's antiapoptotic action. However, there are two important differences between our previous and present studies. First, the concentration of PD98059 was reduced from 50 µM to 25 µM in the present study. Second, PD98059 was administered 15 min prior to serum withdrawal in the previous study and at the time of serum withdrawal in the present study. These two factors could account for MEK 1/2 kinase activity being reduced to a nondetectable level in our previous study. Apparently, the absence of MEK 1/2 kinase activity results in granulosa cell death, which is consistent with the data obtained by Oliver et al. [38]. In the present study, the lower dose of PD98059 used, which was added at the time of serum withdrawal, most likely neutralized the increase in MAP kinase activity without suppressing MAP kinase levels to the point that promotes cell death. The mechanism by which P4 maintains the activity of the MAP kinase cascade at the set point, which promotes viability without inducing apoptosis or mitosis, is presently under investigation.
The conclusion that SIGC apoptosis is dependent in part on a modest increase in MAP kinase activity may seem unusual because the activation of these MAP kinases is often associated with mitosis and/or cell survival [36, 37]. However, modest increases in the activity of MAP kinases have been associated with cell death. For example, Taxol induces an increase in Erk activity and subsequently apoptosis of MCF-7 cells [40, 41]. Further, Taxol-induced apoptosis was inhibited by the MEK inhibitor, PD98059 [40]. Recently, inhibiting either Rac1 or Cdc42 has been shown to induce murine fibroblast apoptosis. This apoptotic event is dependent on the activation of the Erk, but less than a twofold increase in Erk activity is observed [41]. Collectively, these findings lend credence to our finding that SIGCs undergo apoptosis in a manner that is dependent in part on the activation of the MAP kinases.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence. FAX: 860 679 1269; peluso{at}nso2.uchc.edu
3 Current address: Fertility Physicians of Northern California, 540 University Avenue Suite 250, Palo Alto, CA 94301
Received: 21 May 2002.
First decision: 19 June 2002.
Accepted: 5 August 2002.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  F. Stormshak and C. V. Bishop BOARD-INVITED REVIEW: Estrogen and progesterone signaling: Genomic and nongenomic actions in domestic ruminants J Anim Sci, February 1, 2008; 86(2): 299 - 315. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 S. C. Teilmann, C. A. Clement, J. Thorup, A. G. Byskov, and S. T. Christensen Expression and localization of the progesterone receptor in mouse and human reproductive organs J. Endocrinol., December 1, 2006; 191(3): 525 - 535. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
|
|
N. Gava, C. L. Clarke, K. Byth, R. L. Arnett-Mansfield, and A. deFazio Expression of Progesterone Receptors A and B in the Mouse Ovary during the Estrous Cycle Endocrinology, July 1, 2004; 145(7): 3487 - 3494. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
K. A. Dunlap and F. Stormshak Nongenomic Inhibition of Oxytocin Binding by Progesterone in the Ovine Uterus Biol Reprod, January 1, 2004; 70(1): 65 - 69. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
